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SUMOylation of the GTPase Rac1 is required for optimal cell migration
Abstract
The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1-GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion.
Supplementary resources (12)
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... Modification of the carboxylterminal CAAX motif in Rac1, adding either farnesyl or geranylgeranyl isoprenoid lipids, increases its hydrophobicity and facilitates its membrane localization, and hence its activation [13]. Furthermore, Ubiquitinlike (Ubl) modifications to Rac1 in response to cell migration, including ubiquitylation and SUMOylation [14,15], regulate the activity of Rac1. Significantly, Rac1-SUMO1 is required to maintain Rac1 GTPase activity during cell migration required for EMT initiation [14]. ...
... Furthermore, Ubiquitinlike (Ubl) modifications to Rac1 in response to cell migration, including ubiquitylation and SUMOylation [14,15], regulate the activity of Rac1. Significantly, Rac1-SUMO1 is required to maintain Rac1 GTPase activity during cell migration required for EMT initiation [14]. ...
... However, Rac1 is also involved in other biological processes, such as cell cycle regulation and survival, making very difficult its use as a therapeutic target. Nevertheless, Rac1 SUMOylation is only necessary for the maintenance of the activity of Rac1 required during migration/invasion but not for other events in which this GTPase participates (e.g., cell proliferation or apoptosis) [14]. Moreover, inhibiting Rac1 SUMOylation dampens the migratory and invasive behavior of breast cancer (BC) cells [17], although the mechanism by which Rac1-SUMO1 controls cell migration is not well understood. ...
The small GTPase Rac1 (Ras‐related C3 botulinum toxin substrate 1) has been implicated in cancer progression and in the poor prognosis of various types of tumors. Rac1 SUMOylation occurs during epithelial‐mesenchymal transition (EMT), and it is required for tumor cell migration and invasion. Here we identify POTEE (POTE Ankyrin domain family member E) as a novel Rac1‐SUMO1 effector involved in breast cancer malignancy that controls invadopodium formation through the activation of Rac1‐SUMO1. POTEE activates Rac1 in the invadopodium by recruiting TRIO‐GEF (Triple functional domain protein), and it induces tumour cell proliferation and metastasis in vitro and in vivo . We found that the co‐localization of POTEE with Rac1 is correlated with more aggressive breast cancer subtypes. Given its role in tumour dissemination, the leading cause of cancer‐related deaths, POTEE could represent a potential therapeutic target for these types of cancer.
... Rac1 is a member of the Rho family of small GTPases, which regulates a wide array of cellular functions, such as cell-cell adhesion [30], cell migration [31] and invasion [32]. A more recent study showed that SUMOylation of Rac1 is necessary for optimal Rac1 mediated cell migration and invasion [33]. Inhibiting Rac1 SUMOylation reduces breast cancer cell invasion [34]. ...
... In vitro invasion assays ( Supplementary Fig. S3B) and in vivo metastasis ( Supplementary Fig. S3C) further confirmed that the metastasis ability of MDA-MB-231 cells enhanced by PACT overexpression was abolished by Ubc9 knockout, indicating that Ubc9 is indispensable for metastasis promoted by PACT (Supplementary Fig. S3D). As research suggested, SUMOylation of Rac1 is required for optimal cell migration [33], we supposed that PACT might activate Rac1 through enhancement of SUMOylation. Since PIAS3 was reported as a SUMO E3 ligase of Rac1 [33], perform immunoprecipitation to find out whether PACT regulated Rac1 SUMOylation through Ubc9-PIAS3 axis. ...
... As research suggested, SUMOylation of Rac1 is required for optimal cell migration [33], we supposed that PACT might activate Rac1 through enhancement of SUMOylation. Since PIAS3 was reported as a SUMO E3 ligase of Rac1 [33], perform immunoprecipitation to find out whether PACT regulated Rac1 SUMOylation through Ubc9-PIAS3 axis. Ubc9, PIAS3 and Rac1 were Fig. 2 PACT is correlated with poor prognosis and pathogenesis in breast cancer and is highly expressed specifically in BLBC. ...
Most basal-like breast cancers (BLBCs) are triple-negative breast cancers (TNBCs), which is associated with high malignancy, high rate of recurrence and distant metastasis, and poor prognosis among all types of breast cancer. However, there are currently no effective therapies for BLBC. Furthermore, chemoresistance limits the therapeutic options for BLBC treatment. In this study, we screen out protein activator of the interferon-induced protein kinase (PACT) as an essential gene in BLBC metastasis. We find that high PACT expression level was associated with poor prognosis among BLBC patients. In vivo and in vitro investigations indicated that PACT could regulate BLBC metastasis by interacting with SUMO-conjugating enzyme Ubc9 to stimulate the SUMOylation and thus consequently the activation of Rac1. BLBC patients receiving chemotherapy presents poorer prognosis with PACT high expression, and PACT disruption sensitizes experimental mammary tumor metastases to chemotherapy, thus providing insights to consider PACT as a potential therapeutic target to overcome acquired chemoresistance in BLBC.
... Interestingly, Rac1 can be modified by SUMOylation, and this PTM ultimately affects its function as well as cell behavior [50]. Particularly in immortalized cell lines, Rac1 interacts with the SUMO E3-ligase PIAS3 (Protein Inhibitor of Activated STAT3) promoting Sumo1-conjugation [50]. ...
... Interestingly, Rac1 can be modified by SUMOylation, and this PTM ultimately affects its function as well as cell behavior [50]. Particularly in immortalized cell lines, Rac1 interacts with the SUMO E3-ligase PIAS3 (Protein Inhibitor of Activated STAT3) promoting Sumo1-conjugation [50]. SUMO proteins can be covalently attached to the lysine residues K188, K183, and K184/186 within the C-terminal polybasic region (PBR) of Rac1. ...
... Rac1-null mouse embryonic fibroblasts (MEFs) are unable to form lamellipodia-membrane ruffles and are defective in cell migration. While transfection of Rac1-null MEFs with wildtype Rac1 almost completely rescues these defects, transfection of Rac1∆SUMO (non-SUMOylable) constructs only rescues the failure of lamellipodia-ruffle formation partially [50]. ...
Schwann cell development and peripheral nerve myelination are finely orchestrated multistep processes; some of the underlying mechanisms are well described and others remain unknown. Many posttranslational modifications (PTMs) like phosphorylation and ubiquitination have been reported to play a role during the normal development of the peripheral nervous system (PNS) and in demyelinating neuropathies. However, a relatively novel PTM, SUMOylation, has not been studied in these contexts. SUMOylation involves the covalent attachment of one or more small ubiquitin-like modifier (SUMO) proteins to a substrate, which affects the function, cellular localization, and further PTMs of the conjugated protein. SUMOylation also regulates other proteins indirectly by facilitating non-covalent protein–protein interaction via SUMO interaction motifs (SIM). This pathway has important consequences on diverse cellular processes, and dysregulation of this pathway has been reported in several diseases including neurological and degenerative conditions. In this article, we revise the scarce literature on SUMOylation in Schwann cells and the PNS, we propose putative substrate proteins, and we speculate on potential mechanisms underlying the possible involvement of this PTM in peripheral myelination and neuropathies.
... As such, modification of the C-terminal CAAX motif in RAC1 through the addition of either farnesyl or geranylgeranyl isoprenoid lipids increases its hydrophobicity, facilitating both its membrane localization and activation (Mack et al., 2011). Ubiquitin-like (UBL) modifications of RAC1 have also been shown to regulate its activity, including ubiquitylation and SUMOylation (Castillo-Lluva et al., 2010), adding further complexity to the regulation of RAC1 signalling. ...
... We observed RAC1 GTPase SUMOylation (RAC1-SUMO1) when the epithelial to mesenchymal transition (EMT) was induced by hepatocyte growth factor (HGF) (Castillo-Lluva et al., 2010). EMT involves changes in gene expression, and it is associated with a loss of cell polarity and an increase in cell invasiveness (Brabletz et al., 2018). ...
... Likewise, increased expression of this GTPase is associated with a poor prognosis in different cancer types (Mack et al., 2011). Moreover, we previously showed that RAC1 is SUMOylated in response to stimuli that trigger cell migration (Castillo-Lluva et al., 2010). When we assessed whether the inhibition of cell invasion triggered by blocking the SUMO pathway involves a dampening of RAC1 activation, we found that GA did indeed inhibit RAC1 activity (RAC1-GTP) (Fig. 5F,G). ...
Post-translational modifications directly control protein activity and thus, they represent an important means to regulate the responses of cells to different stimuli. Protein SUMOylation has recently been recognised as one such modification and it has been associated with various diseases, including different types of cancer. However, the precise way that changes in SUMOylation influence the tumourigenic properties of cells remains to be fully clarified. Here, we show that blocking the SUMO pathway by depleting SUMO1 and UBC9, or by exposure to Ginkgolic acid C15:1 or 2-D08 (two different SUMOylation inhibitors), induces cell death, also inhibiting the invasiveness of tumour cells. Indeed, diminishing the formation of SUMO1 complexes induces autophagy-mediated cancer cell death by increasing the expression of Tribbles pseudokinase 3. Moreover, we found that blocking the SUMO pathway inhibits tumour cell invasion by decreasing RAC1 SUMOylation. These findings shed new light on the mechanisms by which SUMO1 modifications regulate the survival, and the migratory and invasive capacity of tumour cells, potentially establishing the bases to develop novel anti-cancer treatments based on the inhibition of SUMOylation.
... Since previous studies have reported that Rac1 can be conjugated to SUMO protein and this Rac1 SUMOylation is crucial to maintain the active Rac1-GTP form and enhance Rac1 activity (Castillo-Lluva et al., 2010;Yue et al., 2017), we therefore investigated whether Cc2d1a deletion activates Rac1 through increasing Rac1 SUMOylation levels. The Rac1 SU-MOylation levels were determined by Rac1 pull-down followed by Western blot analysis using an anti-SUMO2/3 antibody. ...
... SUMOylation is a highly dynamic process that can be reversed by SENPs (Mukhopadhyay and Dasso, 2007;Gareau and Lima, 2010;Hay, 2013). Previous studies have shown that SENP1 can interact with Rac1 and de-SUMOylate Rac1 to downregulate its activity in cells (Castillo-Lluva et al., 2010;Yue et al., 2017). We therefore examined whether Cc2d1a deletion increases basal Rac1 SUMOylation levels by decreasing expression of SENPs. ...
... At the molecular level, we showed that Cc2d1a deletion drastically downregulated SENP1 and SENP3 mRNA and protein expression and consequently repressed SENP-mediated de-SUMOylation of Rac1 to activate Rac1. This SENP-mediated de-SUMOylation of Rac1 has also been demonstrated in human cancer cells, where the de-SUMOylation activity of SENP1 is crucial for the promoting effect of mutant p53 on Rac1 activity to regulate tumor progression (Castillo-Lluva et al., 2010;Yue et al., 2017). Moreover, we observed that SUMO2/3, but not SUMO1 (data not shown), conjugation levels are increased in hippocampal CA1 tissue lysates of cKO mice. ...
