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Hydrophobic modifications of Ras proteins by isoprenoid groups and fatty acids--More than just membrane anchoring
Abstract
During the last years, post-translational modification of peripheral membrane proteins with hydrophobic side groups has been attributed to a couple of additional functions than just simple anchoring into lipid bilayers. In particular isoprenylation and N- and S-acylation did quicken interest in terms of specific recognition elements for protein-protein interactions and as hydrophobic switches that allow for temporal regulated association with distinct target structures. Furthermore new insights into the heterogeneity of natural membranes have connected the physical properties of e.g. farnesyl or palmitoyl side chains with a preference for such sub-compartments as lipid rafts or caveolae. In this review the impact of the two frequently realized modifications by isoprenylation and S-acylation on the process of cellular signal transduction is exemplified with proteins of the Ras and Rab family of small GTP-binding proteins.
... Once bound to the cell membrane, the GTPase superfamily members interact with the upstream stimulators, such as SRC family proteins, and the cytoplasmic molecules that tightly are regulating their activity [34]. The activation of small GTPases requires GDP-GTP exchange, which is catalyzed by various guanine nucleotide exchange factors (GEFs) [35,36]. In contrast, GTPase-activating proteins (GAPs) inactivate small GTPases by stimulating the hydrolysis of GTP [35,36]. ...
... The activation of small GTPases requires GDP-GTP exchange, which is catalyzed by various guanine nucleotide exchange factors (GEFs) [35,36]. In contrast, GTPase-activating proteins (GAPs) inactivate small GTPases by stimulating the hydrolysis of GTP [35,36]. Inactive GTPases are sequestered in the cytosol by the guanine nucleotide dissociation inhibitors (GDI) [35,36,37]. ...
... In contrast, GTPase-activating proteins (GAPs) inactivate small GTPases by stimulating the hydrolysis of GTP [35,36]. Inactive GTPases are sequestered in the cytosol by the guanine nucleotide dissociation inhibitors (GDI) [35,36,37]. Due to their ability to increase the activity of small GTPase superfamily, GEFs are potential oncogenes that affect tumorigenesis, e.g. ...
Significance:
Reduction/oxidation (redox) balance could be defined as an even distribution of reduction and oxidation complementary processes and their reaction end products. There is a consensus that aberrant levels of reactive oxygen species (ROS), commonly observed in cancer, stimulate primary cell immortalization and progression of carcinogenesis. However, the mechanism how different ROS regulate redox balance is not completely understood. Recent Advances: In the current review, we have summarized the main signaling cascades inducing NADPH oxidase NOX1-5 and superoxide dismutase (SOD) 1-3 expression and their connection to cell proliferation, immortalization, transformation, and CD34+cell differentiation in thyroid, colon, lung, breast, and hematological cancers.
Critical issues:
Interestingly, many of the signaling pathways activating redox enzymes or mediating the effect of ROS are common, such as pathways initiated from G protein-coupled receptors and tyrosine kinase receptors involving protein kinase A, phospholipase C, calcium, and small GTPase signaling molecules.
Future directions:
The clarification of interaction of signal transduction pathways could explain how cells regulate redox balance and may even provide means to inhibit the accumulation of harmful levels of ROS in human pathologies. Antioxid. Redox Signal. 00, 000-000.
... Once bound to the cell membrane, the GTPase superfamily members interact with the upstream stimulators, such as SRC family proteins, and the cytoplasmic molecules that tightly are regulating their activity [34]. The activation of small GTPases requires GDP-GTP exchange, which is catalyzed by various guanine nucleotide exchange factors (GEFs) [35,36]. In contrast, GTPase-activating proteins (GAPs) inactivate small GTPases by stimulating the hydrolysis of GTP [35,36]. ...
... The activation of small GTPases requires GDP-GTP exchange, which is catalyzed by various guanine nucleotide exchange factors (GEFs) [35,36]. In contrast, GTPase-activating proteins (GAPs) inactivate small GTPases by stimulating the hydrolysis of GTP [35,36]. Inactive GTPases are sequestered in the cytosol by the guanine nucleotide dissociation inhibitors (GDI) [35,36,37]. ...
... In contrast, GTPase-activating proteins (GAPs) inactivate small GTPases by stimulating the hydrolysis of GTP [35,36]. Inactive GTPases are sequestered in the cytosol by the guanine nucleotide dissociation inhibitors (GDI) [35,36,37]. Due to their ability to increase the activity of small GTPase superfamily, GEFs are potential oncogenes that affect tumorigenesis, e.g. ...
Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3-induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3-driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.
... (A) A gene encoding an ortholog of Rab32, a GTPase of the RAB family. RABs are characterized by the presence of two (rarely one) cysteine residues near the very C-terminus of the proteins, most often in the configurations CC*, CXC*, CCX*, or CCXX* (where * indicates the free C-terminus of the protein; Pechlivanis and Kuhlmann 2006). The two Cys residues are geranylgeranylated through the action of a RAB-specific geranylgeranyl transferase II (GGTase II) and this post-translational modification is essential for the proper function of RAB proteins (Pechlivanis and Kuhlmann 2006). ...
... RABs are characterized by the presence of two (rarely one) cysteine residues near the very C-terminus of the proteins, most often in the configurations CC*, CXC*, CCX*, or CCXX* (where * indicates the free C-terminus of the protein; Pechlivanis and Kuhlmann 2006). The two Cys residues are geranylgeranylated through the action of a RAB-specific geranylgeranyl transferase II (GGTase II) and this post-translational modification is essential for the proper function of RAB proteins (Pechlivanis and Kuhlmann 2006). The predicted protein sequence of Rab32 from Blastocystis sp. ...
... Mechanisms underlying Rab11 recruitment to membranes are far from elucidated. Rab11 is prenylated at the C-termini for insertion in membranes [118], and two Rab11-GDIs (alpha and beta) were identified as Rab11 binding partners [119]. The lipid modifying enzyme phosphatidylinositol ...
... Mechanisms underlying Rab11 recruitment to membranes are far from elucidated. Rab11 is prenylated at the C-termini for insertion in membranes [118], and two Rab11-GDIs (alpha and beta) were identified as Rab11 binding partners [119]. The lipid modifying enzyme phosphatidylinositol 4-phosphate kinase type III (PIP4KIII), which is located in the Golgi and catalyzes the synthesis of PI4P, was shown to recruit Rab11 and its effectors, however its role in the ERC homeostasis has not been explored [120,121]. ...
Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral–host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.
... The mobilization of insulin granules and their fusion to the plasma membrane is markedly dependent on a family of proteins that require isoprenylation, the small GTPases (e.g., Rap1, Rac1, and Cdc42). These are involved in vesicle docking, cytoskeletal remodeling, and granule priming and fusion (Li et al. 2004, Fukuda 2005, Pechlivanis & Kuhlmann 2006, Kowluru 2008, Wang & Thurmond 2009, Goalstone et al. 2010). ...
... Geranylgeranylation occurs via the incorporation of GGPP into proteins by a thioester bond to the cysteine of a C-terminal consensus region by geranylgeranyl transferase type 1, which becomes active at high glucose concentrations (Pechlivanis & Kuhlmann 2006, Goalstone et al. 2010. In this work, the acute inhibitory effect of SIM on insulin secretion was restricted to the high-glucose-stimulus group (Fig. 1A). ...
Results from previous investigations have indicated that glucose-stimulated insulin secretion (GSIS) is affected by changes in cholesterol and its intermediates, but the precise link between secretion and cholesterol has not been thoroughly investigated. In this study, we show the contribution of both protein isoprenylation and cholesterol-dependent plasma membrane structural integrity to insulin secretion in INS-1E cells and mouse islets. Acute (2 h) inhibition of hydroxyl-methylglutaryl-CoA reductase by simvastatin (SIM) resulted in inhibition of GSIS without reduction in total cellular cholesterol content. This effect was prevented by cell loading with the isoprenyl molecule geranylgeranyl pyrophosphate. Chronic (24 h) inhibition of cholesterol biosynthesis resulted in inhibition of GSIS with a significant reduction in total cellular cholesterol content, which was also observed after the inhibition of cholesterol biosynthesis downstream of isoprenoid formation. Electron paramagnetic resonance analyses of INS-1E cells showed that the SIM-induced reduction in cholesterol increased plasma membrane fluidity. Thus, the blockade of cholesterol biosynthesis resulted in the reduction of availability of isoprenoids, followed by a reduction in the total cholesterol content associated with an increase in plasma membrane fluidity. Herein, we show the different contributions of cholesterol biosynthesis to GSIS, and propose that isoprenoid molecules and cholesterol-dependent signaling are dual regulators of proper β-cell function.
© 2015 Society for Endocrinology.
... Lipid modifica-tions, such as isoprenylation and N-and S-acylation also play important roles as specific recognition elements for protein-protein interactions, as well as hydrophobic switches that permit the temporal regulation of docking to sub-compartments like lipid rafts, caveolae [232] and other cellular locations [233]. Palmitoylation augments the affinity of Ras proteins for membranes and activates the mitogen-activated protein kinase (MAPK) pathway [234]. In contrast to farnesylation, palmitoylation of Ras and Ras-like proteins is reversible, reflecting its connection to regulatory phenomena [228,234]. ...
... Palmitoylation augments the affinity of Ras proteins for membranes and activates the mitogen-activated protein kinase (MAPK) pathway [234]. In contrast to farnesylation, palmitoylation of Ras and Ras-like proteins is reversible, reflecting its connection to regulatory phenomena [228,234]. Ras activation is the first step in the MAPK pathways, an important and conserved signal transduction mechanisms in eukaryotes [235,236], exemplified by the number of different and interconnected MAPK signal transduction pathways that coexist within cells [235,236]. In this context, Ras activates Raf (MAP kinase kinase kinase, MAPKKK) through interactions that occur at the plasma membrane. ...
Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.
... The HVR has been largely ignored, with much focus instead being centered on the G domain. Considered just a membrane anchor (43), some in vitro studies will replace the HVR with a His tag to Ras proteins of interest and use a lipid derivatized with a Nickel-NTA moiety to anchor the protein to a membrane (44). Even in cases where the HVR clearly determines specificity, inference has been made using the truncated Ras protein. ...
The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1bulletGDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs). Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs.[F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. We hypothesized that the myristate moiety associates with the N-terminal extension to alter its structure, thereby affecting its function. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.
... The nature of the fatty acids is hydrophobic in nature and they prefer to interact with the hydrophobic surface. As the surface of the metallic silver atoms is relatively hydrophobic so the fatty acids can be absorbed onto the surface via van der wall forces and hydrophobic interactions [46]. As compared to the chemical bonding, these interactions are bit weaker [47] but can provide good capping and stabilization to the NPs. ...
Green synthesis of NPs evades many of the deleterious aspects by permitting the synthesis of nanoparticles at relatively low temperatures, pH and pressure, all at a significantly lower cost and short time. A bottom-up approach is used for the synthesis of silver nanoparticles formed due to the reduction of silver ions because of the existence of the phytochemicals contained in the Dracaena Trifasciata plant extract, which is primarily used for the study of green synthesis. X-ray diffraction, scanning electron microscope, Uv-Vis spectroscopy, and Fast Fourier Infrared spectroscopy characteristic techniques are used to analyze the nature of the silver nanoparticles formed by the green synthesis method. To prevent the over-growth of silver nanoparticles and avoid their aggregation, one fatty acids based and one amides based capping agent has been used for the comparison purpose for the preservation of the silver nanoparticles in term of shape, size and coagulation, which is a novel study. Amides interacted with the silver nanoparticles via electrostatic interactions which result in strong stabilization compared to the fatty acids which make van der wall interactions with the surface of the nanoparticles but for bio compatibility and environmentally friendly applications, fatty acid based capping agent can be preferred as these NPs are also suitable for the use in the field of medicine and pharmaceuticals.
... While many members of the Rab-and Rho-subfamilies of small GTPases associate with membranes via the post-translational attachment of a C-terminal prenyl-anchor(s), specific sub-cellular localization has been shown to be dependent on C-terminal polybasic regions (PBRs), protein myristoylation/palmitoylation, GTPase-effector interactions, and biophysical properties of the membrane, e.g. headgroup charge, fatty acid unsaturation, lipid packing etc. (Abe et al., 2003;Kulakowski et al., 2018;Michaelson et al., 2001;Ohya et al., 1993;Pechlivanis and Kuhlmann, 2006;Wu and Brennwald, 2010). ...
The role of sphingolipids in controlling the endolysosomal membrane trafficking remains unclear. Here, we show that in Saccharomyces cerevisiae sphingolipids containing very long-chain fatty-acids (VLCAs) promote homotypic vacuolar fusion. Yeast that lack the C26 VLCA elongase Elo3p display morphological and vacuolar abnormalities. Vacuoles isolated from these cells displayed reduced levels of in vitro fusion, which we traced to a block in tethering and docking. We found that C26 VLCFA deficient yeast mislocalize fusion markers, and the small GTPases Rho1p and Ypt7p fail to selectively concentrate at the boundary and vertex domains of vacuoles isolated from these yeasts. Surprisingly, we only observed mild changes to the localization of other regulatory lipids, but membrane fluidity and solubility was significantly altered. Taken together, these results suggest that sphingolipids containing C26 VLCFAs act as regulatory lipids in the homotypic vacuolar fusion cascade by assembling membrane microdomains that promote the protein and lipid machinery required for the tethering and docking of vacuoles.
Summary
Many sphingolipids contain very-long chain fatty-acids with 26 carbons. The deletion of Elo3, the elongase that adds the final two carbons results in pleiotropic effects that negatively alter membrane fusion at the tethering and docking stages.
... GUVs were formed using a lipid mixture consisting of brain sphingomyelin (BSM), cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (3:1:3 molar ratio) (25) . No binding of unprenylated RAB proteins was observed on these membranes (Fig. S1) which is in good agreement with the commonly accepted view that RAB proteins are incorporated into biological membranes through their C-terminal geranylgeranyl groups (26) . Most RAB GTPases are diprenylated in the cell with the addition of two geranylgeranyl moieties on the two C-terminal cysteines (27) . ...
RAB GTPases are major regulators of vesicular trafficking and localize to specific compartments. Deciphering the molecular mechanisms governing RAB localization is thus critical to understand intracellular transport processes. We have managed, for the first time, to incorporate purified and prenylated RABs into artificial membranes. By doing so, we observed that RAB6, but not RAB1 or RAB5, is able to promote by itself vesicle tethering. We believe that RAB6 is able to interact in trans with itself and to consequently drive homotypic membrane tethering. In the main part of this study, we investigated the physicochemical membrane requirements necessary for RAB recruitment. RAB1, RAB5 and RAB6 were all found to only localize to disordered membrane domains and to preferentially bind to curved membranes. We demonstrated that this specific recruitment of RAB1, RAB5 and RAB6 is primarily dependent on the hydrophobic insertion of their prenyl group into lipid packing defects. In contrast, RAB35 recruitment was primarily dependent on the presence of negatively charged lipids and was found to be modulated, to a lesser extent, by lipid packing defects. Although RAB4 and RAB11 were effectively recruited to purified Golgi fractions, in an effector-independent manner, membrane charges and lipid packing defects were not sufficient to promote their recruitment to synthetic vesicles; suggesting that RAB4 and RAB11 require more demanding membrane physicochemical properties. Our work demonstrates that the properties of membranes are critical for the regulation of RAB specific membrane targeting.
... 34 No binding of unprenylated RAB proteins was observed on these membranes ( Figure S1) which is in good agreement with the commonly accepted view that RAB proteins are incorporated into biological membranes through their C-terminal geranylgeranyl groups. 35 Most RAB GTPases are diprenylated in the cell with the addition of two geranylgeranyl moieties on the 2 C-terminal cysteines. 36 The use of diprenylated proteins and GGTase I. LidA, a RAB6 supereffector from Legionella pneumpophila, outcompetes the REP, while GGTase I shields the prenyl group from the solvent (detailed in Figure S3A). ...
Specific intracellular localization of RAB GTPases has been reported to be dependent on protein factors but the contribution of the membrane physicochemical properties to this process has been poorly described. Here, we show that three RAB proteins (RAB1/RAB5/RAB6) preferentially bind in vitro to disordered and curved membranes, and that this feature is uniquely dependent on their prenyl group. Our results imply that the addition of a prenyl group confers to RAB proteins, and most probably also to other prenylated proteins, the ability to sense lipid packing defects induced by unsaturated conical‐shaped lipids and curvature. Consistently, RAB recruitment increases with the amount of lipid packing defects, further indicating that these defects drive RAB membrane targeting. Membrane binding of RAB35 is also modulated by lipid packing defects but primarily dependent on negatively charged lipids. Our results suggest that a balance between hydrophobic insertion of the prenyl group into lipid packing defects and electrostatic interactions of the RAB C‐terminal region with charged membranes tunes the specific intracellular localization of RAB proteins.
... Some negative regulatory mechanisms were found by which downstream targets of heterotrimeric G-protein signalling inhibit GAPs, what in turn promotes activation of small GTPases (Ishii et al., 2005). GDIs play an important role as they regulate small GTPase's affinity to the plasma membrane thereby being able to further modulate their activation (Kötting and Gerwert, 2015;Pechlivanis and Kuhlmann, 2006). The GTPase-GDI complex cannot be activated by GEFs, which means that a dissociation signal of some sort would appear to be required (Mackay and Hall, 1998). ...
Astrocytes modulate and support neuronal and synapse function via numerous mechanisms that often rely on diffusion of signalling molecules, ions or metabolites through extracellular space. As a consequence, the spatial arrangement and the distance between astrocyte processes and neuronal structures is of functional importance. Likewise, changes of astrocyte structure will affect the ability of astrocytes to interact with neurons. In contrast to neurons, where rapid morphology changes are critically involved in many aspects of physiological brain function, a role of astrocyte restructuring in brain physiology is only beginning to emerge. In neurons, small GTPases of the Rho family are powerful initiators and modulators of structural changes. Less is known about the functional significance of these signalling molecules in astrocytes. Here we review recent experimental evidence for the role of RhoA, Cdc42 and Rac1 in controlling dynamic astrocyte morphology as well as experimental tools and analytical approaches for studying astrocyte morphology changes.
... To elucidate which domain of H-Ras is important for binding with NgBR, we examined the intracellular localization of EGFP fused to 1) the C-terminal 20 residues of H-Ras (H-RasC20), 2) full-length H-Ras that has its CAAX motif (CVLS) replaced with the RhoA CAAX motif (CLVL) (H-Ras-RhoA-CAAX), and 3) full-length H-Ras that has its aa 170 -185 (klnppdesgpgcmsck) replaced with aa 170-185 of K-Ras constituting the polybasic region (mskdgkkkkkksktkc) (H-Ras-K-Ras-PBR) 25,43,66,71 . The diagram of these mutants is presented in Figure 3A. ...
