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GDIs: Central regulatory molecules in Rho GTPase activation
Abstract
The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPase function. GDIs control the access of Rho GTPases to regulatory guanine nucleotide exchange factors and GTPase-activating proteins, to effector targets and to membranes where such effectors reside. We discuss here our current understanding of how Rho GTPase-GDI complexes are regulated by various proteins, lipids and enzymes that exert GDI displacement activity. We propose that phosphorylation mediated by diverse kinases might provide a means of controlling and coordinating Rho GTPase activation.
... Rab cytosol/membrane cycling is tightly regulated. 17,18,[29][30][31] Rabs are synthesized as soluble proteins; their recruitment to membranes requires geranylgeranyl post-translational modification followed by complex formation of the prenylated Rab with RabGDI (Rab Guanosine Dissociation Inhibitor). The latter protein delivers Rabs to and extracts them from membranes, and is thus a key regulator of Rab activity. ...
... The latter protein delivers Rabs to and extracts them from membranes, and is thus a key regulator of Rab activity. 17,18,31 We analyzed the effect of pancreatitis on Rab9-RabGDI complex formation by using gel filtration of pancreatic cytosolic fraction from rats subjected to CER-AP (Figure 2A-D, Figure 3). Pancreas cytosolic proteins were resolved on a Superdex S200 gel-exclusion column using the SMART system 32 as detailed in Methods; the eluted fractions were analyzed by IB with antibodies against Rab9 and the α/β isoforms of RabGDI ( Figure 2A). ...
... However, the results indicate that pancreatitis does not impair Rab9 prenylation, a prerequisite for Rab complex formation with RabGDI. [30][31][32] The levels of both membrane and cytosolic Rabs are regulated by ubiquitin-proteasomal system; 29,33 in particular, proteasomal degradation eliminates protein aggregates formed by Rabs that excessively accumulate in the cytosol (though not shown for Rab9). Notably, CCK-induced Rab9 decrease in ex vivo pancreatitis was entirely prevented by the proteasomal inhibitor MG132 ( Figure 2E,F), implicating the above mechanism. ...
Background
Autophagosome, the central organelle in autophagy process, can assemble via canonical pathway mediated by LC3-II, the lipidated form of autophagy-related protein LC3/ATG8, or noncanonical pathway mediated by the small GTPase Rab9. Canonical autophagy is essential for exocrine pancreas homeostasis, and its disordering initiates and drives pancreatitis. The involvement of noncanonical autophagy has not been explored. We examine the role of Rab9 in pancreatic autophagy and pancreatitis severity.
Methods
We measured the effect of Rab9 on parameters of autophagy and pancreatitis responses using transgenic mice overexpressing Rab9 (Rab9TG) and adenoviral transduction of acinar cells. Effect of canonical autophagy on Rab9 was assessed in ATG5-deficient acinar cells.
Results
Pancreatic levels of Rab9 and its membrane-bound (active) form decreased in rodent pancreatitis models and in human disease. Rab9 overexpression stimulated noncanonical and inhibited canonical/LC3-mediated autophagosome formation in acinar cells through upregulation of ATG4B, the cysteine protease that delipidates LC3-II. Conversely, ATG5 deficiency caused Rab9 increase in acinar cells. Inhibition of canonical autophagy in Rab9TG pancreas was associated with accumulation of Rab9-positive vacuoles containing markers of mitochondria, protein aggregates and trans-Golgi. The shift to the noncanonical pathway caused pancreatitis-like damage in acinar cells and aggravated experimental pancreatitis.
Conclusions
The results show that Rab9 regulates pancreatic autophagy and indicate a mutually antagonistic relationship between the canonical/LC3-mediated and noncanonical/Rab9-mediated autophagy pathways in pancreatitis. Noncanonical autophagy fails to substitute for its canonical counterpart in protecting against pancreatitis. Thus, Rab9 decrease in experimental and human pancreatitis is a protective response to sustain canonical autophagy and alleviate disease severity.
... In a resting cell, the majority of the Rac1 GTPase is inactive. In this inactive state, Rac1 is bound to a Guanine nucleotide Dissociation Inhibitor (GDI) which prevents both its interaction with the plasma membrane and its activation by GEFs [4,5]. Rho GTPases require to be associated to a membrane to release the RhoGDI and undergo GEF-mediated activation [5]. ...
... In this inactive state, Rac1 is bound to a Guanine nucleotide Dissociation Inhibitor (GDI) which prevents both its interaction with the plasma membrane and its activation by GEFs [4,5]. Rho GTPases require to be associated to a membrane to release the RhoGDI and undergo GEF-mediated activation [5]. Whereas most models of the Rac1 activation cycle suggest that active Rac1 is not bound by the GDI, several papers have shown an interaction between a constitutively active Rac1 (G12V) and the GDI and a lack of GDI binding by the constitutively inactive mutant of Rac1 (T17N) [6][7][8]. ...
... It is widely accepted that Rac1-GDP binds to the GDI, whereas Rac1-GTP is not GDI bound, and can therefore associate with the plasma membrane and downstream effectors [5]. We investigated GDI binding of the mCherry-Rac1 activating mutants by performing a mCherry Co-IP with endogenous GDI protein in HEK293T cells (Fig 3A and 3B). ...
Signaling by the Rho GTPase Rac1 is key to the regulation of cytoskeletal dynamics, cell spreading and adhesion. It is widely accepted that the inactive form of Rac1 is bound by Rho GDI, which prevents Rac1 activation and Rac1-effector interactions. In addition, GDI-bound Rac1 is protected from proteasomal degradation, in line with data showing that Rac1 ubiquitination occurs exclusively when Rac1 is activated. We set out to investigate how Rac1 activity, GDI binding and ubiquitination are linked. We introduced single amino acid mutations in Rac1 which differentially altered Rac1 activity, and compared whether the level of Rac1 activity relates to Rac1 ubiquitination and GDI binding. Results show that Rac1 ubiquitination and the active Rac1 morphology is proportionally increased with Rac1 activity. Similarly, we introduced lysine-to-arginine mutations in constitutively active Rac1 to inhibit site-specific ubiquitination and analyze this effect on Rac1 signaling output and ubiquitination. These data show that the K16R mutation inhibits GTP binding, and consequently Rac1 activation, signaling and–ubiquitination, while the K147R mutation does not block Rac1 signaling, but does inhibits its ubiquitination. In both sets of mutants, no direct correlation was observed between GDI binding and Rac1 activity or -ubiquitination. Taken together, our data show that a strong, positive correlation exists between Rac1 activity and its level of ubiquitination, but also that GDI dissociation does not predispose Rac1 to ubiquitination.
... RhoGDI is a constitutive inhibitor of RhoA activation that normally prevents PMEC permeability and pulmonary edema (48, 50, 53-57, 67, 81, 107). RhoGDI binds RhoA in the cytoplasm of PMECs and prevents RhoA activation and RhoA prenyl group attachment to the plasma membrane (50,54,59). ...
... In addition, there are many cell signaling pathways that act on RhoGDI to positively or negatively control RhoA or Rac1 activation (50, 52-56, 58, 111, 112). Thus, the roles of RhoGDI in regulating RhoGTPase responses are complicated by signaling pathway phosphorylation of RhoGDI that results in the selective release of RhoA or Rac1 and controls the balance of barrier integrity and permeability (50,(52)(53)(54)(55)(56). RhoGDI is reportedly phosphorylated on S34, S96, S101, and S174, with discrete interactions and GTPase activation responses directed by each (50,52,54,56,58,73,110). P-21-activated kinase (PAK1) reportedly phosphorylates S101 and S174 residues on RhoGDI, selectively reducing the affinity for Rac1 but not RhoA and resulting in Rac1 activation and enhanced EC barrier function (51,52,54,58,61,63,76,110). ...
... Thus, the roles of RhoGDI in regulating RhoGTPase responses are complicated by signaling pathway phosphorylation of RhoGDI that results in the selective release of RhoA or Rac1 and controls the balance of barrier integrity and permeability (50,(52)(53)(54)(55)(56). RhoGDI is reportedly phosphorylated on S34, S96, S101, and S174, with discrete interactions and GTPase activation responses directed by each (50,52,54,56,58,73,110). P-21-activated kinase (PAK1) reportedly phosphorylates S101 and S174 residues on RhoGDI, selectively reducing the affinity for Rac1 but not RhoA and resulting in Rac1 activation and enhanced EC barrier function (51,52,54,58,61,63,76,110). ...
Andes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs) causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients virtually every PMEC is infected, however the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase, RhoA, in primary human PMECs causing VE-Cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA, but co-precipitates RhoGDI (Rho GDP-dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C-terminus (69-204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N-terminus (1-69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and VEGF increase RhoA induced PMEC permeability by directing Protein Kinase Cα (PKCα) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF activated PKCα, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKCα and opposing Protein Kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV induced capillary permeability, edema and HPS.
Importance
HPS causing hantaviruses infect pulmonary endothelial cells causing vascular leakage, pulmonary edema and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses don't lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34 phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC phosphorylated RhoGDI, synergistically enhancing hypoxia directed RhoA activation and PMEC permeability. Our data suggests inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.
... It was identified that RhoGDIs function as a down-regulator of the Rho family GTPases, preventing nucleotide exchange and membrane association [12,26]. In Arabidopsis, three RhoGDI homologs were identified. ...
... In several stages of plant growth and development, such as root growth, leaf morphogenesis, sexual reproduction, and immunity, Rho GTPases play functional switches [3,4,7,46]. Rho GTPase regulators GAPs, GEFs, and GDIs control the cycling between the active GTP-bound and inactive GDP-bound forms [9,10,12]. Although many species have had their GAPs, GEFs, and GDIs functions studied, the Rosaceae species have received relatively little attention. ...
The primary regulators of Rho GTPases are GTPase-activating protein (GAP), guanine nucleotide exchange factor (GEF), and GDP dissociation inhibitor (GDI), which function as signaling switches in several physiological processes involved in plant growth and development. This study compared how the Rho GTPase regulators functioned in seven Rosaceae species. Seven Rosaceae species, divided into three subgroups, had a total of 177 regulators of Rho GTPases. According to duplication analysis, the expansion of GEF, GAP, and GDI families was facilitated by whole genome duplication or a dispersed duplication event. The balance of cellulose deposition to control the growth of the pear pollen tube, as demonstrated by the expression profile and antisense oligonucleotide approach. Moreover, protein-protein interactions indicated that PbrGDI1 and PbrROP1 could directly interact, suggesting that PbrGDI1 regulated the growth of the pear pollen tube through PbrROP1 signaling downstream. These results lay the foundations for future functional characterization of the GAP, GEF, and GDI gene families in Pyrus bretschneideri.
... Only three human RhoGDIs have been found, compared to the large number of GEF and GAP regulators (each with about 60 members): RhoGDI1 (also known as RhoGDI or RhoGDIα), RhoGDI2 (Ly-GDI, D4-GDI, or RhoGDIβ), and RhoGDI3 (RhoGDIγ) [5][6][7]. RhoGDIs were originally considered to be negative regulators of Rho GTPases because they bind to the majority of Rho GTPases in the cytoplasm and keep them inactive, preventing any interaction with the target effector proteins [8,9]. However, RhoGDIs have been associated with the active forms of Rho, Rac, and Cdc42 in other reports, suggesting that they also act as positive regulators of Rho GTPases [10,11]. ...
... The explanation of the disparity in RhoGDI2 s participation in different cancers remains unclear; however, the dual role of RhoGDI in controlling the activity of Rho GTPase during cancer progression may be one of the plausible reasons. RhoGDI was identified as a negative regulator of Rho GTPases at first [8,9]. RhoGDI caused the loss of Rho-dependent cell activity, such as cytoskeletal activity and motility, when exogenously introduced into cells [25]. ...
Simple Summary
Rho GDP dissociation inhibitor 2 (RhoGDI2), a regulator of Rho family GTPase, has been known to promote tumor growth and malignant progression by activating Rac1 in gastric cancer. However, the precise molecular mechanism by which RhoGDI2 activates Rac1 in gastric cancer cells remains unclear. In this study, we found that interaction between RhoGDI2 and Rac1 is a prerequisite for the recruitment of Rac1 to Filamin A. Moreover, we found that Filamin A acts as a scaffold protein that mediates Rac1 activation. Furthermore, we found that Trio, a Rac1-specific GEF, is critical for Rac1 activation in gastric cancer cells. Conclusively, RhoGDI2 increases Rac1 activity by recruiting Rac1 to Filamin A and enhancing the interaction between Rac1 and Trio, which is critical for invasive ability of gastric cancer cells. Our findings suggest that RhoGDI2 might be a potential therapeutic target for reducing gastric cancer cell metastasis.
Abstract
Rho GDP dissociation inhibitor 2 (RhoGDI2), a regulator of Rho family GTPase, has been known to promote tumor growth and malignant progression in gastric cancer. We previously showed that RhoGDI2 positively regulates Rac1 activity and Rac1 activation is critical for RhoGDI2-induced gastric cancer cell invasion. In this study, to identify the precise molecular mechanism by which RhoGDI2 activates Rac1 activity, we performed two-hybrid screenings using yeast and found that RhoGDI2 plays an important role in the interaction between Rac1, Filamin A and Rac1 activation in gastric cancer cells. Moreover, we found that Filamin A is required for Rac1 activation and the invasive ability of gastric cancer cells. Depletion of Filamin A expression markedly reduced Rac1 activity in RhoGDI2-expressing gastric cancer cells. The migration and invasion ability of RhoGDI2-expressing gastric cancer cells also substantially decreased when Filamin A expression was depleted. Furthermore, we found that Trio, a Rac1-specific guanine nucleotide exchange factor (GEF), is critical for Rac1 activation and the invasive ability of gastric cancer cells. Therefore, we conclude that RhoGDI2 increases Rac1 activity by recruiting Rac1 to Filamin A and enhancing the interaction between Rac1 and Trio, which is critical for the invasive ability of gastric cancer cells.
... Understanding the mechanisms by which signaling events are localized and the physiological consequences of spatial restriction are exerted, is one of the major challenges in cell biology. Comprehensive studies in the last three decades have provided insight into the structure and function of these regulators acting as a shuttle for the RHO GTPases [13,[25][26][27]. The shuttling process, which considerably differs from the KRAS4B Far -PDEδ [28][29][30][31], involves the extraction of RHO GTPases from donor membranes, formation of cytosolic GDI-RHO GTPase complexes and delivery of RHO GTPases to the target membranes [13,27]. ...