Coiled-coil and C2 domain containing 1A (CC2D1A) is an evolutionarily conserved protein, originally identified as a nuclear factor-κB activator through a large-scale screen of human genes. Mutations in the human Cc2d1a gene result in autosomal recessive nonsyndromic intellectual disability. It remains unclear, however, how Cc2d1a mutation leads to alterations in brain function. Here, we have taken advantage of Cre/loxP recombinase-based strategy to conditionally delete Cc2d1a exclusively from excitatory neurons of male mouse forebrain to examine its role in hippocampal synaptic plasticity and cognitive function. We confirmed the expression of CC2D1A protein and mRNA in the mouse hippocampus. Double immunofluorescence staining showed that CC2D1A is expressed in both excitatory and inhibitory neurons of the adult hippocampus. Conditional deletion of Cc2d1a (cKO) from excitatory neurons leads to impaired performance in object location memory test and altered anxiety-like behavior. Consistently, cKO mice displayed a deficit in the maintenance of LTP in the CA1 region of hippocampal slices. Cc2d1a deletion also resulted in decreased complexity of apical and basal dendritic arbors of CA1 pyramidal neurons. An enhanced basal Rac1 activity was observed following Cc2d1a deletion, and this enhancement was mediated by reduced SUMO-specific protease 1 (SENP1) and SENP3 expression, thus increasing the amount of Rac1 SUMOylation. Furthermore, partial blockade of Rac1 activity rescued impairments in LTP and object location memory performance in cKO mice. Together, our results implicate Rac1 hyperactivity in synaptic plasticity and cognitive deficits observed in Cc2d1a cKO mice and reveal a novel role for CC2D1A in regulating hippocampal synaptic function.SIGNIFICANCE STATEMENT CC2D1A is abundantly expressed in the brain, but there is little known about its physiological function. Taking advantage of Cc2d1a cKO mice, the present study highlights the importance of CC2D1A in the maintenance of LTP at Schaffer collateral-CA1 synapses and the formation of hippocampus-dependent long-term object location memory. Our findings establish a critical link between elevated Rac1 activity, structural and synaptic plasticity alterations, and cognitive impairment caused by Cc2d1a deletion. Moreover, partial blockade of Rac1 activity rescues synaptic plasticity and memory deficits in Cc2d1a cKO mice. Such insights may have implications for the utility of Rac1 inhibitors in the treatment of intellectual disability caused by Cc2d1a mutations in human patients.
... Lipidation at the carboxyterminal end enables Rho5 and Rac1 to associate with membranes, which is further enhanced by the electrostatic affinity between phospholipids and the PBR regions of the GTPases [26]. Rac1 can also be adenylated at the conserved tyrosine 32 residue [42], reversibly palmitoylated at cysteine 178 [43], or sumoylated at lysine residues within the PBR [44,45], all of which modulate its activity, stability, and/or subcellular localization, with no indications that these modifications occur in yeast Rho5, so far. ...
... Other functions of Rac1 in the regulation of gene expression upon its translocation to the nucleus and to other endomembranes will not be discussed here in more detail, as they also do not seem to be conserved in yeast Rho5 and have been extensively reviewed elsewhere [44,125]. Likewise, an interactive network with other small GTPases as evident from numerous studies on mammalian Rac1 (recently summarized in [126][127][128][129]) has barely been experimentally tackled for yeast Rho5, so far. ...
Small GTPases are molecular switches that participate in many essential cellular processes. Amongst them, human Rac1 was first described for its role in regulating actin cytoskeleton dynamics and cell migration, with a close relation to carcinogenesis. More recently, the role of Rac1 in regulating the production of reactive oxygen species (ROS), both as a subunit of NADPH oxidase complexes and through its association with mitochondrial functions, has drawn attention. Malfunctions in this context affect cellular plasticity and apoptosis, related to neurodegenerative diseases and diabetes. Some of these features of Rac1 are conserved in its yeast homologue Rho5. Here, we review the structural and functional similarities and differences between these two evolutionary distant proteins and propose yeast as a useful model and a device for high-throughput screens for specific drugs.
... 17 PIAS3-mediated GTP-Rac1 SUMOylation prolongs the Rac1-GTP activated state and further stimulates cell migration. 18 In MDA-MB-231 triple-negative breast cancer cells, PIAS1 and/or PIAS4 mediates PYK2 SUMOylation, subsequently triggering autophosphorylation, interaction with tyrosine kinase SRC, phosphorylation of paxillin, activation of ERK1/2, and promotion of cell migration. 19 Our most recent large-scale quantitative SUMO proteomics analysis revealed that PIAS1 SUMOylates intermediate filament protein vimentin, enhances vimentin solubility, and accelerates intermediate filament disassembly and reassembly, further promoting HeLa cell migration and motility. ...
... 1 Several studies have also demonstrated that PIAS-family members participate in tumorigenesis and cancer-related processes, specifically in the processes of cellular proliferation and migration. 12,18,21,28 To understand the physiological functions and differences between PIAS SUMO E3 ligases, knockout cell lines for each PIAS gene were generated in the breast cancer cell line MDA-MB-231 and used to perform phenotypic assays. All PIAS proteins contain four conserved functional domains that are ideal targets for sgRNA by CRISPR/Cas9 to obtain an efficient gene KO. ...
Protein inhibitor of activated STAT (PIAS) proteins are E3 SUMO ligases playing important roles in protein stability and signaling transduction pathways. PIAS proteins are overexpressed in the triple-negative breast cancer cell line MDA-MB-231, and PIAS knockout (KO) results in a reduction in cell proliferation and cell arrest in the S phase. However, the molecular mechanisms underlying PIAS functions in cell proliferation and cell cycle remain largely unknown. Here, we used quantitative SUMO proteomics to explore the regulatory role of PIAS SUMO E3 ligases upon CRISPR/Cas9 KO of individual PIAS. A total of 1422 sites were identified, and around 10% of SUMO sites were regulated following KO of one or more PIAS genes. We identified protein substrates that were either specific to individual PIAS ligase or regulated by several PIAS ligases. Ki-67 and TOP2A, which are involved in cell proliferation and epithelial-to-mesenchymal transition, are SUMOylated at several lysine residues by all PIAS ligases, suggesting a level of redundancy between these proteins. Confocal microscopy and biochemical experiments revealed that SUMOylation regulated TOP2A protein stability, while this modification is involved in the recruitment of Ki-67 nucleolar proteins containing the SUMO interacting motif. These results provide novel insights into both the redundant and specific regulatory mechanisms of cell proliferation and cell cycle mediated by PIAS SUMO E3 ligases.
... In general, 1 × 10 6 cells were transfected using the DharmaFECT 1 Transfection Reagent (Dharmacon catalogue number T-2001) and incubated for up to 72 h before starting the experiment. For plasmid transfection experiments, 5 μg of plasmids pEGFP-RAC wild type (WT), pEGFP-RAC constitutively active (V12) or pEGFP-C3 (empty vector) was used [24]. Cells were transfected using Lipofectamine 2000 (Invitrogen, #11668-027) and incubated for 24 h before starting the experiments. ...
... Cancer Letters 521 (2021) [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] ...
Cancer-associated fibroblasts (CAFs) are highly abundant stromal components in the tumour microenvironment. These cells contribute to tumorigenesis and indeed, they have been proposed as a target for anti-cancer therapies. Similarly, targeting the Rho-GTPase RAC1 has also been suggested as a potential therapeutic target in cancer. Here, we show that targeting RAC1 activity, either pharmacologically or by genetic silencing, increases the pro-tumorigenic activity of CAFs by upregulating IL-1β secretion. Moreover, inhibiting RAC1 activity shifts the CAF subtype to a more aggressive phenotype. Thus, as RAC1 suppresses the secretion of IL-1β by CAFs, reducing RAC1 activity in combination with the depletion of this cytokine should be considered as an interesting therapeutic option for breast cancer in which tumour cells retain intact IL-1β signalling.
... be SUMOylated (Castillo-Lluva et al., 2010). SUMOylation enhances Rac1 activation (Castillo-Lluva et al., 2010), and subsequent work has demonstrated a link between Rac1 SUMOylation, LTP and performance in object location memory tests in vivo (Yang, Yu, Wen, Ling, & Hsu, 2019). ...
... be SUMOylated (Castillo-Lluva et al., 2010). SUMOylation enhances Rac1 activation (Castillo-Lluva et al., 2010), and subsequent work has demonstrated a link between Rac1 SUMOylation, LTP and performance in object location memory tests in vivo (Yang, Yu, Wen, Ling, & Hsu, 2019). Rac1 SUMOylation is at least partly regulated by expression levels of SENP1 and SENP3, which are in turn regulated by the coiled-coil and C2 domain-containing 1A (CC2D1A) protein . ...
In this Review, we highlight advances in the field of neuronal SUMOylation and discuss how SUMOylation regulates processes within pre‐ and post‐synapses. Enhanced SUMOylation of Na⁺ channels increases conductance and SUMO‐modified collapsin response mediator protein 2 stabilizes Na⁺ channel surface expression. DeSUMOylation of metabotropic glutamate receptors mGluR7 promotes internalization. At the post‐synapse, GluK2 activation leads to SUMOylation and internalization. mGluR activation leads to accumulation of Ubc9, but can also enhance SENP1 levels, likely leading to homeostatic SUMO modulation. Downstream effects include regulation of actin dynamics, increasing AMPA receptor expression and local translation. These findings provide new appreciation of the roles of SUMOylation in synaptic function and plasticity. image
... Our recent report revealed that mutp53 activates Rac1 through increasing Rac1 SUMOylation levels [11]. Rac1 SUMOylation is critical in maintaining the active Rac1-GTP form to enhance Rac1 activity in cells [27]. Here, we investigated whether Rac1 SUMOylation promoted by mutp53 contributes to mutp53-induced AKT activation. ...
... The mutp53-Rac1 interaction activates Rac1 through inhibiting de-SUMOylation of Rac1. SUMOylation of Rac1 is critical to maintain Rac1 activity in cells [27]. The inhibition of Rac1 de-SUMOylation by mutp53 leads to increased Rac1 activity in cells [11]. ...
Tumor suppressor p53 is the most frequently mutated gene in human cancer. Mutant p53 (mutp53) not only loses the tumor suppressive activity of wild type p53, but often gains new oncogenic activities to promote tumorigenesis, defined as mutp53 gain of function (GOF). While the concept of mutp53 GOF is well-established, its underlying mechanism is not well-understood. AKT has been suggested to be activated by mutp53 and contribute to mutp53 GOF, but its underlying mechanism is unclear. In this study, we found that the activation of the Rac1 signaling by mutp53 mediates the promoting effect of mutp53 on AKT activation. Blocking Rac1 signaling by RNAi or a Rac1 inhibitor can inhibit AKT activation by mutp53. Importantly, targeting Rac1/AKT can greatly compromise mutp53 GOF in tumorigenesis. Results from this study uncover a new mechanism for AKT activation in tumors, and reveal that activation of AKT by mutp53 via the Rac1 signaling contributes to mutp53 GOF in tumorigenesis. More importantly, this study provides Rac1 and AKT as potential targets for therapy in tumors containing mutp53.
... Rac1 is an important member of the Rho family and is a key factor regulating the assembly of actin in cells, regulating the formation of filamentous pseudopodia, lamellar pseudopodia, and stress fibers [18,19]. WAVE2 is an important effector molecule downstream of Rac1. ...
... Rac1 activation promotes actin aggregation at the cell front, leading to the formation of lamellar and filamentous pseudopodia. The formation of cellular pseudopodia creates a driving force for the cells to move forward, and causes the cells to polarize and elongate, forming a spindle shape [18,19]. WAVE2 is an important effector molecule downstream of Rac1. ...
Background
The purpose of this study was to investigate whether Orai1 plays a role in the metastasis of osteosarcoma.
Material/Methods
The expression of Orai1 was silenced by small interfering RNAs against Orai1 (Orai1 siRNA) in osteosarcoma MG-63 cells. Various experiments were carried out to detect the changes in migration, invasion, and adhesion ability of these osteosarcoma cells. Furthermore, the activity of Rac1, Wave2, and Ras was detected using Western blot analysis. Moreover, the Rac1 and Ras inhibitors were used to confirm whether the Ras-Rac1-WAVE2 signaling pathway was involved in osteosarcoma metastasis promoted by Orai1.