The localization of prenylated Ras at the plasma membrane promotes activation of Ras by receptor tyrosine kinases and stimulates oncogenic signaling by mutant Ras. The Nogo-B receptor (NgBR) is a transmembrane receptor that contains a conserved hydrophobic pocket. Here, we demonstrate that the NgBR promotes the membrane accumulation of Ras by directly binding prenylated Ras at the plasma membrane. We show that NgBR knockdown diminishes the membrane localization of Ras in multiple cell types. NgBR overexpression in NIH-3T3 fibroblasts increases membrane-associated Ras, induces the transformed phenotype in vitro, and promotes the formation of fibrosarcoma in nude mice. NgBR knockdown in human breast cancer cells reduces Ras membrane localization, inhibits EGF-stimulated Ras signaling, and diminishes tumorigenesis of xenografts in nude mice. Our data demonstrate that NgBR is a unique receptor that promotes accumulation of prenylated Ras at the plasma membrane and promotes EGF pathways.
... Rac, Rho, Cdc42, Rab) or a farnesyl pyrophosphate residue (e.g. Ras), which is covalently attached to the target GTPase [323,324]. These enzymatic reactions are catalyzed by respective prenyltransferases, namely geranylgeranyltransferases (GGTase-I and GGT-ase-II) and farnesyltransferase. ...
Due to the fact that chronic inflammation as well as tumorigenesis in the gut is crucially impacted by the fate of intestinal epithelial cells, our article provides a comprehensive overview of the composition, function, regulation and homeostasis of the gut epithelium. In particular, we focus on those aspects which were found to be altered in the context of inflammatory bowel diseases or colorectal cancer and also discuss potential molecular targets for a disease-specific therapeutic intervention.
... [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] It has also become apparent that several factors influence Rac1 downstream signaling, via coupling active Rac1 to specific downstream effectors, thereby selectively activating certain Rac1-driven functions. 3,31,33,[37][38][39][40][41][42][43][44][45][46] Given the diverse modes involved in Rac1 regulation, studies focused on deciphering the underlying mechanisms implicated in governing Rac1 signaling promise to provide insight into novel therapeutic avenues for effective Rac1 targeting. In particular, uncovering additional Rac1 signaling regulatory cascades will help pinpoint key players in Rac1 signaling, thus expanding the repertoire of potential pharmacological targets that could be utilized to antagonize Rac1 signaling deregulation in human diseases. ...
Abnormal Rac1 signaling is linked to a number of debilitating human diseases, including, cancer, cardiovascular diseases and neurodegenerative disorders. As such, Rac1 represents an attractive therapeutic target, yet the search for effective Rac1 inhibitors is still underway. Given the adverse effects associated with Rac1 signaling perturbation, cells have evolved several mechanisms to ensure the tight regulation of Rac1 signaling. Thus, characterizing these mechanisms can provide invaluable information regarding major cellular events that lead to aberrant Rac1 signaling. Importantly, this information can be utilized to further facilitate the development of effective pharmacological modulators that can restore normal Rac1 signaling. In this review, we focus on the pathological role of Rac1 signaling, highlighting the benefits and potential drawbacks of targeting Rac1 in a clinical setting. Additionally, we provide an overview of available compounds that target key Rac1 regulatory mechanisms and discuss future therapeutic avenues arising from our understanding of these mechanisms.
... These enzymes are composed of both heterologous α-and β-subunits, in which α is common for both enzymes (FTα) while β is distinctive (FTβ and GGT-Iβ) (Zhang and Casey, 1996). Small GTP-binding proteins (G-proteins) such as Ras or Rab are known as targets for farnesylation or geranylgeranylation, and this modification is essential not only for the anchoring of these proteins to the cell membrane but also for the proper signal transduction (Pechlivanis and Kuhlmann, 2006;Resh 2006). The C-terminal amino acid sequences of target proteins are composed of CaaX motifs, where C is cysteine, a is usually aliphatic acid, and X is a variety of amino acids. ...
Protein prenyl transferase, that is, farnesyltransferase (FT) and geranylgeranyltransferase (GGT), catalyze the farnesylation or geranylgeranylation of proteins including GTP-binding proteins (G proteins). The post-transla-tional modifcations by prenyl groups are important for proper signal transduction by G proteins. We report the cloning and mRNA expression analysis of genes encoding the α-and β-subunit of a putative FT (Bmftα and Bmftβ) and β-subunit of GGT type I (Bmggt-Iβ) in the silkworm, Bombyx mori. These proteins have important motifs for enzymatic activity, suggesting that they are functional as FT/GGT in the silkworm. A phylogenetic analysis revealed that each of these genes constituted the same clade with D. melanogaster homologue in the phylo-genetic tree. Developmental expression analysis by a quantitative reverse transcription-polymerase chain reaction clarifed that Bmftα, Bmftβ and Bmggt-Iβ were expressed strongly towards adult emergence. Furthermore, all of these genes were expressed strongly in the ovaries of female adults, and especially for Bmggt-Iβ a strong expression was also detected in the Malpighian tubules of the larval stage. Our results suggest that proper signal transduction with factors modifed by these enzymes is essential for the development or physiological regulation of these tissues in the silkworm.
... This is not surprising since generation of farnesol by FPPase is important beyond the CA. Farnesol and farnesal homoeostasis are vital for cells in all insect tissues, and farnesol plays important roles in the regulation of a wide variety of cell functions, including proliferation and apoptosis [83][84][85], while posttranslational modifications by attachment of a farnesyl group to C-terminal cysteines of target proteins by farnesyl-transferases are essential for signal transduction and vesicular transport [86]. The presence of AaFPPase, AaSDR, and AaALDH3 isozymes with several isoforms capable of catalyzing each of the 3 enzymatic reactions in mosquitoes might have facilitated the evolution of more efficient substrate specificities, as well as a better tissue and developmental regulation. ...
Our understanding of JH biosynthesis has significantly changed in the last years. In this review I would like to discuss the following topics: (1) the progresses in understanding the JH biosynthesis pathway. Access to genome sequences has facilitated the identification of all the genes encoding biosynthetic enzymes and the completion of comprehensive transcriptional studies, as well as the expression and characterization of recombinant enzymes. Now the existence of different flux directionalites, feed-back loops and pathway branching points in the JH biosynthesis pathways can be explored; (2) the new concepts in the modulation of JH synthesis by allatoregulators. The list of putative JH modulators is increasing. I will discuss their possible role during the different physiological states of the CA; (3) the new theoretical and physiological frameworks for JH synthesis analysis. I will discuss the bases of the flux model for JH biosynthesis. JH plays multiple roles in the control of ovary development in female mosquitoes; therefore, the CA presents different physiological states, where JH synthesis is altered by gating the flux at distinctive points in the pathway; (4) in the final section I will identify new challenges and future directions on JH synthesis research.
... Viele Ras-ähnliche Proteine agieren an der Plasmamembran, andere lokalisieren an spezifischen, intrazellulären Membrankompartimenten. Die Proteine der Ras Superfamilie sind an den Membranen durch ihre posttranlational modifizierten Lipidresten angeheftet (Seabra und James 1998, Pechlivanis und Kuhlmann 2006 (Hancock et al. 1990, Adamson et al. 1992, Williams 2003. Die Arf Proteine besitzen N-terminal einen Glycinrest statt eines CAAX Motives, der durch Myristinsäure modifiziert wird (D'Souza-Schorey und Stahl 1995). ...
... A wide variety of proteins, including RAS, pre-lamin A, fungal pheromones, and rhodopsin kinase, depend on posttranslational prenylation for proper localization and activity. Prenylated proteins are modified by either farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase), which covalently link a 15-or 20-carbon isoprenoid moiety to a C-terminal cysteine via a thiolether bond, with farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) acting as the lipid donors (Pechlivanis and Kuhlmann, 2006;Nguyen et al., 2010;Zverina et al., 2012). FTase and GGTase type 1 have been proposed to recognize protein substrates containing a C-terminal CAAX box, where C refers to the modified cysteine residue, A is any aliphatic amino acid, and X refers to a subset of amino acids that determine whether the cysteine receives a farnesyl or geranylgeranyl group. ...
The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here, we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores, and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.
© 2015 by The American Society for Cell Biology.
... Studies of farnesyl interaction with phospholipids generally assume its insertion into the lipid bilayer (72,73). Meister et al. (74) showed that the farnesyl moiety inserts into the lipid bilayer when sufficient external pressure is applied. ...
K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. Kras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics (MD) simulations we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, while the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion towards the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids, but the farnesyl's membrane orientation. Phosphorylation of Ser181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... This is not surprising since generation of farnesol by FPPase is important beyond the CA. Farnesol and farnesal homoeostasis are vital for cells in all insect tissues, and farnesol plays important roles in the regulation of a wide variety of cell functions, including proliferation and apoptosis [83][84][85], while posttranslational modifications by attachment of a farnesyl group to C-terminal cysteines of target proteins by farnesyl-transferases are essential for signal transduction and vesicular transport [86]. The presence of AaFPPase, AaSDR, and AaALDH3 isozymes with several isoforms capable of catalyzing each of the 3 enzymatic reactions in mosquitoes might have facilitated the evolution of more efficient substrate specificities, as well as a better tissue and developmental regulation. ...