... Comprehensive studies in the last three decades have provided insight into the structure and function of these regulators acting as a shuttle for the RHO GTPases [13,[25][26][27]. The shuttling process, which considerably differs from the KRAS4B Far -PDEδ [28][29][30][31], involves the extraction of RHO GTPases from donor membranes, formation of cytosolic GDI-RHO GTPase complexes and delivery of RHO GTPases to the target membranes [13,27]. Accordingly, it has been proposed that GDI regulates the isoprenylation process in the cell [32]. ...
Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylger-anylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.
... Several studies in recent decades have provided information about the structure and function of GDIs and proposed that they act as shuttles for RHO GTPase [8,[66][67][68]. The shuttling process is initiated by the release of RHO GTPases from donor membranes, the formation of inhibitory cytosolic GDI-RHO GTPase complexes, and the delivery of RHO GTPases to the membranes of subcellular compartments [66,67]. ...
... Several studies in recent decades have provided information about the structure and function of GDIs and proposed that they act as shuttles for RHO GTPase [8,[66][67][68]. The shuttling process is initiated by the release of RHO GTPases from donor membranes, the formation of inhibitory cytosolic GDI-RHO GTPase complexes, and the delivery of RHO GTPases to the membranes of subcellular compartments [66,67]. ...
Much progress has been made toward deciphering RHO GTPase functions, and many studies have convincingly demonstrated that altered signal transduction through RHO GTPases is a recurring theme in the progression of human malignancies. It seems that 20 canonical RHO GTPases are likely regulated by three GDIs, 85 GEFs, and 66 GAPs, and eventually interact with >70 downstream effectors. A recurring theme is the challenge in understanding the molecular determinants of the specificity of these four classes of interacting proteins that, irrespective of their functions, bind to common sites on the surface of RHO GTPases. Identified and structurally verified hotspots as functional determinants specific to RHO GTPase regulation by GDIs, GEFs, and GAPs as well as signaling through effectors are presented, and challenges and future perspectives are discussed.
... This cycling is controlled mainly by activating RhoGEFs that promote the exchange of GDP for GTP and inactivating RhoGAPs that enhance GTP hydrolysis 6,9,[11][12][13][14][15][16][17] . Additionally, RhoGDIs bind and sequester inactive RhoA in the cytoplasm and extract RhoA from the membrane following GTP hydrolysis 6,[18][19][20][21][22] . Together, these proteins respond to diverse signaling events to finely tune RhoA activity. ...
Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to other kinases. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity, and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.
... The GTP/GDP turnover process is controlled by GTPase activating proteins (GAPs) and guanine exchange factors (GEFs) (Mishra & Lambright, 2016;Toma-Fukai & Shimizu, 2019). In addition, the GDP dissociation inhibitors (GDIs) regulate the interactions between Rho-GTPases and their protein regulators by controlling their cellular membrane trafficking, where GEFs and GAPs exert their function (DerMardirossian & Bokoch, 2005). Anomalies in this sophisticated regulatory mechanism, and, thus an imbalance in the ratio of the GTPases on/off states, lead to pathological conditions. ...
Rho‐GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer‐associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all‐atom molecular dynamics simulations on wild‐type (wt) and oncogenic isoforms of this protein in GDP‐ and GTP‐bound states. Our results unprecedentedly elucidate that P29Q/S‐induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg²⁺ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer‐associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.
... Rho guanine nucleotide dissociation inhibitors (GDIs) and their interactions with Rho family proteins have known involvement in several malignancies, though their specific activity and enrichment differ between cancer types [45]. GDIs are involved in the regulation of Rho activity through inhibition of GTP binding [46] and/or extraction of membrane-bound Rho GTPases for storage of the inactivated protein in the cytosol, while protecting the cytosolic Rho GTPases from proteolytic degradation [47,48]. In our study, we observed that Rho GDI signaling was significantly enriched in USC ET, while RhoA signaling was conversely enriched in USC ES. ...
Background
Although uterine serous carcinoma (USC) represents a small proportion of all uterine cancer cases, patients with this aggressive subtype typically have high rates of chemotherapy resistance and disease recurrence that collectively result in a disproportionately high death rate. The goal of this study was to provide a deeper view of the tumor microenvironment of this poorly characterized uterine cancer variant through multi-region microsampling and quantitative proteomics.
Methods
Tumor epithelium, tumor-involved stroma, and whole “bulk” tissue were harvested by laser microdissection (LMD) from spatially resolved levels from nine USC patient tumor specimens and underwent proteomic analysis by mass spectrometry and reverse phase protein arrays, as well as transcriptomic analysis by RNA-sequencing for one patient’s tumor.
Results
LMD enriched cell subpopulations demonstrated varying degrees of relatedness, indicating substantial intratumor heterogeneity emphasizing the necessity for enrichment of cellular subpopulations prior to molecular analysis. Known prognostic biomarkers were quantified with stable levels in both LMD enriched tumor and stroma, which were shown to be highly variable in bulk tissue. These USC data were further used in a comparative analysis with a data generated from another serous gynecologic malignancy, high grade serous ovarian carcinoma, and have been added to our publicly available data analysis tool, the Heterogeneity Analysis Portal ( https://lmdomics.org/ ).
Conclusions
Here we identified extensive three-dimensional heterogeneity within the USC tumor microenvironment, with disease-relevant biomarkers present in both the tumor and the stroma. These data underscore the critical need for upfront enrichment of cellular subpopulations from tissue specimens for spatial proteogenomic analysis.
... Activation and inhibition of RhoA is mediated through the tightly regulated cycling of accessory proteins: guanine nucleotide dissociation inhibitor (GDI), which retains Rho in a GDP bound (inactive) state; GTPase activating proteins (GAPs), which stimulate hydrolysis of GTP to GDP; and guanine nucleotide exchange factors (GEFs), which activate GTPase to release the GDP bound to RhoA (Ridley and Hall, 1992). One of the factors that mediates RhoA activation/inactivation is cyclic AMP-dependent protein kinase A (PKA) (DerMardirossian and Bokoch, 2005;Qiao et al., 2003;Tkachenko et al., 2011). PKA plays a role in diverse cellular activities and is reported to exhibit negative temporal and spatial correlation with the activation of RhoA (Tkachenko et al., 2011). ...
Cutaneous melanomas harboring a B-RafV600E mutation are treated with immune check point inhibitors or kinase inhibitor combination therapies relying on MAPK inhibitors (MAPKi) Dabrafenib and Trametinib (Curti and Faries, 2021). However, cells become resistant to treatments over the timespan of a few months. Resistance to MAPKi has been associated with adoption of an aggressive amoeboid phenotype characterized by elevated RhoA signaling, enhanced contractility and thick cortical filamentous actin (F-actin) structures (Kim et al., 2016; Misek et al., 2020). Targeting active RhoA through Rho-kinase (ROCK) inhibitors, either alone or in combination with immunotherapies, reverts MAPKi-resistance (Misek et al., 2020; Orgaz et al., 2020). Yet, the mechanisms for this behavior remain largely unknown. Given our recent findings of cytoskeletons role in cancer cell proliferation (Mohan et al., 2019), survival (Weems et al., 2023), and metabolism (Park et al., 2020), we explored possibilities by which RhoA-driven changes in cytoskeleton structure may confer resistance. We confirmed elevated activation of RhoA in a panel of MAPKi-resistant melanoma cell lines, leading to a marked increase in the presence of contractile F-actin bundles. Moreover, these cells had increased glucose uptake and glycolysis, a phenotype disrupted by pharmacological perturbation of ROCK. However, glycolysis was unaffected by disruption of F-actin bundles, indicating that glycolytic stimulation in MAPKi-resistant melanoma is independent of F-actin organization. Instead, our findings highlight a mechanism in which elevated RhoA signaling activates ROCK, leading to the activation of insulin receptor substrate 1 (IRS1) and P85 of the PI3K pathway, which promotes cell surface expression of GLUT1 and elevated glucose uptake. Application of ROCK inhibitor GSK269962A results in reduced glucose uptake and glycolysis, thus impeding cell proliferation. Our study adds a mechanism to the proposed use of ROCK inhibitors for long-term treatments on MAPKi-resistant melanomas.
... As with our previous work [28], here we adopt the biochemical reaction-diffusion equations introduced in [63,64] to describe the dynamics of active and inactive GTPases. Since the active forms of the GTPases are predominantly associated with the cell membrane, which also serves as a major site for the conversion between active and inactive forms, we track the volume fractions of the signalling proteins in three forms [65,66]: the active membrane-bound form ðG i a ðtÞ ; fR i a ðtÞ, r i a ðtÞgÞ, the inactive membrane-bound form ðG i in ðtÞ ; fR i in ðtÞ, r i in ðtÞgÞ, and the inactive cytosolic form (G cp (t) ≡ {R cp (t), ρ cp (t)}). Given the rapid diffusion of inactive proteins in the cytosol, we assume G cp (t) remains uniformly distributed in the cytosol at all times. ...
We present a chemomechanical whole-cell theory for the spreading and migration dynamics of mesenchymal cells that can actively reinforce their adhesion to an underlying viscoelastic substrate as a function of its stiffness. Our multiscale model couples the adhesion reinforcement effect at the subcellular scale with the nonlinear mechanics of the nucleus–cytoskeletal network complex at the cellular scale to explain the concurrent monotonic area–stiffness and non-monotonic speed–stiffness relationships observed in experiments: we consider that large cell spreading on stiff substrates flattens the nucleus, increasing the viscous drag force on it. The resulting force balance dictates a reduction in the migration speed on stiff substrates. We also reproduce the experimental influence of the substrate viscosity on the cell spreading area and migration speed by elucidating how the viscosity may either maintain adhesion reinforcement or prevent it depending on the substrate stiffness. Additionally, our model captures the experimental directed migration behaviour of the adhesion-reinforced cells along a stiffness gradient, known as durotaxis, as well as up or down a viscosity gradient (viscotaxis or anti-viscotaxis), the cell moving towards an optimal viscosity in either case. Overall, our theory explains the intertwined mechanics of the cell spreading, migration speed and direction in the presence of the molecular adhesion reinforcement mechanism.
... GEF activates a monomeric Rho GTPase by stimulating the release of GDP to allow the binding of GTP. RhoGDI binds to a GDP-bound ''off'' state Rho GTPase and prevents the conversion of the ''off'' state to the ''on'' state (11)(12)(13)(14)(15). Also, it prevents the Rho GTPases from localizing at the membrane, which is the place of their action. ...
... Rho GTPase GDP dissociation inhibitors (RhoGDIs) play significant roles in many cellular responses and are conserved in many species. RhoGDIs are typically negative regulators of Rho GTPases, preventing GTP-GDP nucleotide exchange and affecting targeting and activation of downstream effectors (17,18). However, they also play a positive role by stabilizing inactive Rho GTPases in the cytoplasm and protecting them from degradation (19). ...
To ensure optimal growth, plants actively regulate their growth and development based on environmental changes. Among these, salt stress significantly influences growth and yield. In this study, we demonstrate that the growth of root hairs of salt-stressed Arabidopsis thaliana seedlings is regulated by the SALT OVERLY SENSITIVE 2 (SOS2)-GUANOSINE NUCLEOTIDE DIPHOSPHATE DISSOCIATION INHIBITOR 1 (RhoGDI1)-Rho GTPASE OF PLANTS 2 (ROP2) module. We show here that the kinase SOS2 is activated by salt stress and subsequently phosphorylates RhoGDI1, a root hair regulator, thereby decreasing its stability. This change in RhoGDI1 abundance resulted in a fine-tuning of polar localization of ROP2 and root hair initiation followed by polar growth, demonstrating how SOS2-regulated root hair development is critical for plant growth under salt stress. Our results reveal how a tissue-specific response to salt stress balances the relationship of salt resistance and basic growth.
... Thus, β8-mediated mechanotransduction does not seem to use talin or kindlin for RhoA regulation. Rho GDP dissociation inhibitor (RhoGDI) is an important cytoplasmic protein that inactivates RhoGTPases [35]. Upon RhoGDI binding, Rho GTPases are present in the cytoplasm; however, phosphorylation of RhoGDI by Sac or other kinases releases RhoGDI from RhoGTPases and allows RhoGTPase to be inserted into the plasma membrane for functional exertion. ...
Solid tumor cells live in a highly dynamic mechanical microenvironment. How the extracellular-matrix-generated mechanotransduction regulates tumor cell development and differentiation remains an enigma. Here, we show that a low mechanical force generated from the soft matrix induces dedifferentiation of moderately stiff tumor cells to soft stem-cell-like cells. Mechanistically, integrin β8 was identified to transduce mechano-signaling to trigger tumor cell dedifferentiation by recruiting RhoGDI1 to inactivate RhoA and subsequently Yes-associated protein (YAP). YAP inactivation relieved the inhibition of v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG), allowing MAFG to transactivate the stemness genes NANOG, SOX2, and NESTIN. Inactivation also restored β8 expression, thereby forming a closed mechanical loop. Importantly, MAFG expression is correlated with worse prognosis. Our findings provide mechanical insights into the regulation of tumor cell dedifferentiation, which has therapeutic implications for exploring innovative strategies to attack malignancies.
... Rac GTPases are activated by guanosine nucleotide exchange factors (GEFs) with a GTP-bound state and are inactivated by GTPases activating proteins (GAPs) with a GDP-bound state [107]. Rac GTPase activity is negatively controlled by guanine nucleotide dissociation inhibitors (GDIs) via the sequestration of inactive Rac GTPases in the cytoplasm [108]. This cycle is presumably regulated by these GTPase effectors through the translocation to membranes, relief of auto-inhibitory intramolecular interactions, and conformational change [109]. ...
Animals are required to handle daily massive amounts of information in an ever-changing environment, and the resulting memories and experiences determine their survival and development, which is critical for adaptive evolution. However, intrinsic forgetting, which actively deletes irrelevant information, is equally important for memory acquisition and consolidation. Recently, it has been shown that Rac1 activity plays a key role in intrinsic forgetting, maintaining the balance of the brain's memory management system in a controlled manner. In addition, dysfunctions of Rac1-dependent intrinsic forgetting may contribute to memory deficits in neurological and neurodegenerative diseases. Here, these new findings will provide insights into the neurobiology of memory and forgetting, pathological mechanisms and potential therapies for brain disorders that alter intrinsic forgetting mechanisms.