Results
We found that the migration, invasion, and adhesion ability of MG-63 cells were significantly reduced after silencing Orai1 expression (p<0.05). Moreover, the activity of the Rac1-WAVE2 signaling pathway was significantly inhibited after silencing of Orai1 expression (p<0.05). After the Rac1 inhibitor was added, Orai1 siRNA could not further inhibit migration, invasion, and adhesion of the osteosarcoma cells. Further experiments showed that Ras activity was significantly inhibited after silencing Orai1 expression (p<0.05). Moreover, Orai1 siRNA did not further inhibit the activity of the Rac1-WAVE2 signaling pathway nor did it further inhibit the migration, invasion, and adhesion ability of osteosarcoma cells following the addition of Ras inhibitors.
Conclusions
Orai1 activates the Ras-Rac1-WAVE2 signaling pathway to promote metastasis of osteosarcoma. Abnormal expression or function of Orai1 may be an important cause of osteosarcoma metastasis.
... Rac1 is a signaling G protein that regulates a number of cellular events, among which, besides cytoskeletal reorganization, there are cell growth and activation of kinases. Rac1 signaling is also modulated through post-translational modifications [2][3][4][5][6][7][8][9] (Table 1). In fact, Rac1 is prenylated and subsequently palmitoylated at Cys178, and these post-translational modifications affect Rac1 localization and activity [2]. ...
... Among the Rac1 post-translational modifications, there is SUMOylation by a small ubiquitin-like protein (SUMO) that is covalently linked to lysine residues. SUMOylation of the C-terminal domain of Rac1 leads to increased activity of this GTPase, and it is mediated by the SUMO E3-ligase PIAS3, which, interacting with Rac1, regulates cell migration [6]. Finally, Rac1 is also phosphorylated on Ser71 by Akt and on Thr108 by ERK [7,8]. ...
The small GTPases of the Rho family regulate many aspects of actin dynamics, but are functionally connected to many other cellular processes. Rac1, a member of this family, besides its known function in the regulation of actin cytoskeleton, plays a key role in the production of reactive oxygen species, in gene transcription, in DNA repair, and also has been proven to have specific roles in neurons. This review focuses on the cooperation between Rac1 and Rab proteins, analyzing how the coordination between these GTPases impact on cells and how alterations of their functions lead to disease.
... RhoGDI interacts with X-linked inhibitor of apoptosis protein (XIAP) and negatively modulates RhoGDI SUMOylation and cancer cell migration, invasion and metastasis (180). The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements and it interacts with PIAS3, which is required for increased Rac activation and optimal cell migration and invasion (181). The expression of galectin-1 belongs to the lectin family, participating in malignant tumor development through the regulation of HIF for the cellular response to hypoxia. ...
SUMOylation is a reversible post-translational modification which has emerged as a crucial molecular regulatory mechanism, involved in the regulation of DNA damage repair, immune responses, carcinogenesis, cell cycle progression and apoptosis. Four SUMO isoforms have been identified, which are SUMO1, SUMO2/3 and SUMO4. The small ubiquitin-like modifier (SUMO) pathway is conserved in all eukaryotes and plays pivotal roles in the regulation of gene expression, cellular signaling and the maintenance of genomic integrity. The SUMO catalytic cycle includes maturation, activation, conjugation, ligation and de-modification. The dysregulation of the SUMO system is associated with a number of diseases, particularly cancer. SUMOylation is widely involved in carci-nogenesis, DNA damage response, cancer cell proliferation, metastasis and apoptosis. SUMO can be used as a potential therapeutic target for cancer. In this review, we briefly outline the basic concepts of the SUMO system and summarize the involvement of SUMO proteins in cancer cells in order to better understand the role of SUMO in human disease.
... Interestingly, mutating the pTINCR SIMdomain or treating with a SUMOylation inhibitor impair the ability of pTINCR to activate CDC42, suggesting that CDC42 SUMOylation is required for its activation. These observations are consistent with other small GTPases, such as RAC, that are also activated by SUMOylation 73 . We propose a molecular model in which pTINCR acts as a scaffold to enhance the SUMOylation of CDC42, which in turn promotes its activation. ...
The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.
... Previous data are consistent with these results, as UBC9 protein has been found in the cytosol, nucleus, membranes, and endoplasmic reticulum in a variety of cell types (Gupta et al., 2014), and UBC9 binding has been shown to be crucial for the correct localization at the plasma membrane of different proteins (Collec et al., 2007;Xiong et al., 2017). It has been previously shown that, both UBC9 and SUMO1, promote cell migration, increasing invasion and metastasis in different tumors (Castillo-Lluva et al., 2010;Zhu et al., 2010;LI et al., 2013). Interestingly, here we show that loss of SUMO1 confers a decreased mobility only in HPV-negative HNC cell lines with change in E-cadherin transcription, consistent with the role of the SUMO pathway in affecting E-cadherin transcription through secondary effects (Bogachek et al., 2015). ...
Functional loss of E-cadherin is frequent during tumor progression and occurs through a variety of mechanisms, including proteolytic cleavage. E-cadherin downregulation leads to the conversion of a more malignant phenotype promoting Epithelial to Mesenchymal Transition (EMT). The UBC9/SUMO pathway has been also shown to be involved in the regulation of EMT in different cancers. Here we found an increased expression of UBC9 in the progression of Head and Neck Cancer (HNC) and uncovered a role for UBC9/SUMO in hampering the HPV-mediated E-cadherin cleavage in HNC.
... The GTPase Rac1 is a SUMOylation substrate of PIAS3; it is very essential for cell migration that can induce cytoskeletal rearrangements [189]. When the expression of PIAS3 is down-regulated, the migration ability of cells is impaired compared with the control group. ...
Small ubiquitin-like modifier (SUMO)ylation is a reversible post-translational modification that plays a crucial role in numerous aspects of cell physiology, including cell cycle regulation, DNA damage repair, and protein trafficking and turnover, which are of importance for cell homeostasis. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases recruit substrates and accelerate the transfer of SUMO onto targets, modulating their interactions, localization, activity, or stability. Accumulating evidence highlights the critical role of dysregulated SUMO E3 ligases in processes associated with the occurrence and development of cancers. In the present review, we summarize the SUMO E3 ligases, in particular, the novel ones recently identified, and discuss their regulatory roles in cancer pathogenesis.
... Recent studies have identified two pharmacological inhibitors of the SUMO pathway, namely, ginkgolic acids C15:1 (GA C15:1) that interact with SUMO E1 activating enzymes to abrogate the formation of the E1-SUMO1 complex [152], and 2-D08, which suppresses sumoylation by inhibiting the transfer of SUMO from SUMO E2 conjugating enzyme to target substrate [153]. These two inhibitors play a pivotal anti-cancer role in breast cancer cell lines, such as MDA-MB-231, MCF-7, and BT474, not only by inducing the expression of autophagy modulator Tribbles pseudokinase 3 (TRIB3) and the transcription of various autophagy-related genes, such as ATG1, ATG7, and BECN1 to accelerate autophagy-dependent cancer cell death, but also by inhibiting the sumoylation of RAC1 (a member of Rho GTPase family), and thus suppress the activation RAC1 and repress the RAC1mediated cell migration and invasion [154,155]. Moreover, Triptolide, a component extracted from the Chinese herb, Tripterygium wilfordii Hook F, acts as a natural SENP1 inhibitor that downregulates the expression of the androgen receptor (AR) and c-Jun to restore the balance between sumoylation and deSUMOylation and consequently inhibits prostate cancer [156]. ...
Breast cancer is the most prevalent malignant tumor and a leading cause of mortality among females worldwide. The tumorigenesis and progression of breast cancer involve complex pathophysiological processes, which may be mediated by post-translational modifications (PTMs) of proteins, stimulated by various genes and signaling pathways. Studies into PTMs have long been dominated by the investigation of protein phosphorylation and histone epigenetic modifications. However, with great advances in proteomic techniques, several other PTMs, such as acetylation, glycosylation, sumoylation, methylation, ubiquitination, citrullination, and palmitoylation have been confirmed in breast cancer. Nevertheless, the mechanisms, effects, and inhibitors of these unconventional PTMs (particularly, the non-histone modifications other than phosphorylation) received comparatively little attention. Therefore, in this review, we illustrate the functions of these PTMs and highlight their impact on the oncogenesis and progression of breast cancer. Identification of novel potential therapeutic drugs targeting PTMs and development of biological markers for the detection of breast cancer would be significantly valuable for the efficient selection of therapeutic regimens and prediction of disease prognosis in patients with breast cancer.
... This is particularly key for RHOH, RHOU, RND, and RHOBTB subfamilies (often referred to as "atypical" Rho GTPases), which are predominantly GTP bound (13). For example, sumoylation of RAC1 increased RAC1-GTP levels and MDCKII cancer cell migration and invasion (14). Rho GTPase function is also controlled by gene expression and posttranscriptional regulation by miRNAs (5). ...
Rho GTPases are a family of small G proteins that regulate a wide array of cellular processes related to their key roles controlling the cytoskeleton. On the other hand, cancer is a multi-step disease caused by the accumulation of genetic mutations and epigenetic alterations, from the initial stages of cancer development when cells in normal tissues undergo transformation, to the acquisition of invasive and metastatic traits, responsible for a large number of cancer related deaths. In this review, we discuss the role of Rho GTPase signalling in cancer in every step of disease progression. Rho GTPases contribute to tumour initiation and progression, by regulating proliferation and apoptosis, but also metabolism, senescence and cell stemness. Rho GTPases play a major role in cell migration, and in the metastatic process. They are also involved in interactions with the tumour microenvironment and regulate inflammation, contributing to cancer progression. After years of intensive research, we highlight the importance of relevant models in the Rho GTPase field, and we reflect on the therapeutic opportunities arising for cancer patients.
... The SUMO pathway regulates angiogenesis in response to hypoxia through modulation of vascular endothelial growth factor (VEGF) expression levels [44,141,142]. Furthermore, SUMOylation is involved in cellular migration and epithelial mesenchymal transition (EMT) via multiple mechanisms [143][144][145][146][147][148]. The SUMO pathway is also implicated in regulation of stem-like cell properties of cancer cells e.g., via PIAS3 and STAT3 [129,149]. ...
SUMOylation is a dynamic and reversible post-translational modification, characterized more than 20 years ago, that regulates protein function at multiple levels. Key oncoproteins and tumor suppressors are SUMO substrates. In addition to alterations in SUMO pathway activity due to conditions typically present in cancer, such as hypoxia, the SUMO machinery components are deregulated at the genomic level in cancer. The delicate balance between SUMOylation and deSUMOylation is regulated by SENP enzymes possessing SUMO-deconjugation activity. Dysregulation of SUMO machinery components can disrupt the balance of SUMOylation, contributing to the tumorigenesis and drug resistance of various cancers in a context-dependent manner. Many molecular mechanisms relevant to the pathogenesis of specific cancers involve SUMO, highlighting the potential relevance of SUMO machinery components as therapeutic targets. Recent advances in the development of inhibitors targeting SUMOylation and deSUMOylation permit evaluation of the therapeutic potential of targeting the SUMO pathway in cancer. Finally, the first drug inhibiting SUMO pathway, TAK-981, is currently also being evaluated in clinical trials in cancer patients. Intriguingly, the inhibition of SUMOylation may also have the potential to activate the anti-tumor immune response. Here, we comprehensively and systematically review the recent developments in understanding the role of SUMOylation in cancer and specifically focus on elaborating the scientific rationale of targeting the SUMO pathway in different cancers.