The adult female mosquito Aedes aegypti is an excellent model to study JH biosynthesis. The genome is sequenced, molecular tools such as RNAi are available, and its JH-dependent reproductive physiology is well understood. All the genes encoding biosynthetic enzymes have been identified, comprehensive transcriptional studies have been completed, and several recombinant enzymes have been characterized. Several allatoregulatory factors are able to modify JH biosynthesis in vitro by corpora allata (CA) of adult female mosquitoes, including allatotropin (AT), allatostatin-C (AST-C), ecdysis triggering hormone (ETH) and insulin (INS). Receptors for all these allatoregulatores are expressed in the corpora allata-corpora cardiac complex (CA-CC). These different allatoregulatory factors might have modulatory effects during specific times throughout the development and gonotrophic cycle of female mosquitos. With the advent of new methods that facilitate the analysis of transcripts, enzymes and metabolites in the small CA, we were able to provide comprehensive descriptions of the mevalonic (MVA) and JH synthesis pathway by integrating information on changes in the basic components of those pathways. Our studies provide a previously unseen glimpse into the metabolomics of the MVA and juvenile hormone (JH) synthesis pathways in the CA of insects. Our results revealed remarkable dynamic changes in JH synthesis and exposed part of a complex mechanism that regulates CA activity. Principal component (PC) analysis validated that both pathways (MVAP and JH branch) are transcriptionally co-regulated as a single unit, and coordinate fashion. Metabolite studies showed that global fluctuation in the intermediate pool sizes in the MVAP and JH-branch were often inversely related. PC analyses of metabolites suggest that in female mosquitoes, there are at least 4 developmental switches that alter JH synthesis by modulating the flux at distinctive points in both pathways. The fine tuning of JH synthesis to different physiological stages in mosquito reproduction seems to occur through changes in the size of the precursor pools, rather than by changes in mRNA and enzyme capacity levels.
... Many Ras-related proteins act on the plasma membrane, but in addition or instead, some localize to specific intracellular membrane compartments. Ras superfamily proteins attach to membranes via their posttranslationally modified lipid moieties (reviewed in Seabra 1998;Pechlivanis et al. 2006;Wright et al. 2006). The primary translation product of most Ras and Rho GTPases terminates in a CAAX (C, cysteine; A, aliphatic acid; X, any amino acid) motif. ...
... subtype 7. Many members of this superfamily undergo functionally essential posttranslational prenylation of one or two cysteine residues close to the C-terminus (Pechlivanis and Kuhlmann 2006). However, we noticed that some of the predicted Blastocystis Ras superfamily GTPases lack the expected C-terminal Cyscontaining motifs ( fig. ...
Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However,
in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from
transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear
genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3′-ends of hundreds of genes. From sequence data
we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus,
the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3′-untranslated regions to the point that, in many genes, only one or two nucleotides of the
termination codon are left.
... Ras proteins are modified with a farnesyl moiety to target the protein to the cell membrane. However, additional interactions are needed to enhance membrane localization; H-Ras and N-Ras contain both farnesyl and palmitoyl modifications, while K-Ras membrane association is enhanced by the electrostatic interaction between a positively charged basic sequence and the negatively charged plasma membrane (46)(47)(48)(49). For dually lipid modified proteins, the first modification (e.g. ...
Protein palmitoylation is a widespread lipid modification in which one or more cysteine thiols on a substrate protein are modified to form a thioester with a palmitoyl group. This lipid modification is readily reversible; a feature of protein palmitoylation that allows for rapid regulation of the function of many cellular proteins. Mutations in palmitoyltransferases (PATs), the enzymes that catalyze the formation of this modification, are associated with a number of neurological diseases and cancer progression. This review summarizes the crucial role of palmitoylation in biological systems, the discovery of the DHHC protein family that catalyzes protein palmitoylation, and the development of methods for investigating the catalytic mechanism of PATs.
... Farnesol acts as a signaling molecule in cell proliferation and apoptosis [25,26,27]. Posttranslational modifications by attachment of a farnesyl group to C-terminal cysteine of target proteins by farnesyl-transferases are essential for signal transduction and vesicular transport [28]. Farnesal dehydrogenases play key roles in the generation of fatty alcohols and fatty acids as well as in the elimination of toxic biogenic and xenobiotic aldehydes, such as those produced by oxidative damage of glycerolipids or during protein deprenylation [29,30,31]. ...
The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti.
A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis.
AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.
... Lipidation involves modification of small GTPases with either geranylgeranyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP), and is required for targeting small GTPases to the cell membrane, although the functional consequences of GTPase lipidation are not fully understood [12]. In this process, small GTPase proteins are covalently attached to GGPP (for Rac, Rho and Cdc42) or FPP ( for Ras), primarily at C-terminal cysteine residues, to become lipid avid [13,14]. Since GGPP and FPP are are down-stream products of HMG-CoA reductase, lipidation is also blocked by statins [15]. ...
Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.
Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.
In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.
... MCD, which disrupts lipid rafts by scavenging cholesterol, potentiated the effect of ODPC on LAT2 abundance. These results emphasize the importance of the post-translational modification palmitoylation for stabilizing adaptor proteins in lipid rafts, as previously reported (58,59). Our data suggest that the main effect of ODPC on membranes appears to be interference with the interaction of lipid rafts and palmitoylated proteins, which leads to an inefficient scaffold for maintaining signal transduction proteins. ...
Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.
... Lipid modification of a protein involves the permanent or temporary covalent attachment of lipid-based groups at various positions within the polypeptide chain and serves a variety of roles, with the most obvious being to enhance the membrane affinity of the modified protein. However, numerous other roles have been assigned to such PTM, including modulation of protein-protein interactions, signal transduction, embryogenesis, pattern formation, protein trafficking through the secretory pathway, evasion of the immune response by infectious parasites, and enzyme activation (for review, see Sinensky, 2000;Mann & Beachy, 2004;Pechlivanis & Kuhlmann, 2006;Nadolski & Linder, 2007;Charollais & Van Der Goot, 2009;Greaves et al., 2009;Aicart-Ramos et al., 2011). In efforts aimed at understanding the modes, pathways, and functions of lipid modification in archaea, studies on H. volcanii have proven to be central. ...
As an ever-growing number of genome sequences appear, it is becoming increasingly clear that factors other than genome sequence impart complexity to the proteome. Of the various sources of proteomic variability, post-translational modifications most greatly serve to expand the variety of proteins found in the cell. Likewise, modulating the rates at which different proteins are degraded also results in a constantly changing cellular protein profile. While both strategies for generating proteomic diversity are adopted by organisms across evolution, the responsible pathways and enzymes in Archaea are often less well described than are their eukaryotic and bacterial counterparts. Studies on halophilic archaea, in particular Haloferax volcanii, originally isolated from the Dead Sea, are helping to fill the void. In this review, recent developments concerning post-translational modifications and protein degradation in the haloarchaea are discussed. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
... Interestingly, many important cellular regulators, such as protein kinases, receptors, G-proteins and small GTPases are lipidmodified , highlighting the importance of post-translational protein modifications in intra-and extra-cellular cell signalling events (Resh, 1999 ). The addition of myristoyl or prenyl groups is essential for membrane association, and therefore function, of several small GTPases including most members of the Ras protein family, which are prenylated and targeted to the plasma membrane (Magee & Marshall, 1999; Pechlivanis & Kuhlmann, 2006 ). In contrast to transmembrane proteins, however, the molecular mechanisms that regulate the traffic for lipid-modified proteins are relatively poorly understood. ...
The traffic of proteins to the outer segment of photoreceptors is a fundamentally important process, which when perturbed results in photoreceptor cell death. Recent reports have revealed a novel pathway for the traffic of lipid-modified proteins involving the small GTPase Arl3 and its effectors PDEδ and Unc119. The retinitis pigmentosa protein RP2 is a GTPase activating protein (GAP) for Arl3 and also appears to regulate the assembly and traffic of membrane associated protein complexes. We recently identified the Gβ subunit of transducin (Gβ1) as a novel RP2 interacting protein. Our data support a role for RP2 in facilitating membrane association and traffic of Gβ1, potentially prior to the formation of the obligate Gβ:Gγ heterodimer. Here, we review the recent evidence that suggests that RP2 co-operates with Arl3 and its effectors in protein complex assembly and membrane specification for lipid-modified proteins. This is exemplified by the co-ordination of cilia associated traffic for heterotrimeric G proteins and we propose a model for the role of Arl3 and RP2 in this process.
... Many G proteins function through transient associations with various organelles. For example , Ras reversibly binds to plasma membranes, Ran helps to shuttle cargos between the cytoplasm and nucleoplasm, and Rab proteins provide specificity in targeting vesicles to membrane organelles (Kuersten et al. 2001; Pfeffer 2001; Pechlivanis and Kuhlman 2006). Using differential centrifugation , we found that pea DRG2 occurs predominantly in P150 and S150 fractions (Devitt et al. 1999). ...
DRGs are highly conserved GTP binding proteins. All eukaryotes examined contain DRG1 and DRG2 orthologs. The first experimental evidence for GTP binding by a plant DRG1 protein and by DRG2 from any organism is presented. DRG1 antibodies recognized a single ;43-kDa band in plant tissues, whereas DRG2 antibodies recognized ;45-, 43-, and 30-kDa bands. An in vitro transcription and translation assay suggested that the 45-kDa band represents full-length DRG2 and that the smaller bands are specific proteolytic products. Homogenates from pea roots and root apices were used to produce fractions enriched in cytosolic and microsomal monosomes and polysomes. DRG1 and the 45-and 43-kDa DRG2 bands occurred in the cytosol and associated with cytosolic monosomes. In contrast, the 30-kDa form of DRG2 was strongly enriched in polysome fractions. Thus, DRG1 and the larger forms of DRG2 may be involved in translational initiation, and the 30-kDa form of DRG2 may be involved in translational elongation. DRG1 and the 45-and 43-kDa forms of DRG2 can reassociate with ribosomes in vitro, a process that is partially inhibited by GTP-g-S. Cells expressing FLAG-tagged ribosomal proteins from transgenic lines of Arabidopsis and yeast also demonstrated DRG-ribosome interactions.