... Correspondingly, overexpression of RhoGDIα inhibited Rac1 activity. Aligning with our results, RhoGDIα reportedly exists in a heterodimer with Rac1 and RhoA in several nonmuscle cells (24,31,56,57) and GTP-bound Rac1 is increased in kidneys of RhoGDIα −/− mice (58). Mechanistically, our findings suggest that insulin-induced Rac1-RhoGDIα dissociation in response to insulin is mediated by phosphorylation of RhoGDIα S101. ...
The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.
... CDK1 is the checkpoint protein for the cell cycle from G2 to the M phase and is involved in various tumorigenic processes (Nigg 2001). RhoA, a small molecule G protein/GTPase, can modulate the cytoskeleton and affect cell migration by activating Rho-associated protein kinase (ROCK) and other effectors (Hall 1998;DerMardirossian and Bokoch 2005). Overexpression of circ-NOLC1 can bind to ESRP1 and upregulate the expression of CDK1 and RhoA, thereby promoting OC proliferation, migration, and invasion . ...
Background
The nucleolus is considered the center of metabolic control and an important organelle for the biogenesis of ribosomal RNA (rRNA). Nucleolar and coiled-body phosphoprotein 1(NOLC1), which was originally identified as a nuclear localization signal-binding protein is a nucleolar protein responsible for nucleolus construction and rRNA synthesis, as well as chaperone shuttling between the nucleolus and cytoplasm. NOLC1 plays an important role in a variety of cellular life activities, including ribosome biosynthesis, DNA replication, transcription regulation, RNA processing, cell cycle regulation, apoptosis, and cell regeneration.
Purpose
In this review, we introduce the structure and function of NOLC1. Then we elaborate its upstream post-translational modification and downstream regulation. Meanwhile, we describe its role in cancer development and viral infection which provide a direction for future clinical applications.
Methods
The relevant literatures from PubMed have been reviewed for this article.
Conclusion
NOLC1 plays an important role in the progression of multiple cancers and viral infection. In-depth study of NOLC1 provides a new perspective for accurate diagnosis of patients and selection of therapeutic targets.
... GDP dissociation inhibitors (GDIs, another type regulators of Rho GTPases) can extract the Cdc42 from membrane to cytosol by binding the geranylgeranyl group of the nonstandard Cys188 to control its subcellular distribution and its cycle between membrane-associated (active) and cytosolic (inactive) states [12]. In cytosol, GDIs bind the inactive GDP-bound form of Cdc42 to maintain Cdc42 in an inactive state. ...
Cell division control protein 42 homolog (Cdc42), which controls a variety of cellular functions including rearrangements of the cell cytoskeleton, cell differentiation and proliferation, is a potential cancer therapeutic target. As an endogenous negative regulator of Cdc42, the Rho GDP dissociation inhibitor 1 (RhoGDI1) can prevent the GDP/GTP exchange of Cdc42 to maintain Cdc42 into an inactive state. To investigate the inhibition mechanism of Cdc42 through RhoGDI1 at the atomic level, we performed molecular dynamics (MD) simulations. Without RhoGDI1, Cdc42 has more flexible conformations, especially in switch regions which are vital for binding GDP/GTP and regulators. In the presence of RhoGDI1, it not only can change the intramolecular interactions of Cdc42 but also can maintain the switch regions into a closed conformation through extensive interactions with Cdc42. These results which are consistent with findings of biochemical and mutational studies provide deep structural insights into the inhibition mechanisms of Cdc42 by RhoGDI1. These findings are beneficial for the development of novel therapies targeting Cdc42-related cancers.
... Notably, actin cytoskeleton signaling and RhoA signaling were upregulated after 6 h, whereas RhoGDI was downregulated owing to an increase in the key regulator F-actin. RhoA acts as an on/off switch, whereas RhoGDI acts to stop this switch (DerMardirossian and Bokoch, 2005). Thus, suppression of RhoGDI signaling was predicted to activate the RhoA switch, indicating enhanced turnover within the brain. ...
Organisms adapt to changes in their environment to survive. The emergence of predators is an example of environmental change, and organisms try to change their external phenotypic systems and physiological mechanisms to adapt to such changes. In general, prey exhibit different phenotypes to predators owing to historically long-term prey-predator interactions. However, when presented with a novel predator, the extent and rate of phenotypic plasticity in prey are largely unknown. Therefore, exploring the physiological adaptive response of organisms to novel predators is a crucial topic in physiology and evolutionary biology. Counterintuitively, Xenopus tropicalis tadpoles do not exhibit distinct external phenotypes when exposed to new predation threats. Accordingly, we examined the brains of X. tropicalis tadpoles to understand their response to novel predation pressure in the absence of apparent external morphological adaptations. Principal component analysis of fifteen external morphological parameters showed that each external morphological site varied nonlinearly with predator exposure time. However, the overall percentage change in principal components during the predation threat (24 h) was shown to significantly ( p < 0.05) alter tadpole morphology compared with that during control or 5-day out treatment (5 days of exposure to predation followed by 5 days of no exposure). However, the adaptive strategy of the altered sites was unknown because the changes were not specific to a particular site but were rather nonlinear in various sites. Therefore, RNA-seq, metabolomic, Ingenuity Pathway Analysis, and Kyoto Encyclopedia of Genes and Genomes analyses were performed on the entire brain to investigate physiological changes in the brain, finding that glycolysis-driven ATP production was enhanced and ß -oxidation and the tricarboxylic acid cycle were downregulated in response to predation stress. Superoxide dismutase was upregulated after 6 h of exposure to new predation pressure, and radical production was reduced. Hemoglobin was also increased in the brain, forming oxyhemoglobin, which is known to scavenge hydroxyl radicals in the midbrain and hindbrain. These suggest that X. tropicalis tadpoles do not develop external morphological adaptations that are positively correlated with predation pressure, such as tail elongation, in response to novel predators; however, they improve their brain functionality when exposed to a novel predator.
... GEF activates a monomeric Rho GTPase by stimulating the release of GDP to allow the binding of GTP. RhoGDI binds to a GDP-bound "off" state Rho GTPase and prevents the conversion of the "off" state to the "on'' state (11)(12)(13)(14)(15). Also, it prevents the Rho GTPases from localizing at the membrane which is the place of their action. ...
Rho–specific guanine dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP–bound state of GTPase. Release of Rho GTPase from the RhoGDI–bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code", where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42 etc.). This work attempts to understand the molecular mechanism behind this phosphorylation code. Using several microseconds long atomistic molecular dynamics (MD) simulations of the wild–type and phosphorylated states of the RhoA–RhoGDI complex, we propose a molecular–interaction–based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N–terminal region of RhoGDI that contribute most to the binding affinity. MM–PBSA binding free energy calculations show a significant weakening of interaction on phosphorylation at the RhoA–specific site of RhoGDI. In contrast, phosphorylation at a Rac1–specific site leads to the strengthening of the interaction confirming the presence of a phosphorylation code. RhoA–specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N–terminal region of RhoGDI, which manifests reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.
... The dissociation of GDP from Rho proteins is inhibited by GDIs, preventing GTPase activation by GEFs. Finally, the GDIs are able to interact with the GTP-bound form of the GTPase to prevent interactions with effector targets [41,42]. ...
The cell membranes consist of lipid bilayers that are semipermeable. The semipermeable nature enables the cell membranes to regulate the transport of materials entering and exiting the cell. Apart from providing protection and a fixed environment to the cell, the cell membrane has several functions. The covalently linked proteins to lipids on the surface of the cell membranes are the Lipid-anchored proteins. The function of the protein to which the lipid is attached depends on the type of the lipid. Prenylated proteins, fatty acylated proteins, and glycosylphosphatidylinositol-linked proteins (GPI) are the three main types of lipid-anchored proteins on the cell membrane. In particular, the prenylated proteins are very important for cell growth, differentiation, and morphology. The dynamic interaction of prenylated proteins with the cell membrane is important for their signaling functions and is often deregulated in disease processes, such as cancer. An understanding of the prenylated proteins and their mechanisms is important for drug development efforts to combat cancer.
... Notably, actin cytoskeleton signaling and RhoA signaling were upregulated, while RhoGDI was downregulated after 6 hr, since the key regulator F-actin was increased. RhoA acts as an on/off switch, while RhoGDI acts to stop this RhoA switch [35] . Thus, suppression of RhoGDI signaling activates RhoA switching, indicating that brain turnover may be enhanced. ...
Predator-induced adaptive phenotypic plasticity is essential for evolution. However, Xenopus tropicalis tadpoles do not exhibit distinct phenotypes when exposed to new predation threats. Here, we investigated adaptions within their brain. Principal component analysis using morphological parameters indicated that short-term predation threats (24 hr) altered tadpole morphology unlike the control or 5 day-out treatment (exposure to predation for 5 days and then no exposure for 5 days). Whole-brain ingenuity pathway and metabolome analyses revealed that free radicals, superoxide dismutase, glycogenesis, and pyruvate were elevated after 6 hr of predation pressure. Hemoglobin was also synthesized in the brain, forming oxyhemoglobin in the midbrain and hindbrain to reduce radical production. Furthermore, ATP production through glycolysis was promoted, while β-oxidation and the tricarboxylic acid cycle were downregulated. We also predicted increases in microtubule dynamics, neuronal branching, and neuritogenesis. Therefore, X. tropicalis tadpoles can adapt to predation stress through changes within their central nervous system.
... RhoGDIβ protein belongs to the family of RHO guanosine diphosphate dissociation inhibitors (12,13). Rho GTPases widely participate in a number of cellular responses, particularly in the cell motility (14). ...
Objective
Four and a half Lin-11, Isl-1, Mac-3 (LIM) protein 1 (FHL1) is one of the FHL protein family, which is regarded as a tumor suppressor in the multiple malignant tumors. In this study, we aimed to explore the regulatory effects and mechanisms of FHL1 on lung cancer cell invasion.
Materials and Methods
In this experimental study, bioinformatics analysis of FHL1 transcripts in human lung adenocarcinomas of TCGA database was performed. Quantitative real-time polymerasechain reaction (PCR) was performed to detect FHL1 mRNA expression in 15 paired human lung cancer tissues and their adjacent normal lung tissues, or lung cancer cell lines (A549 and H1299) in comparison with human bronchial epithelial cell line (Beas- 2B). Moreover, western blot was used to analyze FHL1 and rho GDP-dissociation inhibitor beta (RhoGDIβ) protein expression in the indicated cell lines. Also, transwell assays were employed to measure the migrated, and invaded of indicated cell lines.
Results
FHL1 transcripts were downregulated in the human lung adenocarcinoma. The impaired FHL1 transcripts were positively correlated with advanced tumor node metastasis (TNM) stage. Moreover, as compared to the adjacent normal lung tissues, FHL1 mRNA was low expressed in 15 paired human lung cancer tissues than their adjacent normal lung tissues. Besides, FHL1 mRNA and protein expression were also reduced in H1299 and A549 cell lines in comparison with Beas-2B cell line. Overexpressed FHL1 protein inhibited the invasive ability of H1299 and A549 cell lines. Mechanically, FHL1 protein overexpression increased the RhoGDIβ protein and mRNA abundance, while knockdown of RhoGDIβ protein, completely restored the invasion ability of A549 (Flag-FHL1) cell line.
Conclusion
Our findings indicated that as a key FHL1 downstream regulator, RhoGDIβ is in charge of FHL1 inhibiting lung cancer cell invasion abilities, providing a critical insight into understanding the role of FHL1 for lung cancer development.
... The interaction between Ras-GTP and its specific downstream effectors, such as Rafs and PI3K, stimulate signaling cascades that regulate proliferation, differentiation, and malignant transformation [71][72][73][74] (Figure 3). ...
Metabolic reprogramming represents a hallmark of tumorigenesis to sustain survival in harsh conditions, rapid growth and metastasis in order to resist to cancer therapies. These metabolic alterations involve glucose metabolism, known as the Warburg effect, increased glutaminolysis and enhanced amino acid and lipid metabolism, especially the cholesterol biosynthesis pathway known as the mevalonate pathway and these are upregulated in several cancer types, including acute myeloid leukemia (AML). In particular, it was demonstrated that the mevalonate pathway has a pivotal role in cellular transformation. Therefore, targeting this biochemical process with drugs such as statins represents a promising therapeutic strategy to be combined with other anticancer treatments. In the last decade, several studies have revealed that amino-bisphosphonates (BP), primarily used for bone fragility disorders, also exhibit potential anti-cancer activity in leukemic cells, as well as in patients with symptomatic multiple myeloma. Indeed, these compounds inhibit the farnesyl pyrophosphate synthase, a key enzyme in the mevalonate pathway, reducing isoprenoid formation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. This, in turn, inhibits the prenylation of small Guanosine Triphosphate-binding proteins, such as Ras, Rho, Rac, Rab, which are essential for regulating cell survival membrane ruffling and trafficking, interfering with cancer key signaling events involved in clonal expansion and maturation block of progenitor cells in myeloid hematological malignancies. Thus, in this review, we discuss the recent advancements about bisphosphonates’ effects, especially zoledronate, analyzing the biochemical mechanisms and anti-tumor effects on AML model systems. Future studies will be oriented to investigate the clinical relevance and significance of BP treatment in AML, representing an attractive therapeutic strategy that could be integrated into chemotherapy.
... The activation and inhibition of Rho GTPases are mediated respectively by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) [36]. The inactive Rho GTPases are sequestered in the cytosol by guanine nucleotide dissociation inhibitors (GDIs), that prevent the association of Rho GTPases with the plasma membrane [6,17,32]. Rho GTPases' based signal networks are thought to control cellular dynamics by coordinating protrusions and retractions during the process of cell migration [15]. ...
Recent experimental observations reveal that local cellular contraction pulses emerge via a combination of fast positive and slow negative feedbacks based on a signal network composed of Rho, GEF and Myosin interactions [22]. As an examplary, we propose to study a plausible, hypothetical temporal model that mirrors general principles of fast positive and slow negative feedback, a hallmark for activator-inhibitor models. The methodology involves (ⅰ) a qualitative analysis to unravel system switching between different states (stable, excitable, oscillatory and bistable) through model parameter variations; (ⅱ) a numerical bifurcation analysis using the positive feedback mediator concentration as a bifurcation parameter, (ⅲ) a sensitivity analysis to quantify the effect of parameter uncertainty on the model output for different dynamic regimes of the model system; and (ⅳ) numerical simulations of the model system for model predictions. Our methodological approach supports the role of mathematical and computational models in unravelling mechanisms for molecular and developmental processes and provides tools for analysis of temporal models of this nature.