... Four distinct paralogs exist in humans. While recent reports show that various members of the Ras small GTPase family are targeted for SUMO conjugation [58,59], the only Rho GTPase described as SUMOylation substrate is Rac1, whose activity is enhanced upon SUMOylation at its C-terminal region [60]. Indeed, blocking SUMOylation decreases Rac1-dependent breast tumor cell migration and promotes autophagymediated cell death [61]. ...
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.
... This increase in activity is remarkable given that it occurs in an already constitutively active Rac1 protein. Since lysine 183 and 184 are targeted for SUMOylation [48], the loss of SUMOylation at this location may explain the increase in activity [49]. Furthermore, orientation of the C-terminus of Rac1 with respect to the rest of the molecule is not resolved in the 3D structure. ...
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.
... Sumoylation has, for example, been found to affect structural cytoskeletal proteins, including intermediate filament proteins and actin (Hofmann et al., 2009;Kaminsky et al., 2009;Snider et al., 2011;Boudreau et al., 2012;Alonso et al., 2015). In addition, sumoylation controls cytoskeletal regulatory proteins, including the actin regulators Rho1 and Rac1 (Castillo-Lluva et al., 2010;Yu et al., 2012), as well as tau and other microtubule associated proteins (Luo et al., 2014;Abrieu and Liakopoulos, 2019). Beyond these effects of SUMO2 on structural and regulatory cytoskeletal proteins, however, we also observed significant changes at the level of gene expression for genes involved in the epithelial to mesenchymal transition (EMT), the extracellular matrix, integrin-cell surface interactions and genes coding for Wnt-family proteins in the SUMO2 KO cells, which could also contribute to changes in cellular morphology. ...
The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogs with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogs, and also the best studied. To what extent SUMO1, SUMO2 and the other paralogs impart unique and non-redundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogs during development and studies in cell culture have relied largely on transient paralog overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralog gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralog-specific functions in adult human tissues. To systematically identify and characterize unique and non-redundant functions of the SUMO paralogs in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, PML nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal non-redundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.
... All the data obtained in this study suggest that the decreased expression of SNAI2 by CAFs reduces cytokine production and influences the tumor-host microenvironment by modifying the ERK/AKT pathways. Other unknown elements of the EMT program are also likely to be affected (62). Thus, more studies will be necessary to clarify the mechanism by which SNAI2 expression in CAFs affects metastasis in the luminal breast tumors. ...
SNAI2 overexpression appears to be associated with poor prognosis in breast cancer, yet it remains unclear in which breast cancer subtypes this occurs. Here we show that excess SNAI2 is associated with a poor prognosis of luminal B HER2⁺/ERBB2⁺ breast cancers in which SNAI2 expression in the stroma but not the epithelium correlates with tumor proliferation. To determine how stromal SNAI2 might influence HER2⁺ tumor behavior, Snai2-deficient mice were crossed with a mouse line carrying the ErbB2/Neu protooncogene to generate HER2⁺/ERBB2⁺ breast cancer. Tumors generated in this model expressed SNAI2 in the stroma but not the epithelium, allowing for the role of stromal SNAI2 to be studied without interference from the epithelial compartment. The absence of SNAI2 in the stroma of HER2⁺/ERBB2⁺ tumors is associated with: (i) lower levels of cyclin D1 (CCND1) and reduced tumor epithelium proliferation; (ii) higher levels of AKT and a lower incidence of metastasis; (iii) lower levels of angiopoietin-2 (ANGPT2), and more necrosis. Together, these results indicate that the loss of SNAI2 in cancer-associated fibroblasts limits the production of some cytokines, which influences AKT/ERK tumor signaling and subsequent proliferative and metastatic capacity of ERBB2⁺ breast cancer cells. Accordingly, SNAI2 expression in the stroma enhanced the tumorigenicity of luminal B HER2⁺/ERBB2⁺ breast cancers. This work emphasizes the importance of stromal SNAI2 in breast cancer progression and patients' prognosis.
Significance: Stromal SNAI2 expression enhances the tumorigenicity of luminal B HER2⁺ breast cancers and can identify a subset of patients with poor prognosis, making SNAI2 a potential therapeutic target for this disease.
Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/80/23/5216/F1.large.jpg.
... In response to hepatocyte growth factor treatment (HGF), the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3 interacts with Rac1 and triggers SUMO conjugation at Lys 188, 183, and 184 or 186 within the C-terminal polybasic region (PBR) of Rac1. This SUMOylation is required for increased Rac1 activation and for stimulating lamellipodia, cell migration and invasion [30]. In human colorectal cancers, Rac1 SUMOylation and activation correlates with mutant TP53 status. ...
The small GTPase Rac1 has been implicated in a variety of dynamic cell biological processes, including cell proliferation, cell survival, cell-cell contacts, epithelial mesenchymal transition (EMT), cell motility, and invasiveness. These processes are orchestrated through the fine tuning of Rac1 activity by upstream cell surface receptors and effectors that regulate the cycling Rac1-GDP (off state)/Rac1-GTP (on state), but also through the tuning of Rac1 accumulation, activity, and subcellular localization by post translational modifications or recruitment into molecular scaffolds. Another level of regulation involves Rac1 transcripts stability and splicing. Downstream, Rac1 initiates a series of signaling networks, including regulatory complex of actin cytoskeleton remodeling, activation of protein kinases (PAKs, MAPKs) and transcription factors (NFkB, Wnt/β-catenin/TCF, STAT3, Snail), production of reactive oxygen species (NADPH oxidase holoenzymes, mitochondrial ROS). Thus, this GTPase, its regulators, and effector systems might be involved at different steps of the neoplastic progression from dysplasia to the metastatic cascade. After briefly placing Rac1 and its effector systems in the more general context of intestinal homeostasis and in wound healing after intestinal injury, the present review mainly focuses on the several levels of Rac1 signaling pathway dysregulation in colorectal carcinogenesis, their biological significance, and their clinical impact.
... These results were unforeseen since the stimulation of cells with FBS or growth factors is generally expected to increase Rac1 activation, as we observed in LNCaP cells and as previously reported in other cell types [48,49]. Furthermore, calcium-signaling events in other cell types and contexts has been shown to indirectly regulate the activation of Rac1 [50][51][52]. It is therefore remarkable that a rise in intracellular calcium results in the deactivation of Rac1 and Cdc42 in PC3 cells and may indicate the presence of a potent calcium-stimulated GAP or the calcium dependent dissociation of a binding partner that otherwise sustains hyperactivation of these GTPases. ...
The GTPase Rac1 is a well-established master regulator of cell motility and invasiveness contributing to cancer metastasis. Dysregulation of the Rac1 signaling pathway, resulting in elevated motile and invasive potential, has been reported in multiple cancers. However, there are limited studies on the regulation of Rac1 in prostate cancer. Here, we demonstrate that aggressive androgen-independent prostate cancer cells display marked hyperactivation of Rac1. This hyperactivation is independent of P-Rex1 activity or its direct activators, the PI3K product PIP3 and Gβγ subunits. Furthermore, we demonstrate that the motility and invasiveness of PC3 prostate cancer cells is independent of P-Rex1, supporting the analysis of publicly available datasets indicating no correlation between high P-Rex1 expression and cancer progression in patients. Rac1 hyperactivation was not related to the presence of activating Rac1 mutations and was insensitive to overexpression of a Rac-GAP or the silencing of specific Rac-GEFs expressed in prostate cancer cells. Interestingly, active Rac1 levels in these cells were markedly reduced by elevations in intracellular calcium or by serum stimulation, suggesting the presence of an alternative means of Rac1 regulation in prostate cancer that does not involve previously established paradigms.
... We presently focused on the resident macrophages and their related polarization program, but it will be of future interest to clarify the mechanisms leading to the impaired migratory capacity. Given the facts that Rac1 is a SUMOylation target relevant to cell mobility 53 and that ablation of its SUMOylation is associated with reduced cell movement in other cell types, such as tumor cells and synoviocytes 54 , the impaired migratory capability in the KO macrophages could be caused by the lack of Rac1 SUMOylation. Second, in the present study we focused on the effects of Ubc9-mediated SUMOylation of IRF4, a key transcription factor for the M2 program in macrophages. ...
Type 1 diabetes (T1D) is characterized by the selective autoimmune destruction of the islet β cells, and macrophages play a significant role in this process. Small ubiquitin-like modification (SUMOylation) is an important posttranslational modification involved in T1D pathogenesis, but its function in macrophages remains unexplored. We presently developed and used macrophage-specific ubiquitin-conjugating enzyme E2 (Ubc9) knockout (LyzM-Cre-Ubc9fl/fl, KO) mice to address the impact of SUMOylation on macrophage function in a T1D model. We observed that blocking Ubc9 in macrophages exacerbated multiple-low dose streptozotocin (MLD-STZ)-induced diabetes. Specifically, after STZ treatment, blood glucose levels were consistently elevated in the KO mice. The KO mice exhibited a higher diabetes incidence than WT controls (85% vs. 55%, P < 0.01) along with a higher insulitis severity. The loss of Ubc9 impaired macrophage energy metabolism and attenuated macrophage M2 program, thereby enhancing T cell activation. Pancreas-resident macrophages, rather than migrant macrophages, played a predominant role in MLD-STZ-induced diabetes. Mechanistically, Ubc9-mediated SUMOylation of interferon regulator factor 4 (IRF4) enhanced its nuclear localization and stability, thereby transcribing IL-4 and arginase 1 (Arg1) to promote the macrophage M2 program. Ubc9-mediated SUMOylation modulates T1D risk at least in part by regulating macrophage function. Modulation of disturbed SUMOylation process in macrophages, either through cell adoptive transfer or targeted drug-delivery, could help to establish a tolerant pancreatic microenvironment and promote inflammation resolution in early insulitis stage, thus hindering T1D progression.
... Several studies have reported the involvement of HDAC4 in SUMOylation of the transcription factor MEF2 (Gregoire and Yang, 2005;Zhao et al., 2005), suggesting that synaptic proteins may also be targeted. Synaptically localized HDAC4 may SUMOylate Rac1, which is typically required for Rac1 activation and proper cell motility (Castillo-Lluva et al., 2010). As CXCR4 increases dendritic spine density, HDAC4 may also be involved in Rac1 activation. ...
... Furthermore, the prenylation enzyme is increased in hyperglycemic condition, and inhibition of FNTA prevents the activation of Rac1-Nox2-ROS signaling, suggesting prenylation is also an important posttranslational modification for Rac1 activation and the development and progression of diabetic retinopathy [15,63]. Although not examined in the context of the onset of diabetic retinopathy, Rac1 functions have also been shown to be regulated by RNA splicing and other post-translational modifications such as ubiquitination, adenylation, phosphorylation and SUMOylation [64,65], and mutations of Rho GTPases (e.g., Cdc42, Rho and Rac1) have also been reported in pathological states, including immonodeficiency and cancer [64]. ...
Diabetic retinopathy, a microvascular complication of diabetes, remains the leading cause of vision loss in working age adults. Hyperglycemia is considered as the main instigator for its development, around which other molecular pathways orchestrate. Of these multiple pathways, oxidative stress induces many metabolic, functional and structural changes in the retinal cells, leading to the development of pathological features characteristic of this blinding disease. An increase in cytosolic reactive oxygen species (ROS), produced by cytosolic NADPH oxidase 2 (Nox2), is an early event in the pathogenesis of diabetic retinopathy, which leads to mitochondrial damage and retinal capillary cell apoptosis. Activation of Nox2 is mediated through an obligatory small molecular weight GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1), and subcellular localization of Rac1 and its activation are regulated by several regulators, rendering it a complex biological process. In diabetes, Rac1 is functionally activated in the retina and its vasculature, and, via Nox2-ROS, contributes to mitochondrial damage and the development of retinopathy. In addition, Rac1 is also transcriptionally activated, and epigenetic modifications play a major role in this transcriptional activation. This review focusses on the role of Rac1 and its regulation in the development and progression of diabetic retinopathy, and discusses some possible avenues for therapeutic interventions.