... Prenylation is a covalent posttranslational modification that tightly controls the signaling activities of several cellular proteins. 98 Examples of prenylated proteins include the small GTPases (including Ras, Rac1, Rab) 99 and the phosphatase of regenerating liver (PRL) family of PTPs. 100 Protein prenylation involves the addition of a 15-carbon (farnesyl) or a 20-carbon (geranylgeranyl) isoprenoid moiety via a thioether linkage to one or more cysteines located at or near the C terminus of a protein. 101 This type of reaction can be catalyzed by three different protein prenyl transferases: protein farnesyltransferase (FTase), protein geranylgeranyl transferase-I (GGTase-I), and Rab geranylgeranyl transferase (RabGGTase or GGTase-II). ...
Tyrosine phosphorylation of proteins serves as an exquisite switch in controlling several key oncogenic signaling pathways involved in cell proliferation, apoptosis, migration, and invasion. Since protein tyrosine phosphatases (PTPs) counteract protein kinases by removing phosphate moieties on target proteins, one may intuitively think that PTPs would act as tumor suppressors. Indeed, one of the most described PTPs, namely, the phosphatase and tensin homolog (PTEN), is a tumor suppressor. However, a growing body of evidence suggests that PTPs can also function as potent oncoproteins. In this chapter, we provide a broad historical overview of the PTPs, their mechanism of action, and posttranslational modifications. Then, we focus on the dual properties of classical PTPs (receptor and nonreceptor) and dual-specificity phosphatases in cancer and summarize the current knowledge of the signaling pathways regulated by key PTPs in human cancer. In conclusion, we present our perspective on the potential of these PTPs to serve as therapeutic targets in cancer.
Vesicle trafficking is an unceasing and elaborate cellular process that functions in material transport and information delivery. Recent studies have identified the small GTPase, Ras-related protein in brain 11A (RAB11A), as a key regulator in this process. Aberrant RAB11A expression has been reported in several types of cancers, suggesting the important functions and characteristics of RAB11A in cancer. These discoveries are of great significance because therapeutic strategies based on the physiological and pathological status of RAB11A might make cancer treatment more effective, as the molecular mechanisms of cancer development have not been completely revealed. However, these studies on RAB11A have not been reviewed and discussed specifically. Therefore, we summarize and discuss the recent findings of RAB11A involvement in different biological processes, including endocytic recycling regulation, receptors and adhesion molecules recycling, exosome secretion, phagophore formation and cytokinesis, as well as regulatory mechanisms in several tumor types. Moreover, contradictory effects of RAB11A have also been observed in different types of cancers, implying the dual characteristics of RAB11A in cancer, which are either oncogenic or tumor-suppressive. This review on the functions and characteristics of RAB11A highlights the value of RAB11A in inducing multiple important phenotypes based on vesicle trafficking and therefore will offer insights for future studies to reveal the molecular mechanisms, clinical significance, and therapeutic targeting of RAB11A in different cancers.
Pollen tubes are tip-growing cells that create safe routes to convey sperm cells to the embryo sac for double fertilization. Recent studies have purified and biochemically characterized detergent-insoluble membranes from tobacco pollen tubes. These microdomains, called lipid rafts, are rich in sterols and sphingolipids and are involved in cell polarization in organisms evolutionarily distant, such as fungi and mammals. The presence of actin in tobacco pollen tube detergent-insoluble membranes and the preferential distribution of these domains on the apical plasma membrane encouraged us to formulate the intriguing hypothesis that sterols and sphingolipids could be a “trait d’union” between actin dynamics and polarized secretion at the tip. To unravel the role of sterols and sphingolipids in tobacco pollen tube growth, we used squalestatin and myriocin, inhibitors of sterol and sphingolipid biosynthesis, respectively, to determine whether lipid modifications affect actin fringe morphology and dynamics, leading to changes in clear zone organization and cell wall deposition, thus suggesting a role played by these lipids in successful fertilization.
Powdery mildew (Pm), caused by Blumeria graminis f.sp. tritici (Bgt), is one of the most important wheat diseases. Plants employ diverse mechanisms, such as maintaining metal homeostasis, to promote adaptation to abiotic or biotic stresses. One of such mechanisms include the use of metallochaperone subfamily protein, heavy‐metal‐associated isoprenylated plant protein (HIPP1). As such, in present study, we proved that the HIPP1 from Haynaldia villosa plays a positive role in Pm resistance. Bgt induced rapidly upregulation of HIPP1‐V. Transiently or stably heterologous overexpression of HIPP1‐V suppressed haustorium formation, and enhanced Pm resistance. HIPP1‐V isoprenylation is critical for plasma membrane (PM) localization, physical interaction with the E3‐ligase CMPG1‐V and functioned in Pm resistance. Bgt infection recruited isoprenylated HIPP1‐V and CMPG1s on the PM. Blocking HIPP1‐V isoprenylation or tipifarnib treatment reduced such recruitment. Transiently or stably overexpression of HIPP1‐VC148G did not enhance Pm resistance. Tipifarnib treatment compromised the Pm resistance of OE‐CMPG1‐V and OE‐HIPP1‐V. These indicated the dependence of Pm resistance on HIPP1‐V isoprenylation. In OE‐HIPP1‐V, the differentially expressed genes of the reactive oxygen species and salicylic acid pathways were remarkable enriched, revealing their involvement in HIPP1‐mediated Pm resistance. Our results demonstrated an important defense pathway mediated by protein isoprenylation. This article is protected by copyright. All rights reserved.
The small GTPase CDC42 plays essential roles in neurogenesis and brain development. Previously, we showed that a CDC42 splice variant that has a ubiquitous tissue distribution specifically stimulates the formation of neural progenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neural progenitor cells to neurons. These specific roles of CDC42 and CDC42b in neurogenesis are ascribed to their opposing effects on mTORC1 activity. Specifically, the ubiquitous form of CDC42 stimulates mTORC1 activity and thereby up-regulates tissue-specific transcription factors that are essential for neuroprogenitor formation, whereas CDC42b works together with activated CDC42-associated kinase (ACK) to down-regulate mTOR expression. Here we demonstrate that the EGF receptor (EGFR) is an additional and important target of CDC42b and ACK which is down-regulated by their combined actions in promoting neurogenesis. The activation status of the EGFR determines the timing by which neural progenitor cells derived from P19 embryonal carcinoma terminally differentiate into neurons. By promoting EGFR degradation, we found that CDC42b and ACK stimulate autophagy, which protects emerging neurons from apoptosis and helps trigger neural progenitor cells to differentiate into neurons. Moreover, our results reveal that CDC42b is localized in phosphatidylinositol (3,4,5)-triphosphate (PIP3)-enriched microdomains on the plasma membrane, mediated through its polybasic sequence ¹⁸⁵KRK¹⁸⁷, which is essential for determining its distinct functions. Overall, these findings now highlight a molecular mechanism by which CDC42b and ACK regulate neuronal differentiation and provide new insights into the functional interplay between EGFR degradation and autophagy which occurs during embryonic neurogenesis.
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme of the mevalonate pathway, which generates cholesterol and non-sterol compounds such as isoprenoid, which are involved in key steps of tumorigenesis such as cell growth and proliferation. Our aim was to evaluate the role of the mevalonate pathway in adrenocortical tumors (ACTs). Expression pattern of HMGCR, FDFT1, LDLR, SCARB1, StAR, TSPO, CYP11A1, CYP11B1, CYP17A1, CYP21A1, and HSD3B1 genes, involved in the mevalonate pathway and steroidogenesis, was quantified by real-time RT-PCR in 46 ACT [14 adenomas (ACA) and 11 carcinomas (ACC) from adults and 13 ACA and 8 ACC from pediatric patients]. Effects of the mevalonate pathway inhibition on NCI-H295A cell viability was assessed by colorimetric assay. HMGCR was overexpressed in most adult ACT. The expression of TSPO, STAR, CYP11B1, CYP21A1, and HSD3B1 in adult ACC was significantly lower than in ACA (p<0.05). Regarding pediatric ACT, the expression of genes involved in steroidogenesis was not different between ACA and ACC. Inhibition of isoprenoid production significantly decreased the viability of NCI-H295A cells (p<0.05). However, cholesterol synthesis blockage did not show the same effect on cell viability. Low expression of TSPO , StAR, CYP11B1, CYP21A1, and HSD3B1 characterized a signature of adult ACCs. Our data suggest that HMGCR overexpression in adult ACC might lead to intracellular isoprenoid accumulation and cell proliferation. Therefore, the mevalonate pathway is a potential target for ACC treatment.
The spatial organization of plasma membrane proteins is a key factor in the generation of distinct signal outputs, especially for PKC/Ras/ERK signalling. Regulation of activation of the membrane-bound Ras, critical for neuronal differentiation and highly specialized functions, is controlled by exchanges in nucleotides catalyzed by nucleotide exchange factors (GEFs) for GTP loading and Ras activation, and by Ras GTPase Activated Proteins (RasGAPs) that lead to activation of the intrinsic GTPase activity of Ras and thus its inactivation. PKCs are potent Ras activators yet the mechanistic details of these interactions, or the involvement of specific PKC isoforms are now beginning to be addressed. Even less known is the topology where RasGAPs terminate Ras activation. Towards this aim, we isolated lipid rafts from chick embryo neural tissue and primary neuronal cultures when PKCε is the prominent isoform and in combination with in vitro kinase assays, we now show that, in response the PKCε-specific activating peptide ψεRACK, an activated PKCε is recruited to lipid rafts; similar mobility was established when PKCε was physiologically activated with the Cannabinoid receptor 1 (CB1) agonist methanandamide. Activation of H-Ras for both agents was then established for the first time using in vivo RasGAP activity assays, which showed similar temporal profiles of activation and lateral mobility. Moreover, we found that the GEF SOS1, and the major neuronal RasGAP neurofibromin, a specific PKCε substrate, were both transiently significantly enriched in the rafts. Finally, our in silico analysis revealed a highly probable, conserved palmitoylation site adjacent to a CARC motif on neurofibromin, both of which are included only in the RasGAP related domain type I (GRDI) with the known high H-RasGAP activity. Taken together, these results suggest that PKCε activation regulates the spatial plasma membrane enrichments of both SOS1 and neurofibromin, thus controlling the output of activated H-Ras available for downstream signalling in neurons.