... Rho GDIs also prevent Rac1 activation by binding directly to Rac1GDP and preventing GEF-mediated exchange at the plasma membrane [47,48]. However, HUVECs transfected with a point mutant Rac1 construct, R66A, which decreases Rac1 binding to Rho GDI, did not increase active Rac1-mediated lamellipodia formation compared to transfection with a wild-type Rac1 construct [49]. ...
Age-related macular degeneration (AMD) is one of the leading causes of blindness worldwide. Vision loss from the neovascular form is associated with the invasion of choroidal endothelial cells into the neural retina to form vision-threatening macular neovascularization (MNV). Anti-angiogenic agents are the current standard of care but are effective in only ~50% of AMD cases. The molecular mechanisms involved in invasive MNV point to the importance of regulating signaling pathways that lead to pathologic biologic outcomes. In studies testing the effects of AMD-related stresses, activation of the Rho GTPase, Rac1, was found to be important for the choroidal endothelial cell invasion into the neural retina. However, current approaches to prevent Rac1 activation are inefficient and less effective. We summarize active Rac1-mediated mechanisms that regulate choroidal endothelial cell migration. Specifically, we discuss our work regarding the role of a multidomain protein, IQ motif containing GTPase activating protein 1 (IQGAP1), in sustaining pathologic Rac1 activation and a mechanism by which active Rap1, a Ras-like GTPase, may prevent active Rac1-mediated choroidal endothelial cell migration.
... RhoGDIs are cytosolic proteins that exist in a 1:1 stoichiometric association with the small G protein Rho and function to preclude GDP dissociation from inactive Rho. RhoGDIs also inhibit the hydrolysis of GTP by guanine-nucleotide exchange factors or GTPase activating proteins (GAPs) (18). Rho-GDIs are β-sandwich proteins that contain a flexible, N-terminal helix-loop-helix structure that interacts with the switch regions of the small G protein (19). ...
Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB1 cannabinoid receptor G-protein coupling in part by altering the selectivity for Gαi subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at 1.55 Å resolution. CRIP1a exhibits a 10-stranded, antiparallel β-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The β-barrel has a gap between strands β8 and β10, which deviates from β-sandwich fatty acid binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor (GDI) predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes PDE6δ and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like PDE6δ, cargo with a farnesyl moiety that enters from the top of the β-barrel to occupy the hydrophobic interior, or (ii) like Unc119, cargo with a palmitoyl or myristoyl moiety that enters from the side where the missing β-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gαi N-terminus. However, CRIP1a could not bind the non-myristolyated Gαi peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB1 receptor.
... terminal domain, what disturbs its interaction with the lipid bilayer and maintains the Rho protein at the cytosol (DerMardirossian and Bokoch, 2005). In contrast to other yeast, S. ...
Cdc42 rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and by Guanine nucleotide Exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its down-regulation. We describe here that oxidative stress causes Sty1 kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at poles and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles by either eliminating two Cdc42 GAPs or through the constitutive tethering of a GEF to the cell tips, and biochemically demonstrate that Rga3 is a direct substrate of Sty1. We propose that stress-activated Sty1 promotes GTP hydrolysis and prevents GEF activity at the cell tips, thus leading to the inhibition of Cdc42 and polarized growth cessation.
... This additional level of regulation for Rho proteins was known for a long time; however, the exact mechanism by which the GTPase-GDI dissociation is regulated is not well understood. For the Rac1-RhoGDI interaction, it was shown that phosphorylation of RhoGDI by the Rac1/Cdc42 effector kinase Pak1 could release Rac1 [80,81]. Interestingly, this mechanism appears only relevant for Rac1 and RhoGDI because the same phosphorylation of RhoGDI by Pak1 does not impair its capacity to bind Cdc42 or RhoA, two other prominent members of the Rho family. ...
Cells and tissues are continuously exposed to both chemical and physical stimuli and dynamically adapt and respond to this variety of external cues to ensure cellular homeostasis, regulated development and tissue-specific differentiation. Alterations of these pathways promote disease progression-a prominent example being cancer. Rho GTPases are key regulators of the re-modeling of cytoskeleton and cell membranes and their coordination and integration with different biological processes, including cell polarization and motility, as well as other signaling networks such as growth signaling and proliferation. Apart from the control of GTP-GDP cycling, Rho GTPase activity is spatially and temporally regulated by post-translation modifications (PTMs) and their assembly onto specific protein complexes, which determine their controlled activity at distinct cellular compartments. Although Rho GTPases were traditionally conceived as targeted from the cytosol to the plasma membrane to exert their activity, recent research demonstrates that active pools of different Rho GTPases also localize to endomembranes and the nucleus. In this review, we discuss how PTM-driven modulation of Rho GTPases provides a versatile mechanism for their com-partmentalization and functional regulation. Understanding how the subcellular sorting of active small GTPase pools occurs and what its functional significance is could reveal novel therapeutic opportunities.
... When Hhex and RHOGDIA were co-overexpressed, CFL1 phosphorylation were further reduced. RHOGDIA is an RHO-GTPase binding protein which inhibites GDP dissociation from RHOA/CDC42/RAC1 to keep them in inactive state [41]. Although we didn't detect the direct interaction of Hhex with RHOGDIA, Hhex silencing does reduce the interaction of RHOGDIA with RHOA or CDC42 in an unknown manner. ...
Background
Hhex(human hematopoietically expressed homeobox), also known as PRH, is originally considered as a transcription factor to regulate gene expression due to its homebox domain. Increasing studies show that Hhex plays a significant role in development, including anterior–posterior axis formation, vascular development and HSCs self-renewal etc. Hhex is linked to many diseases such as cancers, leukemia, and type-2 diabetes. Although Hhex is reported to inhibit cell migration and invasion of breast and prostate epithelial cells by upregulating Endoglin expression, the effect and molecular mechanism for lung cancer cell motility regulation remains elusive.
Methods
Human non-small cell lung cancer cells and HEK293FT cells were used to investigate the molecular mechanism of Hhex regulating lung cancer cell migration by using Western blot, immunoprecipitation, wound-healing scratch assay, laser confocal.
Results
Our data indicated that Hhex could inhibit cell migration and cell protrusion formation in lung cancer cells. In addition, Hhex inhibited CFL1 phosphorylation to keep its F-actin-severing activity. RHOGDIA was involved in Hhex-induced CFL1 phosphorylation regulation. Hhex enhanced RHOGDIA interaction with RHOA/CDC42, thus maintaining RHOA/CDC42 at an inactive form.
Conclusion
Collectively, these data indicate that Hhex inhibited the activation of RHOA/CDC42 by enhancing interaction of RHOGDIA with RHOA/CDC42, and then RHOA/ CDC42-p-CFL1 signaling pathway was blocked. Consequently, the formation of Filopodium and Lamellipodium on the cell surface was suppressed, and thus the ability of lung cancer cells to migrate was decreased accordingly. Our findings show Hhex plays an important role in regulating migration of lung cancer cells and may provide a potential target for lung cancer therapy.
... A recent study also showed that cholangiocyte-derived H19-exosomes were involved in macrophage activation and hepatic inflammation in cholestatic liver injury [30]. Rho-family GTPases are the key regulators of cell migration and morphogenesis, which are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPaseactivating proteins (GAPs) [31][32][33]. It has been reported that Rhokinase inhibitor Y27632 can reduce BDL-induced cholestatic liver injury [34]. ...
Although macrophages are recognized as important players in the pathogenesis of chronic liver diseases, their roles in cholestatic liver fibrosis remain incompletely understood. We previously reported that long noncoding RNA-H19 (lncRNA-H19) contributes to cholangiocyte proliferation and cholestatic liver fibrosis of biliary atresia (BA). We here show that monocyte/macrophage CD11B mRNA levels are increased significantly in livers of BA patients and positively correlated with the progression of liver inflammation and fibrosis. The macrophages increasingly infiltrate and accumulate in the fibrotic niche and peribiliary areas in livers of BA patients. Selective depletion of macrophages using the transgenic CD11b-diphtheria toxin receptor (CD11b-DTR) mice halts bile duct ligation (BDL)-induced progression of liver damage and fibrosis. Meanwhile, macrophage depletion significantly reduces the BDL-induced hepatic lncRNA-H19. Overexpression of H19 in livers using adeno-associated virus serotype 9 (AAV9) counteracts the effects of macrophage depletion on liver fibrosis and cholangiocyte proliferation. Additionally, both H19 knockout (H19−/−) and conditional deletion of H19 in macrophage (H19ΔCD11B) significantly depress the macrophage polarization and recruitment. lncRNA-H19 overexpressed in THP-1 macrophages enhance expression of Rho-GTPase CDC42 and RhoA. In conclusions, selectively depletion of macrophages suppresses cholestatic liver injuries and fibrosis via the lncRNA-H19 and represents a potential therapeutic strategy for rapid liver fibrosis in BA patients.
... ROCKi is commonly used to reduce dissociation-induced cell death resulting from loss of e-cadherin-mediated cell-cell contact [8]. ARHGDIA inhibits the activation of RHOA by preventing the GDP exchange for GTP [29]. Since RHOA activation is necessary for ROCK activation, we hypothesized that overexpression of ARHGDIA would reduce activation of RHOA and therefore lead to increased single-cell survival conferring selective advantage to hPSCs ( Supplementary Fig. S2). ...
Human pluripotent stem cells (hPSCs) have generated significant interest in the scientific community based on their potential applications in regenerative medicine. However, numerous research groups have reported a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. Work from our laboratory and others has demonstrated that amplification of chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during clinical use. In this study, we focus on a common genomic aberration and a list of candidate genes with increased gene expression to hypothesize a gene that may confer selective advantage when overexpressed. hPSC lines overexpressing ARHGDIA exhibited culture dominance in co-cultures of overexpression lines with non-overexpression lines. Furthermore, during low density seeding, we demonstrate increased clonality of our ARHGDIA lines against matched controls. A striking observation is that we could reduce this selective advantage by varying the hPSC culture conditions with the addition of ROCKi. This work is unique in (a) demonstrating a novel gene that confers selective advantage to hPSCs when overexpressed and may help explain a common trisomy dominance, (b) providing a selection model for studying culture conditions that reduce the appearance of genomically altered hPSCs, and (c) aiding in elucidation of a mechanism that may act as a molecular switch during culture adaptation.
... Rac1 continues to cycle between the cytoplasm and the plasma membrane, and the hydrophilic cytoplasmic Rac1 lacks a transmembrane domain, but posttranslational modifications (e.g., prenylation and lipidation) make it hydrophobic, allowing it to move to the plasma membrane. Post-translational modifications of Rac1 precisely dictate its functional regulation (GDI association) and subcellular localization [32][33][34][35][36][37]. ...
Diabetic retinopathy remains the leading cause of vision loss in working-age adults. The multi-factorial nature of the disease, along with the complex structure of the retina, have hindered in elucidating the exact molecular mechanism(s) of this blinding disease. Oxidative stress appears to play a significant role in its development and experimental models have shown that an increase in cytosolic Reacttive Oxygen Speies (ROS) due to the activation of NADPH oxidase 2 (Nox2), is an early event, which damages the mitochondria, accelerating loss of capillary cells. One of the integral proteins in the assembly of Nox2 holoenzyme, Rac1, is also activated in diabetes, and due to epigenetic modifications its gene transcripts are upregulated. Moreover, addition of hyperlipidemia in a hyperglycemic milieu (type 2 diabetes) further exacerbates Rac1-Nox2-ROS activation, and with time, this accelerates and worsens the mitochondrial damage, ultimately leading to the accelerated capillary cell loss and the development of diabetic retinopathy. Nox2, a multicomponent enzyme, is a good candidate to target for therapeutic interventions, and the inhibitors of Nox2 and Rac1 (and its regulators) are in experimental or clinical trials for other diseases; their possible use to prevent/halt retinopathy will be a welcoming sign for diabetic patients.
Objective
Nonalcoholic fatty liver disease (NAFLD) is an emerging public health threat as the most common chronic liver disease worldwide. However, there remains no effective medication to improve NAFLD. G protein-coupled receptors (GPCRs) are the most frequently investigated drug targets family. The Regulator of G protein signaling 14 (RGS14), as an essential negative modulator of GPCR signaling, plays important regulatory roles in liver damage and inflammatory responses. However, the role of RGS14 in NAFLD remains largely unclear.
Methods and results
In this study, we found that RGS14 was decreased in hepatocytes in NAFLD individuals in a public database. We employed genetic engineering technique to explore the function of RGS14 in NAFLD. We demonstrated that RGS14 overexpression ameliorated lipid accumulation, inflammatory response and liver fibrosis in hepatocytes in vivo and in vitro. Whereas, hepatocyte specific Rgs14-knockout (Rgs14-HKO) exacerbated high fat high cholesterol diet (HFHC) induced NASH. Further molecular experiments demonstrated that RGS14 depended on GDI activity to attenuate HFHC-feeding NASH. More importantly, RGS14 interacted with Guanine nucleotide-binding protein (Gi) alpha 1 and 3 (Giα1/3, gene named GNAI1/3), promoting the generation of cAMP and then activating the subsequent AMPK pathways. GNAI1/3 knockdown abolished the protective role of RGS14, indicating that RGS14 binding to Giα1/3 was required for prevention against hepatic steatosis.
Conclusions
RGS14 plays a protective role in the progression of NAFLD. RGS14-Giα1/3 interaction accelerated the production of cAMP and then activated cAMP-AMPK signaling. Targeting RGS14 or modulating the RGS14-Giα1/3 interaction may be a potential strategy for the treatment of NAFLD in the future.
Protein–protein interactions (PPIs) have been sought as putative therapeutic targets for the advancement of various new treatments. This chapter deals with the various studies that have successfully discovered small-molecule inhibitors (SMIs) associated with particular disease-causing PPI. The employed methodologies in these studies as well as the conclusive results have been thoroughly discussed. Further, other aspects of the discovery like optimization of the process, strategizing drug binding, selection of targets have also been delineated. This chapter thus provides the reader with a comprehensive account of the current state-of-art in the discovery of small molecules inhibiting PPIs. It also throws light on the future potential of these small molecules as commercial drug candidates.