... In Astor et al., we proposed a potential role for Apj1 in either model, based largely on Sahi et al.'s determination that Apj1 is involved in the degradation of sumoylated proteins (Sahi et al. 2013). In the case of the malpartitioning model, we posited that Apj1 may assist in malpartitioning, as cytoskeletal elements such as actin and tubulin are sumoylated (Panse et al. 2004;Castillo-Lluva et al. 2010;Gareau and Lima 2010). In the case of the trimming model, we suggested that Apj1 may be involved in assisting in or promoting the degradation of the prion cores. ...
Prions are self-propagating protein isoforms that are typically amyloid. In Saccharomyces cerevisiae, amyloid prion aggregates are fragmented by a trio involving three classes of chaperone proteins: Hsp40s, also known as J-proteins, Hsp70s, and Hsp104. Hsp104, the sole Hsp100-class disaggregase in yeast, along with the Hsp70 Ssa and the J-protein Sis1, is required for the propagation of all known amyloid yeast prions. However, when Hsp104 is ectopically overexpressed, only the prion [PSI⁺] is efficiently eliminated from cell populations via a highly debated mechanism that also requires Sis1. Recently, we reported roles for two additional J-proteins, Apj1 and Ydj1, in this process. Deletion of Apj1, a J-protein involved in the degradation of sumoylated proteins, partially blocks Hsp104-mediated [PSI⁺] elimination. Apj1 and Sis1 were found to have overlapping functions, as overexpression of one compensates for loss of function of the other. In addition, overexpression of Ydj1, the most abundant J-protein in the yeast cytosol, completely blocks Hsp104-mediated curing. Yeast prions exhibit structural polymorphisms known as “variants”; most intriguingly, these J-protein effects were only observed for strong variants, suggesting variant-specific mechanisms. Here, we review these results and present new data resolving the domains of Apj1 responsible, specifically implicating the involvement of Apj1’s Q/S-rich low-complexity domain.
... Information concerning the involvement of SUMO in cell movement is limited. A morpholino oligonucleotide directed at SUMO1 prevented activin-induced elongation of Xenopus animal caps [15] and SUMOylation of Rac1 GTPase is needed for optimal cell migration in response to hepatocyte growth factor [24]. Since cell movement during gastrulation is primarily internal and cannot be easily observed, we turned to Keller explant assays to determine whether the SUMOylation deficiency in Gam1 embryos impacts this activity [25]. ...
Background
Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development.
Results
We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex, as opposed to housekeeping, processes.
Conclusions
We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies.
Electronic supplementary material
The online version of this article (10.1186/s12864-019-5773-3) contains supplementary material, which is available to authorized users.
... PIAS proteins contain four conserved motifs and domains consisting of a SAP region for binding to chromatin, PINIT motif for localization, and RING for E3-SUMO ligation (a SUMO-binding motif) [69][70][71]. PIAS3, by SUMOylation of Rac1, has been reported to control cell migration [72]. To illustrate the effect of the PIAS family on FLS motility, knockdown of PIAS3 by short hairpin RNA (shRNA) was investigated. ...
Small ubiquitin-like modifier (SUMO) proteins, as a subgroup of post-translational modifiers, act to change the function of proteins. Through their interactions with different targets, immune pathways, and the responses they elicit, can be affected by these SUMO conjugations. Thus, both a change to protein function and involvement in immune pathways has the potential to promote an efficient immune response to either a pathogenic challenge, or the development of an imbalance that could lead to an autoimmune-based disease. Also, a variety of changes such as mutations and polymorphisms can interfere with common functions of these modifications and move an effective immune response in the direction of an autoimmune disease. The present review discusses the general characteristics of SUMO proteins and focuses on their involvement in rheumatoid arthritis as an autoimmune disease.
... Although HACE1 negatively regulates activated Rac1, activated Rac1 also negatively regulates HACE1 via group I PAK kinase-mediated phosphorylation [59]: mutually antagonistic signalling networks such as this have been shown to generate "on-off' switches for cellular functions [60]. Rac1 is also modified by sumoylation, which enhances the activation of Rac1 and promotes lamellipodia formation [61]. The significance of this in glioblastoma or other cancers is not currently known. ...
Glioblastoma is an aggressive and incurable form of brain cancer. Both mutation analysis in human glioblastoma and mouse modelling studies have shown that aberrant activation of the PI 3-kinase pathway is a central driver of glioblastoma malignancy. The small GTPase Rac is activated downstream of this pathway, mediating a subset of the effects of aberrant PI 3-kinase pathway activation. Here I discuss the current state of our knowledge on Rac activation mechanisms in glioblastoma. Current knowledge on roles for specific PI 3-kinase pathway responsive Rac guanine nucleotide exchange factors in glioblastoma is reviewed. Rac is best known for its role in promoting cell motility and invasion, but there is also evidence for roles in multiple other cellular processes with cancer relevance, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis and immunosuppression. I review what is known about the role of Rac in these processes in glioblastoma. Finally, I assess possible strategies to inhibit this pathway in glioblastoma through either direct inhibition of Rac, or inhibition of upstream activators or downstream mediators of Rac signalling.
... Pull down assays were performed as described in Castillo-Lluva et al. (42) and Woodcock et al. (43). Cells were seeded to a density of 500,000 cells per plate in 60 cm diameter tissue culture plates and were grown in complete medium with 5% FBS. ...
Non-typeable Haemophilus influenzae (NTHi) causes persistent respiratory infections in patients with chronic obstructive pulmonary disease (COPD), probably linked to its capacity to invade and reside within pneumocytes. In the alveolar fluid, NTHi is in contact with pulmonary surfactant, a lipoprotein complex that protects the lung against alveolar collapse and constitutes the front line of defense against inhaled pathogens and toxins. Decreased levels of surfactant phospholipids have been reported in smokers and patients with COPD. The objective of this study was to investigate the effect of surfactant phospholipids on the host-pathogen interaction between NTHi and pneumocytes. For this purpose, we used two types of surfactant lipid vesicles present in the alveolar fluid: (i) multilamellar vesicles (MLVs, > 1 μm diameter), which constitute the tensioactive material of surfactant, and (ii) small unilamellar vesicles (SUVs, 0.1 μm diameter), which are generated after inspiration/expiration cycles, and are endocytosed by pneumocytes for their degradation and/or recycling. Results indicated that extracellular pulmonary surfactant binds to NTHi, preventing NTHi self-aggregation and inhibiting adhesion of NTHi to pneumocytes and, consequently, inhibiting NTHi invasion. In contrast, endocytosed surfactant lipids, mainly via the scavenger receptor SR-BI, did not affect NTHi adhesion but inhibited NTHi invasion by blocking bacterial uptake in pneumocytes. This blockade was made possible by inhibiting Akt phosphorylation and Rac1 GTPase activation, which are signaling pathways involved in NTHi internalization. Administration of the hydrophobic fraction of lung surfactant in vivo accelerated bacterial clearance in a mouse model of NTHi pulmonary infection, supporting the notion that the lipid component of lung surfactant protects against NTHi infection. These results suggest that alterations in surfactant lipid levels in COPD patients may increase susceptibility to infection by this pathogen.
... Based on the literatures regarding Rac1 and cell migration, Rac1-GTP can be upregulated by SUMOylation, which maintains Rac1 activity [44], or downregulated by ubiquitination, which degrades Rac1 [45]. In the current study, we first demonstrated that a novel function of intracellular-free ISG15 is involved in cell migration via regulation of Rac1 activity by binding to Rac1-GDP. ...
In an effort to understand the underlying mechanisms of lymph node metastasis in oral squamous cell carcinoma (OSCC), through in vivo selection, LN1-1 cells were previously established from OEC-M1 cells and showed enhanced lymphangiogenesis and lymphatic metastasis capabilities. In the current study, we use a stable isotope labeling with amino acids in cell culture (SILAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic platform to compare LN1-1 to OEC-M1 cells. Interferon-stimulated gene 15 (ISG15) was found highly expressed in LN1-1 cells. Immunohistochemical analysis and meta-analysis of publicly available microarray datasets revealed that the ISG15 level was increased in human OSCC tissues and associated with poor disease outcome. Knockdown of ISG15 had minimal effects on tumor growth but did decrease tumor lymphangiogenesis and lymphatic metastasis of LN1-1 cells. Consistent with the in vivo assay, ISG15 knockdown did not impair cell growth but diminished cell migration, invasion, and transendothelial migration in vitro. ISG15-induced cell migration was independent of ISGylation and associated with membrane protrusion. Ectopic expression of ISG15 increased Rac1 activity and knockdown of Rac1 impaired ISG15-enhanced migration. Furthermore, Rac1 colocalized with ISG15 to a region of membrane protrusion and ISG15 coimmunoprecipitated with Rac1, especially with the Rac1-GDP form. Importantly, as shown by proximity ligation assays, ISG15 and Rac1 physically interacted with each other. Our results indicated that ISG15 affects cell migration by interacting with Rac1 and regulating Rac1 activity and contributes to lymphatic metastasis in OSCC.
Bacteria frequently interfere with the post-translational modifications of host cells to facilitate their survival and growth after invasion. SUMOylation, a reversible post-translational modification process, plays an important role in biological life activities. In addition to being critical to host cell metabolism and survival, SUMOylation also regulates gene expression and cell signal transmission. Moreover, SUMOylation in eukaryotic cells can be used by a variety of bacterial pathogens to advance bacterial invasion. In this minireview, we focused on the role and mechanism of host SUMOylation in the pathogenesis of six important clinical bacterial pathogens ( Listeria monocytogenes , Shigella flexneri , Salmonella Typhimurium , Klebsiella pneumoniae , Staphylococcus aureus , and Escherichia coli ). Taken together, this review provided new insights for understanding the unique pathogen-host interaction based on host SUMOylation and provided a novel perspective on the development of new strategies to combat bacterial infections in the future.
The Rho family GTPases play an important role in mediating signal transduction in multiple fundamental cellular processes. Most of the Rho family members act as molecular switches and cycle between an inactive GDP-bound state and an active GTP-bound state. Upon GTP binding, Rho GTPases undergo conformational changes to interact with a wide variety of downstream effectors. The dynamic cycling of GTP loading/GTP hydrolysis is key for Rho GTPase functions and is tightly regulated by guanine nucleotide exchange factors, GTPase-activating proteins, GDP-dissociation inhibitors, as well as additional posttranslational modifications. Dysregulation of Rho GTPase signaling pathways is involved in multiple human pathological conditions including cancer, inflammation, and cardiovascular diseases. Among the Rho family members, Rac1 and Rac2 are crucial for the assembly and activation of NADPH oxidases NOX1 and NOX2. This chapter provides an overview of the structural mechanisms of the regulation and function of Rho GTPases, with an emphasis on Rac1 and Rac2 where they apply.KeywordsRho GTPasesRacSignaling effectorsNOX2Rho GEFsRho GAPsRho GDIs
Regulation of Rho GTPases remains a topic of active investigation as they are essential participants in cell biology and the pathophysiology of many human diseases. Non-degrading ubiquitination (NDU) is a critical regulator of the Ras superfamily, but its relevance to Rho proteins remains unknown. We show that RhoC, but not RhoA, is a target of NDU by E3 ubiquitin ligase, LNX1. Furthermore, LNX1 ubiquitination of RhoC is negatively regulated by LIS1 (aka, PAFAH1B1). Despite multiple reports of functional interaction between LIS1 and activity of Rho proteins, a robust mechanism linking the two has been lacking. Here, LIS1 inhibition of LNX1 effects on RhoGDI-RhoC interaction provides a molecular mechanism underpinning the enhanced activity of Rho proteins observed upon reduction in LIS1 protein levels. Since LNX1 and RhoC are only found in vertebrates, the LIS1-LNX1-RhoC module represents an evolutionarily acquired function of the highly conserved LIS1. While these nearly identical proteins have several distinct RhoA and RhoC downstream effectors, our data provide a rare example of Rho-isoform specific, upstream regulation that opens new therapeutic opportunities.