Trichogin is a natural peptide endowed with antimicrobial and antitumor activity. A member of the peptaibol family, trichogin possesses a C‐terminal amino alcohol. In the past, this moiety was substituted for a methyl ester for synthetic purposes and it was observed that this apparently slight modification caused significant changes in the peptide bioactivity. With the aim of understanding the reasons behind such observations, a detailed spectroscopic study on a number of trichogin analogues has been performed. Herein, data obtained from synchrotron radiation circular dichroism, NMR spectroscopy, and fluorescence spectroscopy in organic solvents at cryogenic temperatures are compared with those independently acquired by means of EPR spectroscopy at 80 K. It is unambiguously revealed that the presence of a reversible, temperature‐driven, screw‐sense interconversion from a right‐ to left‐handed helix is determined by the C‐terminal capping moiety. Data demonstrate, for the first time, the key role of a C‐terminal methyl ester in promoting peptide screw‐sense inversion.
The covalent attachment of lipids to proteins is a fundamental property of all living cells. These lipidated or lipid-modified proteins are directly targeted to the membranes and display diverse functional roles that are critical to cell function, such as membrane signaling and trafficking. All lipidated proteins have been classified by the type of chemical moieties that are attached to them, majorly, palmitoylation, myristoylation, prenylation, cholesterylation, and addition of the Glycosylphosphatidyl inositol (GPI) anchor. Although the distinct hydrophobic lipid moiety largely dictates the functional compartmentalization, it also facilitates membrane trafficking and triggers a wide range of signaling pathways in cellular growth. In this review, we will focus on mechanistic insights underlying their membrane attachment, with a view to understand the regions that contribute to protein-membrane interface specificity. We also present a computational case study to report how different membrane lipids modulate insertion of the lipidated LC3 protein. Finally, in this review, we demonstrate the potential of regulating lipid modifications that are essential for cell survival and we discuss the concepts regarding their role in disease and therapeutics.
The main aims of this study are (i) to see which stem cell markers are present within prostate cancers and the level of interactions between them and (ii) to determine whether or not Nanog can be used as a stem cell-like marker for pluripotent prostate cancer. Pluripotent stem cells are cells which can both undergo self-renewal and differentiate into cells that are different from the original cell line. Nanog has been observed within many different somatic and germline tumours, but its actual role in cancer is not yet fully known. This study looks at the role of Nanog in prostate tumour cells, in order to elucidate its actual role within stem cells and its effects on pluripotentcy. A variety of artificial neuronal network (ANN) systems are used to determine the strength of the different interactions between Nanog and prostate tumour-negative and tumour-positive cells. The investigation looks at the various stem-cell markers present within prostate tumour cells and their level of accuracy in the diagnosis or prognostic effects of prostate tumour cells. It also aims to look at whether or not Nanog has negative or positive effects on the tumour, aiding in prognosis. The investigation highlights six genes- DLGAP2, TGFβR2, POU, Atatxin-3, PA2G4P2 and FLJ38379 - of which five are found to play some sort of role in prostate cancer. However, one of these five, Ataxin-3, is only present in a small number of cases, while one, FLJ38379, is found to be unclassified and has not been found in any diseases before. These findings do not fully achieve the desired aims of the study, and further research is required.
Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of Rhoa or the gene encoding GGTase-I, Pggt1b, in IECs exhibit spontaneous chronic intestinal inflammation with accumulation of granulocytes and CD4+ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding, ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBDs. As therapeutic triggering of Rho-A signaling suppressed intestinal inflammation in mice with GGTase-I-deficient IECs, our findings suggest new avenues for treatment of epithelial injury and mucosal inflammation in IBD patients.
The organization of cells into interconnected structures such as animal tissues requires a sophisticated system directing receptors and adhesion proteins to the cell surface. The Rab11 small G proteins (Rab11a, b, and Rab25) of the Ras superfamily are master regulators of the surface expression of receptors and adhesion proteins. Acting as a molecular switch, Rab11 builds distinct molecular machinery such as motor protein complexes and the exocyst to transport proteins to the cell surface. Recent evidence reveals Rab11 localization at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome, placing it at the intersection between the endocytic and exocytic trafficking pathways. We review Rab11 in various cellular contexts, and discuss its regulation and mechanisms by which Rab11 couples with effector proteins.
Norovirus is the major cause of acute gastroenteritis worldwide. Between November 2012 and June 2013, 1718 stool samples were requested for norovirus antigen testing in the metropolitan areas of South Korea, and 91 samples were genotyped. The norovirus antigen-positive rate peaked at 52.8% in December 2013. A novel norovirus GII.4 variant, GII.4 Sydney 2012, was the most frequently found genotype (60.4%) during this period. This study demonstrates that norovirus activity increased during the winter of 2012-2013 in South Korea and that norovirus GII.4 Sydney 2012 was the cause of the norovirus epidemic during this period.
A new cysteine anchoring method was developed for the synthesis of peptides containing C-terminal cysteine methyl esters. This method consists of attachment of Fmoc-Cys-OCH(3) to either 2-ClTrt-Cl or Trt-Cl resins (via the side-chain thiol) followed by preparation of the desired peptide using Fmoc-based SPPS. We applied this method to the synthesis of the mating pheromone a-factor and a 5-FAM labeled a-factor analog. The peptides were obtained with high yield and purity and were shown to be bioactive in a growth arrest assay.
Only modest progress has been made in improving the overall survival of pancreatic cancer patients during the past 20 years
with standard cytotoxic chemotherapy drugs. More work needs to be done to address the key biologic characteristics of pancreatic
cancer that make it so aggressive, metastasizing at an early stage, and more refractory to standard treatments than most other
solid tumor types. Here, we discuss the emerging role of targeted therapy in pancreatic cancer and the status of currently
available and tested agents. We also point out the potential pitfalls in current trial designs and recommend new methods for
testing novel compounds in this heterogeneous and difficult-to-treat group of patients.
This paper reviews the chemical and functional aspects of the posttranslational modifications of proteins, which are achieved by the addition of various groups to the side chain of the amino acid residue backbone of proteins. It describes the main prosthetic groups and the interaction of these groups and the apoenzyme in the process of catalysis, using pyridoxal catalysis as an example. Much attention is paid to the role of posttranslational modification of proteins in the regulation of biochemical processes in live organisms, and especially to the role of protein kinases and their respective phosphotases. Methylation and acetylation reactions and their role in the "histone code", which regulates genome expression on the transcription level, are also reviewed. This paper also describes the modification of proteins by large hydrophobic residues and their role in the function of membrane-associated proteins. Much attention is paid to the glycosylation of proteins, which leads to the formation of glycoproteins. We also describe the main non-enzymatic protein modifications such as glycation, homocysteination, and desamida-tion of amide residues in dibasic acids.
PRA1 has been reported as a prenylated Rab acceptor containing GDF activity in human, rat and yeast. Its existence was also proved in plants by sequence analysis, anticipating its important role as a Rab effector, but defined roles of the plant PRA1 homologs remain to be obscure. Here, to get an insight for their role, we performed yeast two-hybrid screen using the OsRab7 as bait and first isolated the OsPRA1, a putative prenylated Rab acceptor, interacting with this protein. Detailed interaction analysis showed that OsPRA1 interacted not only with GDP-bound OsRab7, but also with several other Rab GTPases involved in vacuolar trafficking, in a prenylation-dependent manner. In addition, GFP-fusion analysis demonstrated that OsPRA1 localized to the prevacuolar compartment, and RNA gel blot analysis revealed its significant expression in rice green-aerial tissues such as shoots and mature stems. These results suggest that OsPRA1 may function as a Rab effector for vacuolar trafficking in the plant system.
Simvastatin is a competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway required for the biosynthesis of cholesterol and higher isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Apart from its capacity to lower cholesterol plasma levels and to protect against cardiovascular disease, simvastatin induces apoptosis in various cancer cells. We have generated human Namalwa Burkitt lymphoma cells that display general apoptosis resistance and hyperproliferation due to increased expression and proteolytic activity of 26S proteasomes in response to continuous treatment of the cells with the proteasome inhibitor bortezomib. In these cells, simvastatin does not inhibit proteasome activity, but induces apoptosis, G2/M cell cycle arrest and accumulation of p21Waf1/Cip1, and effectively inhibits hyperproliferation. These effects are reversed by the addition of GGPP. GGPP-dependent plasma membrane localization of the small GTPase RhoA that is required for RhoA-mediated oncogenic signaling is completely inhibited by simvastatin. Finally, bortezomib but not simvastatin induces accumulation and stabilization of the anti-apoptotic protein Mcl-1, which is known to confer resistance to apoptosis in cancer cells. Thus, simvastatin overcomes bortezomib-induced apoptosis resistance by inhibiting synthesis of GGPP and disrupting a GGPP-dependent survival pathway.
Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it.
Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.
RhoB, a small GTP-binding protein, was shown previously to contain farnesyl (C-15) as well as geranylgeranyl (C-20) groups (Adamson, P., Marshall, C. J., Hall, A., and Tilbrook, P. A. (1992) J. Biol. Chem. 267, 20033-20038). The COOH-terminal sequence of the protein is CCKVL. According to current rules of prenylation, the COOH-terminal leucine should render the protein a substrate for CAAX geranylgeranyl transferase (GGTase-1), but not for CAAX farnesyltransferase (FTase). To determine the mechanism of farnesylation, we prepared recombinant RhoB and incubated it with recombinant preparations of either FTase or GGTase-1. RhoB was neither farnesylated nor geranylgeranylated efficiently by FTase, but it was farnesylated as well as geranylgeranylated by GGTase-1. The enzyme attached farnesyl more efficiently than geranylgeranyl to RhoB. Neither farnesylation nor geranylgeranylation required the cysteine at the fifth position from the COOH terminus. However, replacement of the cysteine at the fourth position abolished attachment of both prenyl groups. We conclude that the previously observed farnesylation of RhoB is attributable to the FTase activity of GGTase-1. These data, and other accumulating data, indicate that GGTase-1 is a highly unusual enzyme that efficiently transfers both farnesyl and geranylgeranyl groups and that the choice of prenyl group is dictated by the nature of the protein acceptor.
Geranylgeranylated Rab proteins usually terminate in either Cys-Cys or Cys-Xaa-Cys, where Xaa is Ala, Ser, or Gly. In both classes of proteins, the two cysteines are geranylgeranylated, but only the Cys-Xaa-Cys class has been shown to be carboxyl-methylated on the terminal cysteine in vivo. In the current study, we used recombinant Rab geranylgeranyltransferase and a Rab escort protein (REP-1) to attach geranylgeranyl residues to the two cysteines at the carboxyl terminus of Rab3A (Cys-Ala-Cys) and Rab1A (Cys-Cys). The geranylgeranylated proteins were then incubated with bovine cerebellar membranes that contain an enzyme that transfers [3H]methyl from S-[methyl-3H]adenosyl-L-methionine to geranylgeranylated cysteine. The enzyme transferred [3H]methyl to geranylgeranylated Rab3A but not to geranylgeranylated Rab1A. Replacement of the Cys-Ala-Cys terminus of Rab3A with Cys-Cys abolished methylation, and the opposite result was obtained when the Cys-Cys of Rab1A was replaced with Cys-Ala-Cys. When the Cys-Cys terminus of Rab1A was changed to Ser-Cys, methylation was restored. These studies suggest that the carboxyl-terminal cysteine of Rab proteins terminating in Cys-Xaa-Cys but not Cys-Cys is methylated and that the resistance of Cys-Cys proteins to methylation is attributable to the vicinal geranylgeranylated cysteines.
We have studied whether hSOS1, a mammalian guanine nucleotide exchange factors responsible for activating Ras in response to growth factor stimulation, requires post-translational processing of Ras proteins to promote guanine nucleotide exchange. Our results showed that full-length hSOS1 catalyzed guanine nucleotide exchange on prenylated K-Ras(4B) but with a much lower efficiency on unprocessed K-Ras(4B). The apparent Km of hSOS1 for prenylated K-Ras(4B) was 225 (+/- 25) nM with a Vmax of 0.7 (+/- 0.1) mmol/min/mmol. The activity of hSOS1 against unprocessed K-Ras(4B) was too low to measure Km and Vmax. Consistent with these observations, full-length hSOS1 formed a complex with nucleotide-depleted prenylated K-Ras(4B) but not with unprocessed K-Ras(4B). A geranylgeranylated mutant of K-Ras(4B) was an equally good substrate for hSOS1 as wild-type farnesylated K-Ras. Similarly hSOS1 promoted guanine nucleotide exchange on prenylated Ha-Ras but showed minimal activity toward unprocessed Ha-Ras. Neither the polybasic domain of K-Ras (4B) or palmitoylation of Ha-Ras were required for hSOS1-promoted guanine nucleotide exchange. We attempted to identify a minimal region of hSOS1 capable of promoting guanine nucleotide exchange on both prenylated and unprocessed K-Ras. However, a truncated form of hSOS1 comprising only the CDC25 homology domain retained preferential catalytic activity against prenylated K-Ras, whereas the cognate domain from CDC25 was more active against unprocessed K-Ras.
GTPases of the Ras superfamily regulate many aspects of cell growth, differentiation and action. Their functions depend on their ability to alternate between inactive and active forms, and on their cellular localization. Numerous proteins affecting the GTPase activity, nucleotide exchange rates and membrane localization of Ras superfamily members have now been identified. Many of these proteins are much larger and more complex than their targets, containing multiple domains capable of interacting with an intricate network of cellular enzymes and structures.
GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activation of GTPases and may also be critical for the cellular compartmentalization of GTPases. RhoGDI and GDI/D4 are two currently known GDIs for the Rho-subfamily of GTPases. Using their cDNAs to screen a human brain cDNA library under low stringency, we have cloned a homologous cDNA preferentially expressed at high levels in brain and pancreas. The predicted protein, named RhoGDIgamma, is approximately 50% identical to GDI/D4 and RhoGDI. It binds to CDC42 and RhoA with less affinity compared with RhoGDI and does not bind with Rac1, Rac2, or Ras. RhoGDIgamma functions as a GDI for CDC42 but with approximately 20 times less efficiency than RhoGDI. Immunohistochemical studies showed a diffuse punctate distribution of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. Overexpression of the protein in baby hamster kidney cells caused the cells to round up with loss of stress fibers. A distinct hydrophobic amino terminus in RhoGDIgamma, not seen in the other two RhoGDIs, could provide a mechanism for localization of the GDI to specific membranous compartment thus determining function distinct from RhoGDI or GDI/D4. Our results provide evidence that there is a family of GDIs for the Rho-related GTPases and that they differ in binding affinity, target specificity, and tissue expression. We propose that RhoGDI be renamed RhoGDIalpha and GDID4 be renamed RhoGDIbeta. The new GDI should widen the scope of investigation of this important class of regulatory protein.
A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.
The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.
Cell membrane association by several important peripheral proteins, such as Src, MARCKS, HIV-1 Gag, and K-Ras, requires nonspecific electrostatic interactions between a cluster of basic residues on the protein and acidic phospholipids in the plasma membrane. A simple theoretical model based on the nonlinear Poisson-Boltzmann equation describes well the experimentally measured electrostatic association between such proteins and the cell membrane.
Small Rab GTPases are involved in the regulation of membrane trafficking. They cycle between cytosolic and membrane-bound forms. These membrane association/dissociation are tightly controlled by regulatory proteins. To search for proteins interacting with Rab13, a small GTPase associated with vesicles in fibroblasts and predominantly with tight junctions in epithelial cells, we screened a HeLa two-hybrid cDNA library and isolated a clone encoding a protein of 17.4 kDa. This protein, almost identical to the bovine rod cGMP phosphodiesterase delta subunit, was named human delta-PDE. The delta-PDE binds specifically to Rab13. It exhibits two putative C-terminal sequences necessary for the interaction with PDZ (PSD95, Dlg, ZO-1) domains contained in many proteins localized to specific plasma membrane microdomains. Immunofluorescence microscopic studies revealed that the vesicular stomatitis virus (VSV)-tagged delta-PDE is localized in vesicular structures accumulated near the plasma membrane in epithelial cells. Deletion of the PDZ binding motifs impair VSV-delta-PDE subcellular distribution. Purified recombinant delta-PDE had the capacity to dissociate Rab13 from cellular membranes. Our data support the proposal that delta-PDE, but not GDP dissociation inhibitor, may serve to control the dynamic of the association of Rab13 with cellular membranes.
Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the
reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9
cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and
a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells,
respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To
support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide,
H., and Kaziro, Y. (1998)Oncogene 16, 2781–2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic
Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation
of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although
not sufficient, for Raf-1 activation.
Although its precise function has not yet been established, galectin‐1 seems to play a role in tumor progression. In this study, we investigated galectin‐1 mRNA expression in human glioma specimens and glioma cell lines. Northern blot analysis showed higher galectin‐1 mRNA levels in glioma tissues. The 0.7‐kb galectin‐1 mRNA transcript was detected, and the expression level correlated with the malignant state, from low‐grade astrocytoma to glioblastoma. In several human glioma specimens, immunohistochemical examination with antiserum against a synthetic peptide corresponding to the predicted C‐terminal sequence of the protein showed high levels of galectin‐1 expression. To clarify the correlation between the expression of galectin‐1 and the malignancy of gliomas, we examined whether expression of antisense galectin‐1 would suppress tumor growth in rat 9L cells that express high levels of galectin−1. The cells were transfected with a plasmid DNA that produces antisense galectin‐1 mRNA under the control of the metallothionein promoter, and stable clones expressing low levels of galectin‐1 protein in comparison with control clones were isolated. Cells with low levels of galectin‐1 displayed dramatic phenotypic changes in their morphology and growth properties compared with vector‐transfected control 9L cells. Our data suggest that decreased expression of galectin‐1 may arrest the growth of rat 9L cells. J. Neurosci. Res. 59:722–730, 2000 © 2000 Wiley‐Liss, Inc.