Hypertension is one of the most prominent non communicable disease
suffered by people worldwide. According to WHO, hypertension can be
attributed to 19% of total deaths worldwide. Therapies for hypertension
are continuously being updated and improved to effectively control this
disease. With all the research and development happening, medical cost
increase is inevitable and will act as a barrier for middle & low income
countries to access the latest and most effective therapy to control
hypertension. Acupuncture, a traditional treatment originating from the
East, has gained popularity in the West in recent years. Its reputation for
promoting health and curing illnesses is well-established. Numerous
studies and research have shed light on how acupuncture works in the
human body, leading to the development of medical acupuncture as we
know it today. Medical acupuncture adapts traditional acupuncture points.
Dr. Gary Bokoch’s legacy (Fig. 8.1) continues in each of us who learned about RhoGTPases through his training, publications, collaborations, and presentations. His expertise on RhoGTPases provided the root for the branches of his studies into fields such as chemotaxis, the actin cytoskeleton, p-21 activated kinases, LIM kinase, and NADPH oxidases. A survey of his 217 publications from his graduate work to his final days as professor clearly showed that the topic of neutrophils was special to him since that is how his successful scientific career began. From working on arachidonic metabolism in guinea pig neutrophils during graduate school, to identifying the Gialpha (Giα)-subunit while a post-doc in the laboratory of Nobel Laureate, Dr. Alfred Gilman, to discovering functions for RhoGTPases in NOX2 in neutrophils, Dr. Bokoch’s dedication to this field has taught us how to approach studies on signaling mechanisms applicable to many areas of science. I hope my presentation of Dr. Bokoch (Gary as I shall address him hereon) will paint a vivid picture of his journeys that led him to the path of NADPH Oxidases. As for the many other scientific paths on which he traveled, they would each require a separate chapter.KeywordsGary BokochRhoGTPasesNeutrophilsNADPH oxidases
Synthesis of reactive oxygen species (ROS) by specific NADPH oxidases (Nox) can serve both defense and differentiation signalling roles in animals and plants. Fungi have three subfamilies of NADPH oxidase, NoxA, NoxB and NoxC. NoxA and NoxB have a structure very similar to the human gp91phox whereas NoxC has a Ca2+ binding motif similar to that found in the human Nox5 and plant Rboh families of NADPH oxidases. Specific isoforms of Nox have been shown by genetic analysis to be required for various fungal physiological processes and cellular differentiations, including development of sexual fruiting bodies, ascospore germination, hyphal defense, hyphal growth in both mutualistic and antagonistic plant-fungal interactions.A survey of 65 fungal genomes identified up to four Nox genes in some fungal species, reflecting the diverse morphologies and life cycles of fungal species. The presence of nox genes in fungi from the Chytridiomycota to Ascomycota suggests that Nox is an ancestral enzyme for fungi. This chapter provides an overview of our current knowledge of fungal NADPH oxidases, including Nox distribution in the fungal kingdom, Nox structure and regulation, and known biological functions of this important group of enzymes.KeywordsFungiNADPH oxidaseCellular differentiationsSymbiosisPathogenesisGenomes
Le syndrome néphrotique est une néphropathie glomérulaire fréquente caractérisée par une protéinurie abondante, conséquence d'une perte de l'architecture du podocyte, une des cellules essentielles de la barrière de filtration glomérulaire. L'avènement de la génétique a permis de mettre en évidence des mutations dans une cinquantaine de gènes chez 30% des patients résistants aux corticoïdes ou aux immunosuppresseurs (SNCR). Cependant, l'origine génétique reste indéterminée chez le reste de ces patients. Par séquençage d'exome, nous avons cherché des mutations dans de nouveaux gènes dans des familles atteintes de SNCR. Une fois un gène candidat identifié, nous avons analysé la pathogénicité de la ou des mutations identifiées dans un modèle in vitro de culture cellulaire, et dans un modèle in vivo de poisson-zèbre (ZF). Nous avons d'abord mis en évidence des mutations dans le gène TBC1D8B dans deux familles de SNCR de survenue précoce. TBC1D8B a été très peu étudiée dans la littérature mais est prédite comme une Rab-GTPase Activating Protein (Rab-GAP), c'est à dire ayant la capacité d'inactiver certaines Rab-GTPases (non identifiées). Chez le ZF, l'inactivation de tbc1d8b induit un œdème péricardique, marqueur indirect des lésions rénales, associé à une perte de l'architecture des podocytes. La protéinurie a été confirmée par la détection de fluorescence dans les cellules tubulaires après l'injection d'un dextran fluorescent de 500kDa chez les poissons mutés, témoin d'un passage glomérulaire. Les Rab étant impliquées dans le trafic vésiculaire, nous avons étudié, dans les podocytes ou les fibroblastes de patients, le recyclage du récepteur de la transferrine et avons montré que les mutations p.Q246H et p.F291S étaient associées à un défaut de son recyclage. Nous avons également mis en évidence une interaction entre TBC1D8B et Rab11b, un acteur majeur du recyclage des vésicules provenant du compartiment de recyclage péri-nucléaire. Ces données confirment que les mutations de TBC1D8B sont responsables de SNCR chez nos patients par défaut du recyclage dépendant de Rab11b, et suggèrent que TBC1D8B est une Rab-GAP de Ra11b. Nous avons ensuite identifié le variant p.R28W dans le gène TRIM3 (tripartite motif containing 3) ségrégeant avec la maladie dans une famille dans laquelle 4 personnes ont présenté un SNCR autosomique dominante (AD). Dans les neurones, la protéine TRIM3 joue un rôle dans le trafic vésiculaire dans un complexe protéique comprenant la protéine a-actinine 4. Des mutations du gène ACTN4 codant l'a-actinine 4 ont été décrites dans la survenue de SNCR AD. Les patients porteurs de la mutation p.R28W étaient également porteurs d'un polymorphisme rare et non pathogène p.V801M d'ACTN4. Nous avons d'abord montré que l'injection d'ARNm muté codant pour TRIM3 p.R28W dans des embryons de poisson-zèbre, entraînait un phénotype identique à celui décrit précédemment, avec une désorganisation de l'architecture des podocytes. Dans les podocytes en culture, TRIM3 p.R28W était délocalisé au niveau des fibres d'actine. Malgré une interaction normale entre TRIM3 p.R28W et l'a-actinine 4, le trafic vésiculaire le long des fibres d'actine était altéré. La présence du polymorphisme p.V801M de l'a-actinine 4 ne modifiait pas le phénotype. Un autre variant (p.R433C) du gène TRIM3 a été identifié chez un patient ayant présenté un SNCR sporadique de survenue tardive, sans possibilité d'étudier la ségrégation du variant dans la famille. Les études chez le ZF ont montré le même phénotype après injection d'ARNm muté. Ces résultats suggèrent que des mutations dominantes du gène TRIM3 peuvent, comme celles d'ACTN4, conduire à un SNCR AD. Dans cette thèse, nous avons mis en évidence de nouveaux mécanismes moléculaires conduisant aux lésions podocytaires. En effet, ces données suggèrent que le recyclage dans le podocyte tient une place fondamentale dans le développement de certaines podocytopathies, ce qui n'avait jamais été montré auparavant.
L’asthme est une pathologie chronique des voies aériennes caractérisée par une hyperréactivité bronchique, un remodelage tissulaire et une inflammation chronique. Les formes sévères résistantes aux traitements conventionnels requièrent de nouvelles stratégies thérapeutiques. Mon équipe d’accueil a démontré dans un modèle murin d’asthme sévère une augmentation d’activité de la GTPase Rac1 dans les cellules musculaires lisses (CML) bronchiques qui participe à leur hypercontractilité et leur prolifération. Cette suractivation de Rac1 est aussi observée dans d’autres types cellulaires, dont les cellules inflammatoires. L’objectif principal de ma thèse a été (i) de confirmer chez les asthmatiques sévères la suractivation de Rac1, (ii) d’identifier les populations inflammatoires concernées et (iii) les fonctions effectrices dépendantes de Rac dans ces différents types cellulaires. Titre : Nouvelles stratégies thérapeutiques dans l’asthme sévère Inhibition de la voie de signalisation dépendante de la GTPase Rac Mots clés : Asthme sévère, Rac GTPase, Eosinophiles, Muscle lisse bronchique Dans le cadre du protocole de recherche clinique NARACAS (NCT NCT03325088), j’ai confirmé sur des biopsies bronchiques de patients asthmatiques sévères la suractivation de Rac dans les CML et dans les cellules infiltrantes. Les polynucléaires éosinophiles ont été identifiés comme la principale population inflammatoire suractivant Rac dans le cadre d’un modèle murin d’asthme allergique sévère aux acariens. Par approches biochimiques et cytométriques, mes résultats démontrent une activation de Rac durant la maturation de l’éosinophile. Par ailleurs, l’inhibition de l’activité de Rac réduit la dégranulation des éosinophiles. L’ensemble de ces résultats démontrent que les niveaux d’activité de Rac régulent à la fois la maturation et la dégranulation des éosinophiles. Le développement d’inhibiteurs des voies de signalisation dépendante de Rac pourrait constituer une stratégie innovante de lutte contre le remodelage bronchique et ses déterminants inflammatoires.
A small GTPase, Cdc42 is evolutionarily one of the most ancient members of the Rho family, which is ubiquitously expressed and involved in a wide range of fundamental cellular functions. The crucial role of Cdc42 includes regulation of the actin cytoskeleton, cell polarity, morphology and migration, endocytosis and exocytosis, cell cycle, and proliferation in many different cell types. Many studies have provided compelling yet contradicting evidence that Cdc42 dysregulation plays an important role in cellular and tissue aging. Furthermore, Cdc42 is a critical factor in the development and progression of aging-related pathologies, such as neurodegenerative and cardiovascular disorders, diabetes type 2, and aging-related disorders of the joints and bones, and the inhibition of the Cdc42 demonstrates potentially significant therapeutic and anti-aging effects in animal models of aging and disease. However, regulation of Cdc42 expression and activity is very complex and depends on many factors, such as the origin and complexity of the tissues, hormonal status, etc. Therefore, this review is focused on current advances in understanding the underlying cellular and molecular mechanisms associated with Cdc42 activity and regulation of senescence in different cell types since they may provide a foundation for novel therapeutic strategies and targeted drugs to reverse the aging process and treat aging-associated disorders.
The Rho subfamily members of Rho GTPases, RhoA, RhoB, and RhoC, are key regulators of signal transduction in a variety of cellular processes, including regulation of actomyosin and microtubule dynamics, cell shape, cell adhesion, cell division, cell migration, vesicle/membrane trafficking, and cell proliferation. Traditionally, the focus of research on RhoA/B/C has been on tumor biology, as dysregulation of expression or function of these proteins plays an important role in the pathogenesis of various cancer entities. However, RhoA, RhoB, and RhoC are also important in the context of vascular biology and pathology because they influence endothelial barrier function, vascular smooth muscle contractility and proliferation, vascular function and remodelling as well as angiogenesis. In this context, RhoA/B/C exploit numerous effector molecules to transmit their signals, and their activity is regulated by a variety of guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) that enable precise spatiotemporal activation often in concert with other Rho GTPases. Although their protein structure is very similar, different mechanisms of regulation of gene expression, different localization, and to some extent different interaction with RhoGAPs and RhoGEFs have been observed for RhoA/B/C. In this review, we aim to provide a current overview of the Rho subfamily as regulators of vascular biology and pathology, analyzing database information and existing literature on expression, protein structure, and interaction with effectors and regulatory proteins. In this setting, we will also discuss recent findings on Rho effectors, RhoGEFs, RhoGAPs, as well as guanine nucleotide dissociation inhibitors (RhoGDIs).
Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)‐dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ‐forming human dermal microvascular ECs (HDMECs) and adherens junction only forming‐human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction‐disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF‐induced decrease in trans‐endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA‐GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP‐bound Rap1A after four hours but increased GTP‐bound RhoA after 12 h. In cell‐free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A‐C, RhoB‐C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ‐forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP‐mediated Epac1 activation.
Cell division control protein 42 homolog (Cdc42), which contributes to multiple cellular processes including cell proliferation and migration, is a potential target for cancer therapy, especially in the intervention of tumor migration. Cdc42’s mutants G12V and Q61L are discovered constitutively active, and the overexpression of them exhibits oncogenic activities. Here, using molecular dynamics (MD) simulations and dynamic analysis, we illustrated the activation mechanism of Cdc42G12V and Cdc42Q61L. Without GAP, the two mutations differently elicited state transition from the wild‐type's open “inactive” state 1 to the closed “active” state 2, induced by the introduction of a newly formed water‐mediated T35‐γ‐phosphate hydrogen bond in G12V system and the additional hydrophobic interactions between L61 and T35 together with the direct T35‐γ‐phosphate hydrogen bond in Q61L system. When binding with GAP, both mutations weakened the hydrogen bond interactions between Cdc42‐GTP and GAP's finger loop, and disturbed the catalytically‐competent organizations of GAP's catalytic R305/R306 and Cdc42’s Q61, thereby impairing the GAP‐mediated GTP hydrolysis. Our findings first reveal the activation mechanism of Cdc42’s G12V and Q61L mutants on a molecular basis, which provide new insights into the structural and dynamical characteristics of Cdc42 and its mutants and can be exploited in the further development of novel therapies targeting Cdc42‐related cancers. This article is protected by copyright. All rights reserved.
Rho subfamily of G proteins (e.g., Rac1) have been implicated in glucose-stimulated insulin secretion from the pancreatic β-cell. Interestingly, metabolic stress (e.g., chronic exposure to high glucose) results in sustained activation of Rac1 leading to increased oxidative stress, impaired insulin secretion and β-cell dysfunction. Activation-deactivation of Rho G proteins is mediated by three classes of regulatory proteins, namely the guanine nucleotide exchange factors (GEFs), which facilitate the conversion of inactive G proteins to their active conformations; the GTPase-activating proteins (GAPs), which convert the active G proteins to their inactive forms); and the GDP-dissociation inhibitors (GDIs), which prevent the dissociation of GDP from G proteins. Contrary to a large number of GEFs (82 members) and GAPs (69 members), only three members of RhoGDIs (RhoGDIα, RhoGDIβ and RhoGDIγ) are expressed in mammalian cells. Even though relatively smaller in number, the GDIs appear to play essential roles in G protein function (e.g., subcellular targeting) for effector activation and cell regulation. Emerging evidence also suggests that the GDIs are functionally regulated via post-translational modification (e.g., phosphorylation) and by lipid second messengers, lipid kinases and lipid phosphatases. We highlight the underappreciated regulatory roles of RhoGDI-Rho G protein signalome in islet β-cell function in health and metabolic stress. Potential knowledge gaps in the field, and directions for future research for the identification of novel therapeutic targets to loss of functional β-cell mass under the duress of metabolic stress are highlighted.