SUMOylation is a key post-translational modification that involves the covalent attachment of small ubiquitin-like modifier (SUMO) to the lysine residues of target proteins. The well-balanced SUMOylation is essential for normal cellular behaviors, while disturbance of SUMOylation is associated with various cancers and other diseases. Herein, we summarize the structures and biological functions of proteins involved in the SUMOylation process, their dysregulation in human diseases, and the discovery of small-molecular inhibitors targeting this pathway. In addition, we highlight the emerging trends in this field.
Fibrosis is an abnormal healing process that only repairs the structure of an organ after injury and does not address damaged functions. The pathogenesis of fibrosis is multifactorial and highly complex; numerous signalling pathways are involved in this process, with the transforming growth factor-β (TGF-β) signalling pathway playing a central role. TGF-β regulates the generation of myofibroblasts and the epithelial–mesenchymal transition by regulating transcription and translation of downstream genes and precisely regulating fibrogenesis. The TGF-β signalling pathway can be modulated by various post-translational modifications, of which SUMOylation has been shown to play a key role. In this review, we focus on the function of SUMOylation in canonical and non-canonical TGF-β signalling and its role in fibrosis, providing promising therapeutic strategies for fibrosis.
The rapid healing of impaired intestinal surface plays a role in maintaining intestinal homeostasis. This study investigated the effect of calcium-sensing receptor (CaSR) on the migration and proliferation of intestinal porcine epithelial cells (IPEC-J2). Results showed that cell migration area and width were increased by R568 (CaSR activator) and decreased by NPS2143 (CaSR inhibitor). The protein level of GTP-rac1 and the phosphorylation of phospholipase C gamma 1 (PLCγ1) were increased by 2 µM R568. Furthermore, R568 + 120 µM NSC23766 (Rac1 inhibitor) and R568 + 1 µM U73122 (PLCγ1 inhibitor) decreased the protein level of GTP-rac1 and the phosphorylated PLCγ1, respectively, and both inhibited cell migration compared with R568. In addition, spermine increased the protein expression levels of CaSR and the levels of GTP-rac1 and the phosphorylated PLCγ1 and thereby promoted the migration of IPEC-J2 cells. Moreover, R568 improved the proliferation of the IPEC-J2 cells. Spermine increased cell proliferation, but NPS2143 incubated with spermine decreased cell proliferation compared with the spermine group. This study suggests that CaSR activation increased cell migration by activating Rac1 and PLCγ1 signaling and improved cell proliferation, and both effects were regulated by spermine by activating CaSR.
Dysregulation of glucose homeostasis leading to metabolic syndrome and type 2 diabetes is the cause of an increasing world health crisis. New intriguing roles have emerged for Rho family GTPases and their Rho guanine nucleotide exchange factor (GEF) activators in the regulation of glucose homeostasis. This review summates the current knowledge, focusing in particular on the roles of Rho GEFs in the processes of glucose-stimulated insulin secretion by pancreatic β cells and insulin-stimulated glucose uptake into skeletal muscle and adipose tissues. We discuss the ten Rho GEFs that are known so far to regulate glucose homeostasis, nine of which are in mammals, and one is in yeast. Among the mammalian Rho GEFs, P-Rex1, Vav2, Vav3, Tiam1, Kalirin and Plekhg4 were shown to mediate the insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane and/or insulin-stimulated glucose uptake in skeletal muscle or adipose tissue. The Rho GEFs P-Rex1, Vav2, Tiam1 and β-PIX were found to control the glucose-stimulated release of insulin by pancreatic β cells. In vivo studies demonstrated the involvement of the Rho GEFs P-Rex2, Vav2, Vav3 and PDZ-RhoGEF in glucose tolerance and/or insulin sensitivity, with deletion of these GEFs either contributing to the development of metabolic syndrome or protecting from it. This research is in its infancy. Considering that over 80 Rho GEFs exist, it is likely that future research will identify more roles for Rho GEFs in glucose homeostasis.
Objective:
To investigate whether human RhoA is modified by SUMO.
Methods:
Overlap extension PCR and double digestion technique were used to construct the eukaryotic expression vector pcDNA3-3flag-RhoA, which was identified by sequencing. The plasmid was transfected into HEK293T cells and its expression was detected by Western blotting. Immunofluorescence assay was used to detect whether RhoA is co-localized with SUMO. Co-Immunoprecipitation was used to detect whether RhoA is modified by SUMO.
Results:
The recombinant plasmid pcDNA3-3flag-RhoA was successfully constructed and verified. Western blotting showed that the recombinant plasmid pcDNA3-3flag-RhoA expressed abundant fusion protein in HEK293T cells. Immunofluorescence showed that RhoA was co-localized with SUMO2/3 but not with SUMO1. Co-immunoprecipitation verified that RhoA was modified by SUMO2/3 but not SUMO1.
Conclusions:
Human RhoA is modified by SUMO2/3 and probably participates in the regulation of axon regrowth after nervous system injury.
SUMOylation has emerged as an important post-translational modification that has been shown to modulate protein activity associated with various signaling pathways, and consequently, it has emerged as an important therapeutic target. While several natural products have been shown to inhibit enzymes involved in the SUMOylation process, there has been little progress toward the development of more selective and potent SUMOylation inhibitors. Ginkgolic acid was one of the first natural products discovered to inhibit the SUMO E1 enzyme. Despite its use to mechanistically investigate the SUMOylation process, ginkgolic acid also modulates other pathways as well. In this Letter, preliminary structure-activity relationships for ginkgolic acid as a SUMOylation inhibitor are presented.
Mitochondria play a central role in biological oxidation that inevitably generates reactive oxygen species (ROS) as by-products. Maintenance of mitochondrial redox balance status requires NADPH, which is primarily generated by the mitochondrial matrix protein isocitrate dehydrogenase 2 (IDH2). The activity of IDH2 is regulated by post-translational modifications (PTMs). In this study, we found IDH2 is modified by small ubiquitin-like modifier 1 (SUMO1) at lysine 45. SUMO specific protease 1 (SENP1) is responsible for deSUMOylation of IDH2. SUMOylation of IDH2 is induced by oxidants and enhances the antioxidant activity of IDH2 to protect cells against oxidative stress. Mutation of the SUMOylation site impairs the enzymatic activity of IDH2 and hence decreases levels of α-ketoglutarate (α-KG), NADPH and GSH. Cells with SUMOylation deficient IDH2 suffer more apoptosis than that with wild type IDH2 under oxidative stress. These results indicate that SUMOylation is an important way to regulate IDH2 activity to maintain mitochondrial redox balance.
Rho GTPases play central roles in numerous cellular processes, including cell motility, cell polarity, and cell cycle progression, by regulating actin cytoskeletal dynamics and cell adhesion. Dysregulation of Rho GTPase signaling is observed in a broad range of human cancers, and is associated with cancer development and malignant phenotypes, including metastasis and chemoresistance. Rho GTPase activity is precisely controlled by guanine nucleotide exchange factors, GTPase-activating proteins, and guanine nucleotide dissociation inhibitors. Recent evidence demonstrates that it is also regulated by post-translational modifications, such as phosphorylation, ubiquitination, and sumoylation. Here, we review the current knowledge on the role of Rho GTPases, and the precise mechanisms controlling their activity in the regulation of cancer progression. In addition, we discuss targeting strategies for the development of new drugs to improve cancer therapy.
Glucose-induced (physiological) insulin secretion from the islet β-cell involves interplay between cationic (i.e., changes in intracellular calcium) and metabolic (i.e., generation of hydrophobic and hydrophilic second messengers) events. A large body of evidence affirms support for novel regulation, by G proteins, of specific intracellular signaling events, including actin cytoskeletal remodeling, transport of insulin-containing granules to the plasma membrane for fusion, and secretion of insulin into the circulation. This article highlights the following aspects of GPCR-G protein biology of the islet. First, it overviews our current understanding of the identity of a wide variety of G protein regulators and their modulatory roles in GPCR-G protein-effector coupling, which is requisite for optimal β-cell function under physiological conditions. Second, it describes evidence in support of novel, noncanonical, GPCR-independent mechanisms of activation of G proteins in the islet. Third, it highlights the evidence indicating that abnormalities in G protein function lead to islet β-cell dysregulation and demise under the duress of metabolic stress and diabetes. Fourth, it summarizes observations of potential beneficial effects of GPCR agonists in preventing/halting metabolic defects in the islet β-cell under various pathological conditions (e.g., metabolic stress and inflammation). Lastly, it identifies knowledge gaps and potential avenues for future research in this evolving field of translational islet biology. Published 2020. Compr Physiol 10:453-490, 2020.
Introduction: The HGF/MET axis is a key therapeutic pathway in cancer; it is aberrantly activated because of mutations, fusions, amplification or aberrant ligand production. Extensive efforts have been made to discover predictive factors of anti-MET therapeutic efficacy, but they have mostly unsuccessful. An understanding of the intrinsic and acquired mechanism of MET resistance will be fundamental for the development of new therapeutic interventions.
Areas covered: This article provides a systematic review of phase II randomized and phase III clinical trials investigating the use of MET inhibitors in the treatment of cancer. We discuss preliminary findings on efficacy and methodologic design flaws in these trials.
Expert opinion: MET inhibitors showed poor activity in unselected patients or patients selected by MET expression, p-MET or high HGF basal levels. The efficacy in advanced solid tumors is very modest and in phase III clinical trials, survival differences did not fulfill the stringent requirements of ESMO-Magnitude Clinical Benefit Score (MCBS). Prospective novel liquid biomarker-driven studies and novel trial designs such as Umbrella and Basket trials, are necessary to progress MET inhibitor development.
Protein SUMOylation modification conjugated with small ubiquitin‐like modifiers (SUMOs) is one kind of post‐translational modifications (PTMs), which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses and tumorigenesis. With the development of the peptide enrichment approaches and mass spectrometry (MS) technology, more than 6000 SUMOylated proteins and about 40000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co‐modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially the advent of the clustered regularly interspaced short palindromic repeats ༈CRISPR༉/ Cas9 technique will facilitate the development of MS for SUMOylation identification. This article is protected by copyright. All rights reserved
Numerous experimental studies demonstrate that the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases) Ras homolog family member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42) are important regulators in somatosensory neurons, where they elicit changes in the cellular cytoskeleton and are involved in diverse biological processes during development, differentiation, survival and regeneration. This review summarizes the status of research regarding the expression and the role of the Rho GTPases in peripheral sensory neurons and how these small proteins are involved in development and outgrowth of sensory neurons, as well as in neuronal regeneration after injury, inflammation and pain perception. In sensory neurons, Rho GTPases are activated by various extracellular signals through membrane receptors and elicit their action through a wide range of downstream effectors, such as Rho-associated protein kinase (ROCK), phosphoinositide 3-kinase (PI3K) or mixed-lineage kinase (MLK). While RhoA is implicated in the assembly of stress fibres and focal adhesions and inhibits neuronal outgrowth through growth cone collapse, Rac1 and Cdc42 promote neuronal development, differentiation and neuroregeneration. The functions of Rho GTPases are critically important in the peripheral somatosensory system; however, their signalling interconnections and partially antagonistic actions are not yet fully understood.