Rho GTPases are critically important and are centrally positioned regulators of the actomyosin cytoskeleton. By influencing the organization and architecture of the cytoskeleton, Rho proteins play prominent roles in many cellular processes including adhesion, migration, intra‐cellular transportation, and proliferation. The most important method of Rho GTPase regulation is via the GTPase cycle; however, post‐translational modifications (PTMs) also play critical roles in Rho protein regulation. Relative to other PTMs such as lipidation or phosphorylation that have been extensively characterized, protein oxidation is a regulatory PTM that has been poorly studied. Protein oxidation primarily occurs from the reaction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), with amino acid side chain thiols on cysteine (Cys) and methionine (Met) residues. The versatile redox modifications of cysteine residues exemplify their integral role in cell signalling processes. Here we review prominent members of the Rho GTPase family and discuss how lipidation, phosphorylation, and oxidation on conserved cysteine residues affects their regulation and function. Reactive oxygen species (ROS) can oxidize Rho GTPases on redox‐sensitive cysteine residues, resulting in reduced affinity for guanine nucleotides and consequent increased GDP dissociation rates. Binding of cellular GTP to the nucleotide‐free protein, independent of guanine nucleotide exchange factors (GEF), transitions the GTPases to active conformations that enable downstream signalling.
Background
The breakpoint cluster region (BCR) is a protein that originally forms a fusion protein with c‐Abl tyrosine kinase and induces leukemia. Researchers have shown that BCR is enriched in the central nervous system and may contribute to neurological disorders. We aimed to investigate the physiological function of BCR in neural development in the gastrointestinal (GI) tract and brain.
Methods
Whole‐exome sequencing was used to screen for mutations in the BCR. Bcr knockout mice (Bcr−/−, ΔExon 2–22) were generated using the CRISPR/Cas9 system. Transit of carmine red dye and glass bead expulsion assays were used to record total and proximal GI transit and distal colonic transit.
Key Results
In an infant with pediatric intestinal pseudo‐obstruction, we found a heterozygous de novo mutation (NM_004327.3:c.3072+1G>A) in BCR. Bcr deficiency mice (Bcr−/−) exhibited growth retardation and impaired gastrointestinal motility. Bcr−/− mice had a prolonged average total GI transit time with increased distal colonic transit and proximal GI transit in isolation. Morphology analysis indicated that Bcr−/− mice had a less number of neurons in the submucosal plexus and myenteric plexus. Bcr−/− mice exhibited apparent structural defects in the brain, particularly in the cortex. Additionally, Bcr⁻ depletion in the mouse cortex altered the expression of Ras homologous (Rho) family small GTPases.
Conclusions and Inferences
BCR mutations are associated with intestinal obstruction in children. Loss of Bcr can cause intestinal dysmotility and brain developmental defects may via regulation of Rho GTPases.
Members of the Rho family of GTP-binding proteins are localized in the cytosol of cells by complexation with a protein known as (Rho)GDI. We show by sucrose gradient equilibrium sedimentation analysis that all of the Rac protein present in human neutrophil cytosol exists as a complex with (Rho)GDI under non-activating conditions. This interaction can be disrupted in the presence of various lipids which have been shown to have biological activity in a variety of systems, including NADPH oxidase activation. Particularly effective were arachidonic acid, phosphatidic acid, and phosphatidylinositols. These lipids were active at concentrations from 0.5-50 muM and were capable of disrupting complexation of (Rho)GD1 with both GDP- and GTP-bound forms of Rac, although the latter were more sensitive to lipid. These data suggest that certain lipids generated in chemoattractant-stimulated neutrophils may play a role in modulating the activity of Rac and thus NADPH oxidase activity.
Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.
GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activation of GTPases and may also be critical for
the cellular compartmentalization of GTPases. RhoGDI and GDI/D4 are two currently known GDIs for the Rho-subfamily of GTPases.
Using their cDNAs to screen a human brain cDNA library under low stringency, we have cloned a homologous cDNA preferentially
expressed at high levels in brain and pancreas. The predicted protein, named RhoGDIγ, is ≈50% identical to GDI/D4 and RhoGDI.
It binds to CDC42 and RhoA with less affinity compared with RhoGDI and does not bind with Rac1, Rac2, or Ras. RhoGDIγ functions
as a GDI for CDC42 but with ≈20 times less efficiency than RhoGDI. Immunohistochemical studies showed a diffuse punctate distribution
of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. Overexpression of the protein
in baby hamster kidney cells caused the cells to round up with loss of stress fibers. A distinct hydrophobic amino terminus
in RhoGDIγ, not seen in the other two RhoGDIs, could provide a mechanism for localization of the GDI to specific membranous
compartment thus determining function distinct from RhoGDI or GDI/D4. Our results provide evidence that there is a family
of GDIs for the Rho-related GTPases and that they differ in binding affinity, target specificity, and tissue expression. We
propose that RhoGDI be renamed RhoGDIα and GDID4 be renamed RhoGDIβ. The new GDI should widen the scope of investigation of
this important class of regulatory protein.
Rho GTPases have two interconvertible forms and two cellular localizations. In their GTP-bound conformation, they bind to the cell membrane and are activated. In the inactive GDP-bound conformation, they associate with a cytosolic protein called GDP dissociation inhibitor (GDI). We previously reported that the RhoA component of the RhoA/Rho-GDI complex was not accessible to the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides. We show here that PtdIns, PtdIns4P, PtdIns3,4P2, PtdIns4,5P2 and PtdInsP3 enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA component of the prenylated RhoA/Rho-GDI complex. In contrast, in the nonprenylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exchange are of the same order as those measured on free RhoA and are not modified by phosphoinositides. In both cases, phosphoinositides partially opened, but did not fully dissociate the complex. Upon treatment of the prenylated RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer to neutrophil membranes was evidenced. Using an overlay assay with the prenylated RhoA/Rho-GDI complex pretreated with PtdIns4P and labeled with [32P]GTP, three membrane proteins with molecular masses between 26 and 32 kDa were radiolabeled. We conclude that in the presence of phosphoinositides, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP. The opened GTP-RhoA/Rho-GDI complex acquires the capacity to target specific membrane proteins.
Rho GDP dissociation inhibitors (rhoGDIs) are postulated to regulate the activity of small G proteins of the Rho family by a shuttling process involving the extraction of Rho from donor membranes, the formation of the inhibitory cytosolic Rho/rhoGDI complexes, and delivery of Rho to target membranes. However, the role of rhoGDIs in site-specific membrane targeting or extraction of Rho is still poorly understood. Here we investigated the molecular functions of two rhoGDIs, the specific rhoGDI-3 and the less specific but well studied rhoGDI-1, in HeLa cells using structure-based mutagenesis of the rhoGDI protein. We identified two sites in rhoGDI, which form conserved interactions with their Rho target, whose mutation results in the uncoupling of inhibitory and shuttling functions of rhoGDIs: D66GDI-3 (equivalent to D45GDI-1), a conserved residue in the helix-loop-helixGDI/switch 1Rho interface, and D206GDI-3 (equivalent to D185GDI-1) in the beta-sandwichGDI/switch 2Rho interface. Mutations of both sites result in the loss of rhoGDI-3 or rhoGDI-1 inhibitory activity but not of their ability to form cytosolic complexes with RhoG or Cdc42 in vivo. Remarkably, the mutants were detected at Rho-induced membrane ruffles or protrusions where they co-localized with RhoG or Cdc42, likely identifying for the first time the site of extraction of a Rho protein by a rhoGDI in vivo. We propose that these mutations act by modifying the steady-state kinetics of the shuttling process regulated by rhoGDIs, such that transient steps at the cell membranes now become detectable. They should provide valuable tools for future investigations of the dynamics of membrane extraction or delivery of Rho proteins and their regulation by cellular partners.
The ras-related protein, CDC42Hs, is a 22-kDa GTP-binding protein which is the human homolog of a Saccharomyces cerevisiae yeast-cell-division cycle protein. In attempting to isolate and biochemically characterize mammalian proteins capable of regulating various activities of CDC42Hs, we have identified an activity in bovine brain cytosol which effectively inhibits the dissociation of [3H]GDP from the platelet- or the Spodoptera frugiperda-expressed CDC42Hs protein. The purification of this activity was achieved by a series of steps which included ammonium sulfate fractionation, DEAE-Sephacel, Mono-Q, and Mono-S chromatographies. The purified CDC42Hs regulatory protein has an apparent molecular weight of 28,000, and cyanogen bromide-generated peptide sequences of this protein were identical to sequences from the carboxyl-terminal portion of rho-GDP-dissociation inhibitor (rho-GDI) (Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H., Araki, S., Ueda, T., Kikuchi, A., and Takai, Y. (1990) Oncogene 5, 1321-1328). In addition, an Escherichia coli-expressed, glutathione S-transferase-rho-GDI fusion protein fully substitutes for the GDI which we have purified from bovine brain in its ability to inhibit GDP dissociation from CDC42Hs. These findings suggest either that a common regulatory protein (GDI) is capable of inhibiting GDP dissociation from the rho and CDC42Hs proteins or that these two GTP-binding proteins interact with GDI proteins of very similar structure. The purified brain GDI protein shows little ability to inhibit GDP dissociation from the E. coli-expressed CDC42Hs and is capable of only a very weak inhibition of the dissociation of [35S]guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) from the Spodoptera frugiperda-expressed CDC42. However, brain GDI very effectively inhibits the ability of the human dbl oncogene product to catalyze GDP dissociation from CDC42Hs. In addition to influencing guanine nucleotide association with CDC42Hs, the purified brain GDI protein also appears to catalyze the dissociation of CDC42Hs from the plasma membranes of human placenta and human epidermoid carcinoma (A431) cells. This effect by the GDI protein is observed whether the membrane-associated CDC42Hs is preincubated with GDP, GTP gamma S, or no guanine nucleotides, and occurs over a similar concentration range as that necessary for the inhibition of the intrinsic GDP dissociation.
rac1 and rac2 p21s are ras p21-like small GTP-binding proteins which are implicated in the NADPH oxidase-catalyzed superoxide generation in phagocytes. rac1 and rac2 p21s have a Cys-A-A-Leu (A = aliphatic amino acid) structure in their C-terminal region which may undergo post-translational processing including prenylation, proteolysis, and carboxyl methylation. We studied the function of this post-translational processing of rac p21s in their interaction with the stimulatory and inhibitory GDP/GTP exchange proteins for rac p21s, named smg GDS and rho GDI, and in their NADPH oxidase activation. We produced human recombinant rac1 and rac2 p21s in insect cells and purified them from the membrane and soluble fractions as the post-translationally processed and unprocessed forms, respectively. Post-translationally processed rac1 and rac2 p21s were sensitive to both smg GDS and rho GDI, but post-translationally unprocessed rac1 and rac2 p21s were insensitive to them. The GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-bound form of post-translationally processed rac1 and rac2 p21s stimulated the NADPH oxidase activity, but post-translationally unprocessed rac1 and rac2 p21s were far less effective. These results indicate that both rac1 and rac2 p21s stimulate the NADPH oxidase activity and that their post-translational processing is important not only for their interaction with smg GDS and rho GDI but also for their NADPH oxidase activation.
A novel regulatory protein for the rho proteins (rhoA p21 and rhoB p20), belonging to a ras p21/ras p21-like small molecular weight (Mr) GTP-binding protein (G protein) superfamily, was purified to near homogeneity from bovine brain cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor (GDI) for the rho proteins (rho GDI), inhibited the dissociation of GDP from rhoB p20 and the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. The Mr value of rho GDI was estimated to be about 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S value, indicating that rho GDI is composed of a single polypeptide without a subunit structure. The isoelectric point was about pH 5.7. rho GDI made a complex with the GDP-bound form of rhoB p20 with a molar ratio of 1:1 but not with the GTP gamma S-bound or guanine nucleotide-free form. rho GDI did not stimulate the GTPase activity of rhoB p20 and by itself showed neither GTP gamma S-binding nor GTPase activity. rho GDI was equally active for rhoA p21 and rhoB p20 but was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p25A, and smg p21. rho GDI activity was detected in the cytosol fraction of various rat tissues. These results indicate that, in mammalian tissues, there is a novel type of regulatory protein specific for the rho proteins that interacts with the GDP-bound form of the rho proteins and thereby regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. Since there is a GTPase-activating protein for the rho proteins stimulating the GTPase activity of the rho proteins in mammalian tissues, the rho proteins appear to be regulated at least by GTPase-activating protein and GDI in a dual manner.
The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote
formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now
show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin
microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia,
as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection.
This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes
and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin
were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating
cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance
of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.
GTP-binding proteins of the Rho family are maintained as cytosolic complexes with RhoGDI in resting cells, but are released and translocate to the membrane during the course of cell activation. Membrane association of Rac/Rho/CDC42 was specifically induced by GTP analogs and required a heat- and trypsin-labile membrane component. Translocation was associated with the release of Rho family proteins from RhoGDI, but such release did not occur in the absence of membranes, nor was release in the absence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) sufficient for membrane association. Membrane binding was correlated with exchange of GTP gamma S for GDP on Rac, and only GTP gamma S-bound Rac became membrane localized. We propose that translocation of Rac and other members of the Rho family is controlled by membrane-associated guanine nucleotide exchange factors, providing a mechanism to regulate the release and activation of individual members of the Rho family during cell stimulation.
rho GDI is an inhibitory GDP/GTP exchange protein for the rho family. Recently, rho GDI has been reported to interact with the GTP-bound form of G25K and rac1 p21 and to inhibit their basal and GTPase-activating protein (GAP)-stimulated GTPase activity. Here, we examined whether rho GDI interacts with the GTP-bound form of rho p21 and rac p21 and inhibits their basal and rho GAP-stimulated GTPase activity, rho GDI interacted with both the GDP- and GTP-bound forms of rhoA p21 and rac1 p21 as estimated by measuring its ability to form a complex with both forms and to inhibit the membrane-binding activity of both forms. The efficiency of rho GDI for interaction with the GTP-bound form was, however, about 10% that for interaction with the GDP-bound form. Moreover, rho GDI inhibited both the basal and rho GAP-stimulated GTPase activity of rhoA p21 and rac1 p21 in a dose-dependent manner. The doses of rho GDI necessary for this action were, however, about 10-fold higher than those necessary for the action to inhibit their GDP/GTP exchange reaction. These results indicate that rho GDI interacts with the GTP-bound form of its substrate small G proteins, as well as with the GDP-bound form, but much less efficiently than with the GDP-bound form.