Rac1, a small G protein, regulates physiological insulin secretion from the pancreatic β-cell. Interestingly, Rac1 has also been implicated in the onset of metabolic dysfunction of the β-cell under the duress of hyperglycemia (HG). This study is aimed at the identification of interaction partners of Rac1 in β-cells under basal and HG conditions. Using co-immunoprecipitation and UPLC-ESI-MS/MS, we identified 324 Rac1 interaction partners in INS-1832/13 cells, which represent the largest Rac1 interactome to date. Furthermore, we identified 27 interaction partners that exhibited increased association with Rac1 in β-cells exposed to HG. Western blotting (INS-1832/13 cells, rat islets and human islets) and co-immunoprecipitation (INS-1832/13 cells) further validated the identity of these Rac1 interaction partners including regulators of GPCR-G protein-effector coupling in the islet. These data form the basis for future investigations on contributory roles of these Rac1-specific signaling pathways in islet β-cell function in health and diabetes.
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
Focal adhesion (FA) disassembly required for optimal cell migration is mediated by microtubules (MTs); targeting of FAs by MTs coincides with their disassembly. Regrowth of MTs, induced by removal of the MT destabilizer nocodazole, activates the Rho-like GTPase Rac, concomitant with FA disassembly. Here, we show that the Rac guanine nucleotide exchange factor (GEF) Sif and Tiam1-like exchange factor (STEF) is responsible for Rac activation during MT regrowth. Importantly, STEF is required for multiple targeting of FAs by MTs. As a result, FAs in STEF-knockdown cells have a reduced disassembly rate and are consequently enlarged. This leads to reduced speed of migration. Together, these findings suggest a new role for STEF in FA disassembly and cell migration through MT-mediated mechanisms.
MaxQuant is a quantitative proteomics software package designed for analyzing large mass spectrometric data sets. It is specifically aimed at high-resolution mass spectrometry (MS) data. Currently, Thermo LTQ-Orbitrap and LTQ-FT-ICR instruments are supported and Mascot is used as a search engine. This protocol explains step by step how to use MaxQuant on stable isotope labeling by amino acids in cell culture (SILAC) data obtained with double or triple labeling. Complex experimental designs, such as time series and drug-response data, are supported. A standard desktop computer is sufficient to fulfill the computational requirements. The workflow has been stress tested with more than 1,000 liquid chromatography/mass spectrometry runs in a single project. In a typical SILAC proteome experiment, hundreds of thousands of peptides and thousands of proteins are automatically and reliably quantified. Additional information for identified proteins, such as Gene Ontology, domain composition and pathway membership, is provided in the output tables ready for further bioinformatics analysis. The software is freely available at the MaxQuant home page.
Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
The stress-activated p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that appear
to play a key role in regulating inflammatory responses. Co-expression of constitutively active forms of Rac and Cdc42 leads
to activation of p38 while dominant negative Rac and Cdc42 inhibit the ability of interleukin-1 to increase p38 activity.
p21-activated kinase 1 (Pak1) is a potential mediator of Rac/Cdc42 signaling, and we observe that Pak1 stimulates p38 activity.
A dominant negative Pak1 suppresses both interleukin-1- and Rac/Cdc42-induced p38 activity. Rac and Cdc42 appear to regulate
a protein kinase cascade initiated at the level of Pak and leading to activation of p38 and JNK.
Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.
Acute promyelocytic leukemia arises following a reciprocal chromosome translocation t(15;17), which generates PML-retinoic
acid receptor α fusion proteins (PML-RARα). We have shown previously that wild type PML, but not PML-RARα, is covalently modified
by the sentrin family of ubiquitin-like proteins (Kamitani, T., Nguyen, H. P., Kito, K., Fukuda-Kamitani, T., and Yeh, E.
T. H. (1998) J. Biol. Chem. 273, 3117–3120). To understand the mechanisms underlying the differential sentrinization of PML versus PML-RARα, extensive mutational analysis was carried out to determine which Lys residues are sentrinized. We show that Lys65 in the RING finger domain, Lys160 in the B1 Box, and Lys490 in the nuclear localization signal contributes three major sentrinization sites. The PML mutant with Lys to Arg substitutions
in all three sites is expressed normally, but cannot be sentrinized. Furthermore, the triple substitution mutant is localized
predominantly to the nucleoplasm, in contrast to wild type PML, which is localized to the nuclear bodies. Thus, sentrinization
of PML, in the context of the RING finger and the B1 box, regulates nuclear body formation. Furthermore, we showed that sentrinization
of PML-RARα could be restored by overexpression of sentrin, but not by retinoic acid treatment. These studies provide novel
insight into the pathobiochemistry of acute promyelocytic leukemia and the sentrinization pathway.
SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on
a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian
tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates,125I-SUMO-1-αNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-35S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by
successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from
a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity
of SUMO-1 hydrolase was almost completely inhibited byN-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating
enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly
smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple
SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing
of the SUMO-1-precursor.
Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial-mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events.
The formation of cell-cell adherens junctions is a cadherin-mediated process associated with reorganization of the actin cytoskeleton. Because Rho family GTPases regulate actin dynamics, we investigated whether cadherin-mediated adhesion regulates the activity of RhoA, Rac1, and Cdc42. Confluent epithelial cells were found to have elevated Rac1 and Cdc42 activity but decreased RhoA activity when compared with low density cultures. Using a calcium switch method to manipulate junction assembly, we found that induction of cell-cell junctions increased Rac1 activity, and this was inhibited by E-cadherin function-blocking antibodies. Using the same calcium switch procedure, we found little effect on RhoA activity during the first hour of junction assembly. However, over several hours, RhoA activity significantly decreased. To determine whether these effects are mediated directly through cadherins or indirectly through engagement of other surface proteins downstream from junction assembly, we used a model system in which cadherin engagement is induced without cell-cell contact. For these experiments, Chinese hamster ovary cells expressing C-cadherin were plated on the extracellular domain of C-cadherin immobilized on tissue culture plates. Whereas direct cadherin engagement did not stimulate Cdc42 activity, it strongly inhibited RhoA activity but increased Rac1 activity. Deletion of the C-cadherin cytoplasmic domain abolished these effects.
PIAS (protein inhibitor of activated STAT) proteins interact with and modulate the activities of various transcription factors.
In this work, we demonstrate that PIAS proteins xα, xβ, 1, and 3 interact with the small ubiquitin-related modifier SUMO-1
and its E2 conjugase, Ubc9, and that PIAS proteins themselves are covalently modified by SUMO-1 (sumoylated). PIAS proteins
also tether other sumoylated proteins in a noncovalent fashion. Furthermore, recombinant PIASxα enhances Ubc9-mediated sumoylation
of the androgen receptor and c-Jun in vitro. Importantly, PIAS proteins differ in their abilities to promote sumoylation in
intact cells. The ability to stimulate protein sumoylation and the interaction with sumoylated proteins are dependent on the
conserved PIAS RING finger-like domain. These functions are linked to the activity of PIASxα on androgen receptor-dependent
transcription. Collectively, our results imply that PIAS proteins function as SUMO-1-tethering proteins and zinc finger-dependent
E3 SUMO protein ligases, and these properties are likely to explain their ability to modulate the activities of various transcription
factors.
Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility,
and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and
effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere
with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of
Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol
4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal
Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate
Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show
that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates
intracellular targeting and differential signaling in a unique and non-redundant fashion.
We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small
GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA
in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic
localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box
by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA).
These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasome-mediated degradative pathways involving
the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated
degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling
or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal
sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for
degradation.
Phosphorylation, although necessary, may not be sufficient to fully activate many receptor tyrosine kinases (RTKs). Oligomerization-induced conformational changes may be necessary to modulate the kinetic properties of RTKs and render them fully functional. To investigate this regulatory mechanism, recombinant TPR-MET, a functionally active oncoprotein derivative of the RTK c-MET, has been expressed and purified for quantitative enzymatic analysis. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil motif from the nuclear pore complex (TPR). cytoMET, the monomeric analog of TPR-MET, has also been expressed and purified for comparative enzymatic analysis. ATP and peptide substrates have been kinetically characterized for both TPR-MET and cytoMET. Significantly, phosphorylated TPR-MET has smaller Km values for ATP (Km,ATP) and peptide substrates (Km,peptide) and a larger kcat relative to phosphorylated cytoMET. This provides the first direct evidence that receptor oligomerization and not simply activation loop phosphorylation modulates RTK enzymatic activity. The ATP dissociation constants (Kd,ATP) for the two enzymes also displayed significant differences. In contrast, the KI values for the ATP competitive inhibitor staurosporin are similar for the two phosphorylated enzymes. These results suggest that much of the oligomerization-induced kinetic changes occur with respect to peptide substrate binding or catalytic efficiency. The possibility that oligomerization-induced conformational changes occur within the cytoplasmic domain of receptor tyrosine kinases has significant implications for structure-based design of RTK inhibitors and the development of a detailed mechanistic model of RTK activation.
Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator beta-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with beta-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-beta-Pix interaction is required for Rac1 activation by beta-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the beta-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor beta-Pix.
Previous studies of Rac1 in fibroblasts have used dominant negative constructs, which may have nonspecific effects. We used a conditional Rac1 allele to critically examine Rac1 function in mouse fibroblasts. Lack of Rac1 had dramatic effects on nonconfluent cells, which were elongated and had extensive blebbing, but no lamellipodia or ruffle formation. However, Rac1-null fibroblasts translocated using pseudopodia-like protrusions without lamellipodia, migrating toward a platelet-derived growth factor (PDGF) gradient as efficiently as their wild-type counterparts. Rac1-null fibroblasts closed wounds in vitro and spread on a fibronectin substrate, although at a slower rate than wild-type cells. However, Rac1-null cells were markedly impaired in proliferation, with a defect in G1 to S transition, although they were capable of surviving in culture for more than 2 wk. These results refine our understanding of the functions of Rac1, indicate that lamellipodia formation is not required for cell motility, and show that PDGF-induced chemotaxis can occur in the absence of both lamellipodia and Rac1.
Post-translational modifiers of the SUMO (Small Ubiquitin-related Modifier) family have emerged as key regulators of protein function and fate. While the past few years have seen an enormous increase in knowledge on SUMO enzymes, substrates, and consequences of modification, regulation of SUMO conjugation is far from being understood. This brief review will provide an overview on recent advances concerning (i) the interplay between sumoylation and other post-translational modifications at the level of individual targets and (ii) global regulation of SUMO conjugation and deconjugation.
SUMOs (small ubiquitin-like modifiers) are ubiquitin-related proteins that become covalently conjugated to cellular target proteins that are involved in a variety of processes. Frequently, this modification has a key role in regulating the activities of those targets and, thus, their cellular functions. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of members of the Ubl (ubiquitin-like protein)-specific protease (Ulp) family. The same family of enzymes is also responsible for maturation of newly synthesized SUMOs prior to their initial conjugation. Recent advances in structural, biochemical and cell biological analysis of Ulp/SENPs reveal their high degree of specificity towards SUMO paralogs, in addition to discrimination between processing, deconjugation and chain-editing reactions. The dissimilar sub-nuclear localization patterns of Ulp/SENPs and phenotypes of Ulp/SENP mutants further indicate that different Ulp/SENPs have distinct and non-redundant roles.