The majority of the GTP-binding proteins of the Ras superfamily hydrolyze GTP to GDP very slowly. A notable exception to this are the Rac proteins, which have intrinsic GTPase rates at least 50-fold those of Ras or Rho. A protein (or proteins) capable of inhibiting this GTPase activity exists in human neutrophil cytosol. Since Rac appears to exist normally in neutrophils as a cytosolic protein complexed to (Rho)GDI, we examined the ability of (Rho)GDI to inhibit GTP hydrolysis by Rac. (Rho)GDI produced a concentration-dependent inhibition of GTP hydrolysis by Rac1 that paralleled its ability to inhibit GDP dissociation from the Rac protein. Maximal inhibition occurred at or near equimolar concentrations of the GDI and the Rac substrate. The ability of two molecules exhibiting GTPase activating protein (GAP) activity toward Rac to stimulate GTP hydrolysis was also inhibited by the presence of (Rho)GDI. The inhibitory effect of the GDI could be overcome by increasing the GAP concentration to levels equal to that of the GDI. (Rho)GDI weakly, but consistently, inhibited GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) dissociation from Rac1, confirming an interaction of (Rho)GDI with the GTP-bound form of the protein. These data describe an additional activity of (Rho)GDI and suggest a mechanism by which Rac might be maintained in an active form in vivo in the presence of regulatory GAPs.
Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis, ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 microM DEVD-CHO, a reported selective inhibitor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 microM. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD19S. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein.
The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the GDI to the Rho-like protein, Cdc42Hs. We show here that when the GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence. The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda-expressed Cdc42Hs and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed Cdc42Hs and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying GDI activity. A truncated Cdc42Hs mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to GDI regulation also does not show changes in the fluorescence of its bound Mant-GDP upon GDI addition. Thus, the GDI-induced quenching of Mant-GDP provides a direct read-out for the binding of the GDI to Cdc42Hs. Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for Cdc42Hs-GDI interactions with an apparent Kd value of 30 nM. A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to Cdc42Hs. These results suggest that the GDI can bind to the GDP-bound and GTP-bound forms of Cdc42Hs equally well. We also have used the fluorescence assay for GDI interactions to demonstrate that the differences in functional potency observed between the GDI molecule and a related human leukemic protein, designated LD4, are due to differences in their binding affinities for Cdc42Hs. This, together with the results from studies using GDI/LD4 chimeras, allow us to conclude that a limit region within the carboxyl-terminal domain of the GDI molecule is responsible for its ability to bind with higher affinity (compared with LD4) to Cdc42Hs.
RhoB is a small GTP-binding protein highly homologous to the RhoA protein. While RhoA is known to regulate the assembly of
focal adhesions and stress fibers in response to growth factors, the function of RhoB remains unknown. We have reported that
the transient expression of the endogenous RhoB protein is regulated during the cell cycle, contrasting with the permanent
RhoA protein expression (1). Using the yeast two-hybrid system to characterize proteins interacting with RhoB, we identified a new mouse Rho GDP dissociation
inhibitor, referenced as RhoGDI-3. The NH2-terminal α helix of RhoGDI-3 is strongly amphipatic and differs thus from that found in previously described bovine, human,
and yeast RhoGDI proteins and mouse and human D4/Ly-GDIs. Contrary to the cytosolic localization of all known GDI proteins,
acting on Rab or Rho, RhoGDI-3 is associated to a Triton X-100-insoluble membranous or cytoskeletal subcellular fraction.
In the two-hybrid system, RhoGDI-3 interacts specifically with GDP- and GTP-bound forms of post-translationally processed
RhoB and RhoG proteins, both of which show a growth-regulated expression in mammalian cells. No interaction is found with
RhoA, RhoC, or Rac1 proteins. We show that GDI-3 is able to inhibit GDP/GTP exchange of RhoB and to release GDP-bound but
not GTP-bound RhoB from cell membranes.
Prenylated Rab GTPases occur in the cytosol in their GDP-bound conformations bound to a cytosolic protein termed GDP-dissociation inhibitor (GDI). Rab-GDI complexes represent a pool of active, recycling Rab proteins that can deliver Rabs to specific and distinct membrane-bound compartments. Rab delivery to cellular membranes involves release of GDI, and the membrane-associated Rab protein then exchanges its bound GDP for GTP. We report here the identification of a novel, membrane-associated protein factor that can release prenylated Rab proteins from GDI. This GDI-displacement factor (GDF) is not a guanine nucleotide exchange factor because it did not influence the intrinsic rates of nucleotide exchange by Rabs 5, 7 or 9. Rather, GDF caused the release of each of these endosomal Rabs from GDI, permitting them to exchange nucleotide at their intrinsic rates. GDF displayed the greatest catalytic rate enhancement on Rab9-GDI complexes. However, catalytic rate enhancement paralleled the potency of GDI in blocking nucleotide exchange: GDI was shown to be most potent in blocking nucleotide exchange by Rab9. The failure of GDF to act on Rab1-GDI complexes suggests that it may be specific for endosomal Rab proteins. This novel, membrane-associated activity may be part of the machinery used to localize Rabs to their correct intracellular compartments.
The crystal structure of human rac1, a member of the rho family of small G-proteins, complexed with the non-hydrolysable GTP analogue, guanosine-5'-(beta gamma-imino)triphosphate (GMPPNP), has been determined by X-ray analysis at a resolution of 1.38 A. Comparison with the structure of H-ras indicates that rac1 has an extra alpha-helical domain that is characteristic of the rho G proteins, and may be involved in the signalling pathway of this family.
GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activation of GTPases and may also be critical for the cellular compartmentalization of GTPases. RhoGDI and GDI/D4 are two currently known GDIs for the Rho-subfamily of GTPases. Using their cDNAs to screen a human brain cDNA library under low stringency, we have cloned a homologous cDNA preferentially expressed at high levels in brain and pancreas. The predicted protein, named RhoGDIgamma, is approximately 50% identical to GDI/D4 and RhoGDI. It binds to CDC42 and RhoA with less affinity compared with RhoGDI and does not bind with Rac1, Rac2, or Ras. RhoGDIgamma functions as a GDI for CDC42 but with approximately 20 times less efficiency than RhoGDI. Immunohistochemical studies showed a diffuse punctate distribution of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. Overexpression of the protein in baby hamster kidney cells caused the cells to round up with loss of stress fibers. A distinct hydrophobic amino terminus in RhoGDIgamma, not seen in the other two RhoGDIs, could provide a mechanism for localization of the GDI to specific membranous compartment thus determining function distinct from RhoGDI or GDI/D4. Our results provide evidence that there is a family of GDIs for the Rho-related GTPases and that they differ in binding affinity, target specificity, and tissue expression. We propose that RhoGDI be renamed RhoGDIalpha and GDID4 be renamed RhoGDIbeta. The new GDI should widen the scope of investigation of this important class of regulatory protein.
The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.
Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA‐125, the currently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI‐FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two‐dimensional gel electrophoresis (2‐D PAGE) to identify proteins that may serve as invasive ovarian cancer‐specific biomarkers for early detection and/or new therapeutic targets. We performed 2‐D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two noninvasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2‐D gels created from microdissected lysates with SYPRO‐Ruby stained 2‐D PAGE profiles of the patient‐matched undissected bulk tumor lysates from all five patients. Twenty‐three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or underexpressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and preliminary target validation of the mass spectrometry identified proteins cut from the Ruby‐red stained gels was performed by LCM coupled Western blot and reverse‐phase array technology in a study set of six cases (the aforementioned five cases used in the 2‐D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G‐protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely overexpressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype.
The small G proteins of the Ras family act as bimodal relays in the transfer of intracellular signals. This is a dynamic phenomenon involving a cascade of protein–protein interactions modulated by chemical modifications, structural rearrangements and intracellular relocalisations. Most of the small G proteins could be operationally defined as proteins having two conformational states, each of which interacts with different cellular partners. These two states are determined by the nature of the bound nucleotide, GDP or GTP. This capacity to cycle between a GDP-bound conformation and a GTP-bound conformation enables them to filter, to amplify or to temporise the upstream signals that they receive. Thus the control of this cycle is crucial. Membrane anchoring of the proteins in the Ras family is a prerequisite for their activity. Most of the proteins in the Rho/Rac and Rab subfamilies of Ras proteins cycle between cytosol and membranes. Then the control of membrane association/dissociation is an other important regulation level. This review will describe one family of crucial regulators acting on proteins in the Rho/Rac family—the Rho guanine nucleotide dissociation inhibitors, or RhoGDIs. As yet, only three RhoGDIs have been described: RhoGDI-1, RhoGDI-2 (or D4/Ly-GDI) and RhoGDI-3. RhoGDI 1 and 2 are cytosolic and participate in the regulation of both the GDP/GTP cycle and the membrane association/dissociation cycle of Rho/Rac proteins. The non-cytosolic RhoGDI-3 seems to act in a slightly different way.
Six peaks of small GTP-binding proteins (G proteins) were separated by column chromatographies from the cytosol fraction of the differentiated HL-60 cells: two peaks of rho p21, one peak of p21, two peaks of rac1 p21, and one peak of an unidentified small G protein with a Mr of about 20,000 (20 KG). smg GDS, previously thought to be a stimulatory exchange protein for smg p21, Ki-ras p21, and rho p21, but not for Ha-ras p21 or smg p25A, was also active on rac1 p21. rho GDI, previously thought to be an inhibitory exchange protein specific for rho p21, was also active on rac1 p21. These results indicate that both smg GDS and rho GDI are active on multiple small G proteins.
Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA-125, the currently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI-FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two-dimensional gel electrophoresis (2-D PAGE) to identify proteins that may serve as invasive ovarian cancer-specific biomarkers for early detection and/or new therapeutic targets. We performed 2-D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two noninvasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2-D gels created from microdissected lysates with SYPRO-Ruby stained 2-D PAGE profiles of the patient-matched undissected bulk tumor lysates from all five patients. Twenty-three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or underexpressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and preliminary target validation of the mass spectrometry identified proteins cut from the Ruby-red stained gels was performed by LCM coupled Western blot and reverse-phase array technology in a study set of six cases (the aforementioned five cases used in the 2-D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G-protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely overexpressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype.
The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.
The substrate of the C3 exoenzyme from botulinum toxin is a protein which is particularly abundant in the cytosol of neutrophils [Stasia, M. J., Jouan, A., Bourmeyster, N., Boquet, P., & Vignais, P. V. (1991) Biochem. Biophys. Res. Commun. 180, 615-622]. Optimal conditions for the ADP-ribosylation of the C3 substrate have been established in order to follow the course of its purification from bovine neutrophil cytosol. In particular, phosphoinositides at micromolar concentrations were found to enhance the ADP-ribosylation capacity of the C3 substrate in crude neutrophil cytosol and partially purified fractions. A [32P]ADP-ribosylatable protein, migrating on SDS-PAGE with a mass of 24 kDa, was copurified with a 29-kDa protein by a series of chromatographic steps on DEAE-Sephacel, Biogel P60, and Mono Q. In the case of the C3 substrate, isoelectric focusing revealed two major labeled bands with pI values of 6.2 and 5.6; the pI of the 29-kDa protein was 4.8-5.0. On the basis of the amino acid sequence of peptides resolved after proteolytic digestion, the 24-kDa protein and the 29-kDa protein were identified respectively as rho and the GDP dissociation inhibitor (GDI), suggesting that rho and GDI copurify from bovine neutrophil cytosol in the form of a complex. The presence of a number of amino acid residues specific of rho A in the enzymatic digest originating from rho indicates that, among the rho proteins, at least rho A belongs to the GDI-rho complex.
As with other lipid modifications of proteins, prenylation now appears to be critically important in the regulation of protein function. Recent research has led to an explosion of information concerning prenylation signals, prenyl transferase enzymes and the role of prenylation in protein-membrane interactions. Experiments have examined the role of prenylation in protein function and the results suggest that protein prenylation may be involved in facilitating proper subcellular localization, promoting protein-protein and protein-membrane interactions and regulating protein function.
The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.
Actin stress fibers are one of the major cytoskeletal structures in fibroblasts and are linked to the plasma membrane at focal adhesions. rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells. Readdition of serum produced a similar response, detectable within 2 min. This activity was due to a lysophospholipid, most likely lysophosphatidic acid, bound to serum albumin. Other growth factors including PDGF induced actin reorganization initially to form membrane ruffles, and later, after 5 to 10 min, stress fibers. For all growth factors tested the stimulation of focal adhesion and stress fiber assembly was inhibited when endogenous rho function was blocked, whereas membrane ruffling was unaffected. These data imply that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.
We have recently purified to near homogeneity a novel type of regulatory protein for the rho proteins, ras p21-like small GTP-binding proteins, from bovine brain cytosol. This regulatory protein, named GDP dissociation inhibitor for the rho proteins (rho GDI), regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from them, and the subsequent binding of GTP to them. In the present studies, we have isolated the cDNA of rho GDI from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences of the purified rho GDI and determined its complete nucleotide and deduced amino acid sequences. The cDNA contains an open reading frame encoding a protein of 204 amino acids with a calculated Mr value of 23,421. This Mr value is similar to those of the purified rho GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultra-centrifugation, both of which are about 27,000. The rho GDI cDNA is expressed in Escherichia coli and COS7 cells and the encoded protein exhibits rho GDI activity. The 1.9-kilobase rho GDI mRNA corresponding to the isolated cDNA is detected in various rat tissues by Northern blot analysis. Hydropathy analysis indicates that rho GDI is overall hydrophilic except for one hydrophobic region. Computer homology search has revealed that rho GDI is a novel protein that does not share a high amino acid sequence homology with any known protein.
The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.
The small GTP-binding proteins, rho and rac, control signal transduction pathways that link growth factor receptors to the activation of actin polymerization. In Swiss 3T3 cells, rho proteins mediate the lysophosphatidic acid and bombesin-induced formation of focal adhesions and actin stress fibres, whilst rac proteins are required for the platelet-derived growth factor-, insulin-, bombesin- and phorbol ester (phorbol 12-myristate 13-acetate)-stimulated actin polymerization at the plasma membrane that results in membrane ruffling. To investigate the role of p85/p110 phosphatidylinositol 3-kinase in the rho and rac signalling pathways, we have used a potent inhibitor of this activity, wortmannin. Wortmannin has no effect on focal adhesion or actin stress fibre formation induced by lysophosphatidic acid, bombesin or microinjected recombinant rho protein.
In contrast, it totally inhibits plasma membrane edge-ruffling induced by platelet-derived growth factor and insulin though not by bombesin, phorbol ester or microin-jected recombinant rac protein. We conclude that phos-phatidylinositol 3,4,5 trisphosphate mediates activation of rac by the platelet-derived growth factor and insulin receptors. The effects of lysophosphatidic acid on the Swiss 3T3 actin cytoskeleton can be blocked by the tyrosine kinase inhibitor, tyrphostin. Since tyrphostin does not inhibit the effects of microinjected rho protein, we conclude that lysophosphatidic acid activation of rho is mediated by a tyrosine kinase.
Rho proteins are involved in the regulation of the assembly of the microfilamental cellular network and are known to be specific substrates for the ADP-ribosyltransferase C3 from Clostridium botulinum. Here, we studied the distribution of Rho and Rho-regulating proteins in extracts from various rabbit tissues. The highest amounts of [32P]ADP-ribosylated proteins were detected in cell extracts from lung and kidney. Compared to these tissues, 50-95% reduced labeling of Rho proteins was observed in extracts from liver, spleen, brain, heart and muscle. The level of the C3-mediated [32P]ADP-ribosylation of Rho did not correlate with the amount of RhoA proteins detected by Western analysis. The relative amounts of [32P]ADP-ribosylated proteins located in cytosolic or membrane fractions, respectively, depended on the type of tissue investigated, indicating a tissue-specific variation in the subcellular distribution of Rho proteins. The same was true for the complexation of Rho with other factors and the expression of diverse Rho species. In respect to Rho-regulating proteins, extracts from lung and brain contained the highest amounts of guanine nucleotide dissociation-inhibitor proteins (Rho-GDI). The association of Rho with Rho-GDI however showed tissue specificity and did not correlate with Rho-GDI amounts. The highest Rho-GAP (GAP = GTPase-activating protein) activities were observed in extracts from lung, kidney and spleen, the lowest ones in extracts from muscle and heart. In total, our data demonstrate tissue-specific differences in the expression of RhoA, [32P]ADP-ribosylated proteins and Rho-regulating factors, indicating a tissue-specific variation in the activity and regulation of Rho proteins.
Members of the Rho family of GTP-binding proteins are localized in the cytosol of cells by complexation with a protein known as (Rho)GDI. We show by sucrose gradient equilibrium sedimentation analysis that all of the Rac protein present in human neutrophil cytosol exists as a complex with (Rho)GDI under non-activating conditions. This interaction can be disrupted in the presence of various lipids which have been shown to have biological activity in a variety of systems, including NADPH oxidase activation. Particularly effective were arachidonic acid, phosphatidic acid, and phosphatidylinositols. These lipids were active at concentrations from 0.5-50 microM and were capable of disrupting complexation of (Rho)GDI with both GDP- and GTP-bound forms of Rac, although the latter were more sensitive to lipid. These data suggest that certain lipids generated in chemoattractant-stimulated neutrophils may play a role in modulating the activity of Rac and thus NADPH oxidase activity.
The dbl oncogene product (Dbl) showed the GDP/GTP exchange protein (GEP) activity on all the rho family small GTP-binding protein (G protein) members including RhoA and Rac1 as well as mCdc42. Dbl was active on both the lipid-modified and -unmodified forms of these small G proteins, but was much more active on the former form than on the latter form. In the presence of Rho GDI, an inhibitory GEP for the rho family members, the GEP activity of Dbl was markedly reduced. These properties of Dbl were partly different from those of Smg GDS, another GEP which is active not only on the rho family members but also on Ki-Ras and Rap1 and is active only on their lipid-modified form.
The Ras-related small GTP-binding proteins are involved in diverse cellular events, including cell signaling, proliferation, cytoskeletal organization, and secretion. The interconversion of the active, GTP-bound form of the protein to the inactive, GDP-bound form is influenced by two types of regulatory proteins, those that alter the intrinsic GTPase activity of the GTP-binding protein and those that affect the rate of GDP/GTP exchange. By utilizing a subtractive hybridization approach, we have isolated a human gene encoding Ly-GDI, a protein that has striking homology to the product of a previously cloned gene, Rho-GDI, which inhibits GDP/GTP exchange on the Rho family of GTPases. In contrast to Rho-GDI, which is ubiquitously expressed, Ly-GDI is expressed only in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. Stimulation of T lymphocytes with phorbol ester leads to phosphorylation of Ly-GDI, suggesting an involvement of Ly-GDI in lymphocyte activation pathways. Cell type-specific regulators of the Ras-like GTP-binding proteins may provide one mechanism by which different cell types respond uniquely to signals transduced through the same cell surface receptor or may provide a way by which the GTP-binding proteins can be uniquely engaged by tissue-restricted receptors.
Membrane transport is known to be regulated by protein phosphorylation and by small GTPases of the rab family. Using specific antibodies, we have identified a 55 kDa phosphorylated protein which co-immunoprecipitated with the cytosolic forms of rab5 and other rab proteins. We demonstrate, on the basis of its mobility in two-dimensional electrophoresis gels and its immunological properties, that this protein is rab GDI (p55/GDI). We also found that, a minor fraction of p55/GDI is membrane associated, but, whilst also complexed with rab proteins, it is not phosphorylated. On the basis of these data we suggest that the cycling of rab proteins between membranes and cytosol is regulated by phosphorylation of p55/GDI.
The Drosophila developmental mutation quartet causes late larval lethality and small imaginal discs and, when expressed in
the adult female, has a lethal effect on early embryogenesis. These developmental defects are associated with mitotic defects,
which include a low mitotic index in larval brains and incomplete separation of chromosomes in mitosis in the early embryo.
quartet mutations also have a biochemical effect, i.e., a basic shift in isoelectric point in three proteins. We have purified
one of these proteins, raised an antibody to it, and isolated and sequenced its cDNA. At the amino acid level, the sequence
shows 68% identity and 81% similarity to bovine smg p25a GDP dissociation inhibitor (GDI), a regulator of ras-like small GTPases
of the rab/SEC4/YPT1 subfamily. The correlation between a basic shift in isoelectric point in Drosophila GDI in quartet mutant
tissue and the quartet developmental phenotype raises the possibility that a posttranslational modification of GDI is necessary
for its function and that GDI function is essential for development.
We have identified the mRNA for a human gene, denoted D4, which is expressed at very high levels in hematopoietic cell lines and in normal cells of lymphoid and myeloid origin. The 1.5-kb transcript is absent or detectable only at low levels in nonhematopoietic tissues. D4 encodes a 201-amino acid protein with homology to rhoGDI, an inhibitor of GDP dissociation for the ras-homologous protein rho. D4 might function also as a regulator of guanine nucleotide exchange for small GTP-binding proteins. A homologous transcript of similar size is also preferentially expressed in murine hematopoietic tissues. When totipotent murine embryonic stem cells develop in vitro into hematopoietic cells, the gene is activated with the onset of hematopoiesis. When hematopoietic cell lines are induced to differentiate, the expression of D4 is modulated. Thus, D4 appears to be a developmentally regulated gene. Its preferential expression in hematopoietic cells indicates that D4 likely plays some significant role in the growth and differentiation processes of hematopoietic cells. This significance is underscored by increasing evidence for the involvement of regulators of G proteins in clinical diseases.
The GDP dissociation inhibitor Rho GDI from bovine neutrophil cytosol was purified in association with prenylated Rho A. Upon treatment of this complex with alkaline phosphatase, the Rho A and Rho GDI components were released to their free forms. Following migration in 2D-PAGE and specific immunodetection, the shape of the spot of Rho GDI was found to depend markedly on whether Rho GDI subjected to electrophoresis was present in a Rho A-Rho GDI complex or in a free form. In the first case Rho GDI focused as an elongated spot between pI 5.2 and pI 4.6 whereas in the later case it focused at a pI of 5.0-5.2 as a round spot. Activation of neutrophils by anaphylatoxin C5a in a [32Pi] supplemented medium resulted in radiolabeling of Rho GDI. In vitro incubation of Rho GDI with a neutrophil homogenate in the presence of [gamma 32P] ATP led also to radiolabeling of Rho GDI. Taken together these results suggest that Rho GDI in the Rho A-Rho GDI complex is phosphorylated and that the stability of the complex depends on the phosphorylation state of Rho GDI.
We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.
GTPases of the Rho family regulate many aspects of inflammatory cell activity, including motility, formation of toxic oxygen metabolites, and generation of proinflammatory cytokines. Defective regulation of such signaling pathways leads to a variety of acute and chronic inflammatory disorders, although the mechanisms by which this occurs have not been well defined. We describe in this work specific proteolytic cleavage of D4 GDI, a critical regulator of Rho GTPase activity in inflammatory leukocytes, by IL-1 beta-converting enzyme (ICE). Cleavage of D4 GDI by ICE occurs at Asp55, leading to the formation of the truncated D4 that is unable to effectively bind and regulate GTPases of the Rho family. Our data suggest that activation of ICE protease(s) at inflammatory sites leads to defective Rho GTPase regulation. Release of these critical regulatory proteins may contribute substantially to the inflammatory response at these sites, exacerbating and perpetuating the resulting tissue damage.
Prenylation is a class of lipid modification involving covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. Known prenylated proteins include fungal mating factors, nuclear lamins, Ras and Ras-related GTP-binding proteins (G proteins), the subunits of trimeric G proteins, protein kinases, and at least one viral protein. Prenylation promotes membrane interactions of most of these proteins, which is not surprising given the hydrophobicity of the lipids involved. In addition, however, prenylation appears to play a major role in several protein-protein interactions involving these species. The emphasis in this review is on the enzymology of prenyl protein processing and the functional significance of prenylation in cellular events. Several other recent reviews provide more detailed coverage of aspects of prenylation that receive limited attention here owing to length restrictions (1-4).
The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes, and from inhibition of GTPase cycling between the GTP- and GDP-bound states. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (M[r] 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a beta-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.
The rho family of small G proteins, including rho, rac and cdc42, are involved in many cellular processes, including cell transformation by ras and the organization of the actin cytoskeleton. Additionally, rac has a role in the regulation of phagocyte NADPH oxidase. Guanine nucleotide dissociation inhibitors (GDIs) of the rhoGDI family bind to these G proteins and regulate their activity by preventing nucleotide dissociation and by controlling their interaction with membranes.
We report the structure of rhoGDI, determined by a combination of X-ray crystallography and NMR spectroscopy. NMR spectroscopy and selective proteolysis show that the N-terminal 50-60 residues of rhoGDI are flexible and unstructured in solution. The 2.5 A crystal structure of the folded core of rhoGDI, comprising residues 59-204, shows it to have an immunoglobulin-like fold, with an unprecedented insertion of two short beta strands and a 310 helix. There is an unusual pocket between the beta sheets of the immunoglobulin fold which may bind the C-terminal isoprenyl group of rac. NMR spectroscopy shows that the N-terminal arm is necessary for binding rac, although it remains largely flexible even in the complex.
The rhoGDI structure is notable for the existence of both a structured and a highly flexible domain, both of which appear to be required for the interaction with rac. The immunoglobulin-like fold of the structured domain is unusual for a cytoplasmic protein. The presence of equivalent cleavage sites in rhoGDI and the closely related D4/Ly-GDI (rhoGDI-2) suggest that proteolytic cleavage between the flexible and structured regions of rhoGDI may have a role in the regulation of the activity of members of this family. There is no detectable similarity between the structure of rhoGDI and the recently reported structure of rabGDI, which performs the same function as rhoGDI for the rab family of small G proteins.
Proteins of the rho subfamily of ras GTPases have been shown to be crucial components of pathways leading to cell growth and the establishment of cell polarity and mobility. Presented here is the solution structure of one such protein, Cdc42Hs, which provides insight into the structural basis for specificity of interactions between this protein and its effector and regulatory proteins. Standard heteronuclear NMR methods were used to assign the protein, and approximately 2100 distance and dihedral angle constraints were used to calculate a set of 20 structures using a combination of distance geometry and simulated annealing refinement. These structures show overall similarity to those of other GTP-binding proteins, with some exceptions. The regions corresponding to switch I and switch II in H-ras are disordered, and no evidence was found for an alpha-helix in switch II. The 13-residue insertion, which is only present in rho-subtype proteins and has been shown to be an important mediator of binding of regulatory and target proteins, forms a compact structure containing a short helix lying adjacent to the beta4-alpha3 loop. The insert forms one edge of a "switch surface" and, unexpectedly, does not change conformation upon activation of the protein by the exchange of GTP analogs for GDP. These studies indicate the insert region forms a stable invariant "footrest" for docking of regulatory and effector proteins.
Ly-GDI (lymphoid-specific guanosine diphosphate (GDP) dissociation inhibitor), also called D4-GDI, is preferentially expressed in hematopoietic tissues including bone marrow, thymus, spleen and lymph nodes. It binds to the small guanosine triphosphate (GTP)-binding protein Rho and inhibits GDP dissociation from Rho proteins. To explore the function of Ly-GDI in lymphocytes, we have generated Ly-GDI-deficient mice by gene targeting. These mice showed no striking abnormalities of lymphoid development or thymocyte selection. The mice also exhibited, for the most part, normal immune responses including lymphocyte proliferation, IL-2 production, cytotoxic T lymphocyte activity, antibody production, antigen processing and presentation, immune cell aggregation and migration, and protection against an intracellular protozoan. However, Ly-GDI-deficient mice exhibited deregulated T and B cell interactions after in vitro cultivation of mixed lymphocyte populations in concanavalin A (Con A) leading to overexpansion of B lymphocytes. Further studies revealed that Ly-GDI deficiency decreased IL-2 withdrawal apoptosis of lymph node cells while dexamethasone- and T cell receptor-induced apoptosis remained intact. These data implicate the regulation of the Rho GTPase by Ly-GDI in lymphocyte survival and responsiveness, but suggest that these functions may be partially complemented by other Rho regulatory proteins when the Ly-GDI protein is deficient.