Most protein complexes are inaccessible to high resolution structural analysis. We report the results of a combined approach of cross-linking, mass spectrometry, and bioinformatics to two human complexes containing large coiled-coil segments, the NDEL1 homodimer and the NDC80 heterotetramer. An important limitation of the cross-linking approach, so far, was the identification of cross-linked peptides from fragmentation spectra. Our novel approach overcomes the data analysis bottleneck of cross-linking and mass spectrometry. We constructed a purpose-built database to match spectra with cross-linked peptides, define a score that expresses the quality of our identification, and estimate false positive rates. We show that our analysis sheds light on critical structural parameters such as the directionality of the homodimeric coiled coil of NDEL1, the register of the heterodimeric coiled coils of the NDC80 complex, and the organization of a tetramerization region in the NDC80 complex. Our approach is especially useful to address complexes that are difficult in addressing by standard structural methods.
Transcriptional activity of signal-dependent transcription factors, including nuclear receptors, relies on interacting co-regulator proteins, many of which possess protein-modifying activity. SUMOs (small ubiquitin-related modifiers) and their conjugation pathway components act as co-regulator proteins for numerous transcription factors that also are often targets for SUMO modification. PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] proteins promote SUMOylation in a manner that resembles the action of RING-type ubiquitin E3 ligases. PIAS proteins were initially named for their ability to interact with STAT proteins and inhibit their activity, but their interactions and functions are not restricted to the STATs. Moreover, PIAS proteins do not operate merely as SUMO E3s, since their co-regulator effects are often independent of their RING finger but dependent on their SIM (SUMO-interacting motif) or SAP (scaffold attachment factor-A/B/acinus/PIAS) domain capable of interacting with DNA. The modulator activity imparted by the PIAS/SUMO system involves altered subnuclear targeting and/or assembly of transcription complexes. PIAS proteins may act as platforms that facilitate both removal and recruitment of other regulatory proteins in the transcription complexes.
Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle-blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division.
Many small GTPases in the Ras and Rho families have a C-terminal polybasic region (PBR) comprised of multiple lysines or arginines. The PBR controls diverse functions of these small GTPases, including their ability to associate with membranes, interact with specific proteins, and localize in subcellular compartments. Different signaling pathways mediated by Ras and Rho family members may converge when the small GTPases are directed by their PBRs to shared binding sites in specific proteins or at cell membranes. The PBR promotes the interactions of small GTPases with SmgGDS, which is a nucleocytoplasmic shuttling protein that stimulates guanine nucleotide exchange by small GTPases. The PBR of Rac1 was recently found to have a functional nuclear localization signal (NLS) sequence, which enhances the nuclear accumulation of protein complexes containing SmgGDS and Rac1. Sequence analysis demonstrates that canonical NLS sequences (K-K/R-x-K/R) are present in the PBRs of additional Ras and Rho family members, and are evolutionarily conserved across several phyla. These findings suggest that the PBR regulates the nucleocytoplasmic shuttling of some Ras and Rho family members when they are in protein complexes that are too large to diffuse through nuclear pores. These diverse functions of the PBR indicate its critical role in signaling by Ras and Rho family GTPases.
The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiple cellular processes including proliferation, apoptosis and adherens junction maintenance. Here we describe a modified tandem affinity purification (TAP) technique that we have applied to specifically enrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunction with LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seen with the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors. We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitation experiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interaction is largely dependent on the N-terminal region of Tiam1; within this region, there are four putative phospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231) are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at three of these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins to this region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity; significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is required for the degradation of Tiam1. Thus, we have established and proven methodology that allows the identification of additional protein-protein interactions in mammalian cells, resulting in the discovery of a novel mechanism of regulating Tiam1 stability.
The small ubiquitin-like modifiers (SUMOs) are posttranslationally conjugated to eukaryotic cellular proteins with generally unpredictable consequences. SUMO substrates are found in many cellular systems, and functional analysis has revealed that substrate SUMOylation often has an important role in their regulation. Here we describe a cell-based protocol which can be used to detect the SUMOylation of a protein that relies on the enrichment of SUMO conjugates by purification of 6His-SUMO under denaturing conditions, followed by western blotting for the protein of interest. By purifying under denaturing conditions this method not only reduces the risk of false-positive identifications by non-covalent interactions, but also preserves SUMO-substrate conjugates by inhibiting SUMO-specific proteases--two caveats that may complicate other less stringent purification methods. In preliminary form, this protocol takes 4-5 d to perform, and it can be further elaborated to provide a multi-angled approach to investigate protein conjugation by SUMO.
The Rac activator Tiam1 is required for adherens junction (AJ) maintenance, and its depletion results in AJ disassembly. Conversely, the oncoprotein Src potently induces AJ disassembly and epithelial-mesenchymal transition (EMT). Here, we show that Tiam1 is phosphorylated on Y384 by Src. This occurs predominantly at AJs, is required for Src-induced AJ disassembly and cell migration, and creates a docking site on Tiam1 for Grb2. We find that Tiam1 is associated with ERK. Following recruitment of the Grb2-Sos1 complex, ERK becomes activated and triggers the localized degradation of Tiam1 at AJs, likely involving calpain proteases. Furthermore, we demonstrate that, in human tumors, Y384 phosphorylation positively correlates with Src activity, and total Tiam1 levels are inversely correlated. Thus, our data implicate Tiam1 phosphorylation and consequent degradation in Src-mediated EMT and resultant cell motility and establish a paradigm for regulating local concentrations of Rho-GEFs.
Rho GTPases are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking and cytokinesis. These proteins are conserved from plants and yeast to mammals, and function by interacting with and stimulating various downstream targets, including actin nucleators, protein kinases and phospholipases. The roles of Rho GTPases have been extensively studied in different mammalian cell types using mainly dominant negative and constitutively active mutants. The recent availability of knockout mice for several members of the Rho family reveals new information about their roles in signalling to the cytoskeleton and in development.
Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.
Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.
Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2
share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates
that they both contain the sequence ψKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric
chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detectedin vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery,
modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.
The activity of the p53 tumor suppressor protein and the c-Jun protooncogene is regulated by posttranslational modifications, such as phosphorylation or ubiquitination. In addition, covalent attachment of the ubiquitin-like modifier SUMO appears to modulate their transcriptional activity. Sumoylation proceeds via an enzymatic pathway that is mechanistically analogous to ubiquitination, but requires a different E1-activating enzyme and Ubc9, a SUMO-specific E2-conjugating enzyme. Here, we show that two members of the PIAS family, PIAS1 and PIASxbeta, act as specific E3-like ligases that promote sumoylation of p53 and c-Jun in vitro and in vivo. The PIAS proteins physically interact with both p53 and c-Jun. In addition, they bind to Ubc9, suggesting that they recruit the E2 enzyme to their respective substrate. The SUMO ligase activity requires the conserved zinc-finger domain, which is distantly related to the essential RING-finger motif, found in a subset of ubiquitin ligases. Furthermore, similar to RING-type ubiquitin ligases, PIASxbeta can catalyze its own modification. Hence, these data further extend the analogy between the ubiquitin and SUMO pathway. Strikingly, PIAS proteins strongly repress the transcriptional activity of p53, suggesting that the PIAS-SUMO pathway plays a crucial role in the regulation of p53 and presumably other transcription factors.
The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.
Despite the complete determination of the genome sequence of several higher eukaryotes, their proteomes remain relatively poorly defined. Information about proteins identified by different experimental and computational methods is stored in different databases, meaning that no single resource offers full coverage of known and predicted proteins. IPI (the International Protein Index) has been developed to address these issues and offers complete nonredundant data sets representing the human, mouse and rat proteomes, built from the Swiss-Prot, TrEMBL, Ensembl and RefSeq databases.
The small ubiquitin-like modifier (SUMO) is covalently linked to a variety of proteins and is deconjugated by SUMO-specific proteases. A characteristic of SUMO modification is that the biological consequences of conjugation do not appear proportionate to the small fraction of substrate that is modified. SUMO conjugation appears to alter the long-term fate of the modified protein even though the SUMO may be rapidly deconjugated. Thus an unmodified protein with a history of SUMO modification may have different properties from a protein that never has been modified. Here, the diverse effects of SUMO modification are discussed and models proposed to explain SUMO actions.
Duplin was originally isolated as a negative regulator of β-catenin–dependent T-cell factor (Tcf) transcriptional activity
in the Wnt signaling pathway. However, Duplin knockout mice exhibit embryonic lethality at 5.5-em day, suggesting that Duplin
has important roles other than as a negative regulator of the Wnt signal. To identify new roles of Duplin, the Duplin-binding
proteins were screened. PIAS3, which is a SUMO E3 ligase and acts as an inhibitor of signal transducer and activator of transcription
(STAT3), was identified as a Duplin-binding protein. Duplin was sumoylated, but PIAS3 affected neither the sumoylation of
Duplin nor its ability to inhibit Tcf-4 activity. Like PIAS3, Duplin suppressed the leukemia-inhibitory factor (LIF)–induced
STAT3 transcriptional activity. Duplin did not affect the LIF-dependent tyrosine phosphorylation or nuclear localization of
STAT3 but inhibited the formation of complex between STAT3 and DNA. Although STAT3 is not modified with SUMO, PIAS3 inhibited
the STAT3 activity in a manner partially depending on its SUMO E3 ligase activity. Duplin suppressed the LIF-dependent STAT3
activity independently of sumoylation. These results demonstrate that Duplin inhibits not only Tcf-4 but also STAT3, suggesting
that Duplin may act as a repressor for multiple transcriptional factors.
Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.
Rho/Rac proteins constitute a subgroup of the Ras superfamily of GTP hydrolases. Although originally implicated in the control of cytoskeletal events, it is currently known that these GTPases coordinate diverse cellular functions, including cell polarity, vesicular trafficking, the cell cycle and transcriptomal dynamics. In this review, we will provide an overview on the recent advances in this field regarding the mechanism of regulation and signaling, and the roles in vivo of this important GTPase family.
Rho GTPases are small proteins that act as binary molecular switches in a wide range of signalling pathways upon stimulation of cell surface receptors. Three different classes of regulatory proteins control their activity. In the activated state small GTPases are able to bind a variety of effector proteins and initiate downstream signalling. Rho GTPases regulate important cellular processes ranging from cytoskeletal remodelling and gene expression to cell proliferation and membrane trafficking. Therefore it is not surprising that deregulated Rho signalling can contribute to disturbed cellular phenotypes in a wide range of diseases. The main focus of this review will be the diversity of functions of Rho GTPases and the effects of aberrant Rho GTPase signalling in various aspects of cancer.
Met is a tyrosine kinase receptor, encoded by an oncogene, whose crucial role has been elucidated during the last two decades. The complex biological program triggered by Met has been dissected and its biological relevance in both physiology and pathology has been proven. Met supports a morphogenetic program, known as invasive growth, taking place both during embryogenesis and adulthood. In tumors Met is often aberrantly activated, giving rise to the pathological counterpart of the invasive growth program: cancer progression towards metastasis. Several approaches have been recently developed to interfere with the tumorigenic and metastatic processes triggered by Met.
SUMO-1–Rac1 was localized simi-larly to wild-type GFP–Rac1 (Supplementary Information, Fig. S4i, j)
- Furthermore
- Gfp
Furthermore, GFP–SUMO-1–Rac1 was localized simi-larly to wild-type GFP–Rac1 (Supplementary Information, Fig. S4i, j).
Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity
- D Schmidt
- S Muller
Schmidt, D. & Muller, S. Members of the PIAS family act as SUMO ligases for c-Jun and
p53 and repress p53 activity. Proc. Natl Acad. Sci. USA 99, 2872-2877 (2002).
In-gel digestion for mass spectrometric characterization of proteins and proteomes
- A Shevchenko
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- M Mann
Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass
spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856-2860
(2006).