Mutational analysis of p21ras has shown that plasma membrane targeting requires the combination of a CAAX motif with a polybasic domain of six lysine residues or a nearby palmitoylation site. However, it is not known from these studies whether these signals alone target p21ras to the plasma membrane. We now show that these C‐terminal sequences are sufficient to target a heterologous cytosolic protein to the plasma membrane. Interestingly, the key feature of the p21K‐ras(B) polybasic domain appears to be a positive charge, since a polyarginine domain can function as a plasma membrane targeting motif in conjunction with the CAAX box and p21K‐ras(B) with the polylysine domain replaced by arginines is biologically active. Since some ras‐related proteins are modified by geranylgeranyl rather than farnesyl we have investigated whether modification of p21ras with geranylgeranyl affects its subcellular localization. Geranylgeranyl can substitute for farnesyl in combining with a polybasic domain to target p21K‐ras(B) to the plasma membrane, but such geranylgeranylated proteins are more tightly bound to the membrane. This increased avidity of binding is presumably due to the extra length of the geranylgeranyl alkyl chain.
Remodeling of the endothelial cell (EC) cytoskeleton is central to many functions of the endothelium. The Rho family of small GTP-binding proteins have been identified as key regulators of F-actin cytoskeletal dynamics in a variety of cell types. They integrate signals from soluble mediators interacting with cytokine, growth factor tyrosine kinase, and G-protein–coupled receptors (GPCRs); as well as signals from cell–cell, and cell–matrix protein adhesion molecules. Recently, it has become appreciated that effector molecules downstream of Rho GTP-binding proteins also modulate several other well described cell signaling pathways. We review the role these molecules play in the cell, with a particular focus on the EC. HISTORY The Rho family of small GTP-binding proteins, which consists of 22 members, is part of the larger Ras GTP-binding protein superfamily. These approximately 21-kDa proteins cycle between inactive GDP-and active GTP-bound forms to act as a molecular switch in signal transduction pathways. The members of this family are grouped by virtue of a shared structural motif, the Rho insert loop, that is present in the GTPase domain and contributes to the binding specificity for downstream effector molecules (1,2). In addition to this shared structural feature, most Rho family members undergo post-translational modification to link farnesyl or geranylgeranyl groups to the cysteine in a CAAX motif at the C-terminus of the molecule. Subcellular localization of the molecule is directed by the lipid moiety and, in some family members, is also influenced by additional domains in the C-terminus.
There has been considerable recent interest in the possibility that the plasma membrane contains lipid “rafts,” microdomains enriched in cholesterol and sphingolipids. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. However, rafts have proven difficult to visualize in living cells. Most of the evidence for their existence and function relies on indirect methods such as detergent extraction, and a number of recent studies have revealed possible problems with these methods. Direct studies of the distribution of raft components in living cells have not yet reached a consensus on the size or even the presence of these microdomains, and hence it seems that a definitive proof of raft existence has yet to be obtained.
This chapter discusses the Rab13 interaction with the phosphodiesterase (PDE) delta subunit (δPDE). The small GTPase Rab13 protein is highly homologous to the mammalian Rab8 and Rab10 and to the yeast Sec4 protein involved in polarized secretion during the budding process. Rab13 associates with vesicles in fibroblasts and accumulates in tight junctions of polarized epithelial cells. This subcellular localization depends on the integrity of tight junctions, which constitute a specialized subdomain of the lateral plasma membrane separating the apical from the basolateral cell surface in epithelial cells. Results indicate that the intercellular junctions may constitute a specific targeting site on the plasma membrane for delivery of specialized cargo vesicles. To investigate the function of Rab13, protein partners are searched. Different techniques are presented in the chapter, such as solubilization assay and immunofluorescence localization studies that are used to identify δPDE, a Rab13 interacting protein, and to show that it is required for the recycling of Rab13 from membranes to the cytosol.
The farnesyl group and a number of its analogs were enzymatically incorporated into H-Ras and K-Ras in vitro in order to study the prenyl group dependence of the hSOS1-catalyzed nucleotide exchange of Ras. Farnesylation of H-Ras and K-Ras in the absence of further processing was sufficient to facilitate the exchange although fully processed Ras showed a somewhat higher rate of exchange. H-Ras containing 15-carbon analogs of the farnesyl group were good substrates for hSOS1, but geranylated-H-Ras was not.
Protein (lectin)-carbohydrate (cellular glycoconjugate) recognition is operative in biochemical information transfer. Galectins
constitute a family of endogenous galactoside-binding lectins with conserved features in the binding site. The members of
this lectin category are assumed to be involved in cell adhesion and growth regulation. To assess to what extent the different
modes of binding-site presentation and/or carbohydrate fine-specificities will affect aspects of galectin behavior, homodimeric
cross-linking galectin-1 and monomeric chimeric galectin-3, with its collagenase-sensitive stalk linked to the carbohydrate-recognition
domain, were investigated. Cell-surface expression of the two galectins and accessible galectin-binding sites on various tumor
cell lines was ascertained by FACScan analysis. In particular, ligand accessibility for the two galectins differed for the
tested cell line types. Binding of tumor cells to laminin and plasma or placental fibronectin was generally reduced by treatment
of cells or matrix with galectins. Galectin-3 was more efficient than galectin 1 at impairing laminin's potency as matrix.
Cell binding of galectin-1, on the other hand, proved on average more effective for blocking cell association to fibronectins
after its preincubation with cell suspensions. Differences were also apparent in the biodistribution of the galectins, where
an avian homolog of galectin-1 served as the control to distinguish effects of spatial and sugar-binding features. Histopathological
analysis of lymph-node-negative and -positive breast and colorectal carcinomas (n = 180 including 60 metastatic lesions) indicated a correlation of either increased galectin-1 binding and reduced galectin-3
expression or reduced binding of both galectins with the occurrence of lymph node lesions. Together with data on the heparin-binding
lectin, revealing reduced expression to be associated with a positive lymph-node status in the breast cancer group, these
results can be interpreted to reflect cell-type-dependent requirements of galectin ligand presentation during the metastatic
cascade. By introducing mammalian lectins to lectin-histochemical studies, the detection of quantitative differences in glycosylation
brings an understanding of its cell biological significance one step closer.
Seeing is believing! Semisynthetic Ras-proteins with a fluorophore introduced at an appropriate site have been developed as probes for live-cell imaging experiments. They are processed by the cellular machinery, and their intracellular location can then be determined by confocal laser fluorescence microscopy.
Mutational analysis of p21ras has shown that plasma membrane targeting requires the combination of a CAAX motif with a polybasic domain of six lysine residues or a nearby palmitoylation site. However, it is not known from these studies whether these signals alone target p21ras to the plasma membrane. We now show that these C-terminal sequences are sufficient to target a heterologous cytosolic protein to the plasma membrane. Interestingly, the key feature of the p21K-ras(B) polybasic domain appears to be a positive charge, since a polyarginine domain can function as a plasma membrane targeting motif in conjunction with the CAAX box and p21K-ras(B) with the polylysine domain replaced by arginines is biologically active. Since some ras-related proteins are modified by geranylgeranyl rather than farnesyl we have investigated whether modification of p21ras with geranylgeranyl affects its subcellular localization. Geranylgeranyl can substitute for farnesyl in combining with a polybasic domain to target p21K-ras(B) to the plasma membrane, but such geranylgeranylated proteins are more tightly bound to the membrane. This increased avidity of binding is presumably due to the extra length of the geranylgeranyl alkyl chain.
The products of rap genes (rap1A, rap1B and rap2) are small molecular weight GTP-binding proteins that share approximately 50% homology with ras-p21s. It had previously been shown that a rap1 protein (also named Krev-1 or smg p21) could be phosphorylated on serine residues by the cAMP-dependent protein kinase (PKA) in vitro as well as in intact platelets stimulated by prostaglandin E1. We show here that the rap1A protein purified from recombinant bacteria is phosphorylated in vitro by the catalytic subunit of PKA and that the deletion of the 17 C-terminal amino acids leads to the loss of this phosphorylation. This suggests that the serine residue at position 180 constitutes the site of phosphorylation of the rap1A protein by PKA. The rap1 protein can also be phosphorylated by PKA in intact fibroblasts; this phenomenon is independent of their proliferative state. In contrast, protein kinase C (PKC) does not phosphorylate the rap1 proteins, neither in vitro nor in vivo. Finally, the 60% homologous rap2 protein is neither phosphorylated in vitro nor in vivo by PKA or PKC.
The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.
The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.
To investigate the possible role of ras proteins in the differentiation process signaled by nerve growth factor, we have microinjected the proto-oncogenic and oncogenic (T24) forms of the human H-ras protein into living rat pheochromocytoma cells (PC12). PC12 cells, which have the phenotype of replicating chromaffin-like cells under normal growth conditions, respond to nerve growth factor by differentiating into nonreplicating sympathetic neuron-like cells. Microinjection of the ras oncogene protein promoted the morphological differentiation of PC12 cells into neuron-like cells. In contrast, microinjection of similar amounts of the proto-oncogene form of the ras protein had no apparent effect on PC12 cells. The induction of morphological differentiation by the ras oncogene protein occurred in the absence of nerve growth factor, was dependent on protein synthesis, and was accompanied by cessation of cell division. Treatment of PC12 cells with nerve growth factor or cAMP analogue prior to injection did not alter the phenotypic changes induced by the ras oncogene protein.
Hydrophobic insertion of the acyl chain into the bilayer is necessary but not sufficient for the membrane binding of a myristoylated protein. The myristoylated alanine-rich C kinase substrate (MARCKS), Src, ADP-ribosylation factor and human immunodeficiency virus-1 matrix proteins also contain a cluster of basic residues that bind to acidic phospholipids; the hydrophobic and electrostatic interactions act together to anchor the protein to a membrane. For MARCKS, and perhaps other proteins, phosphorylation of serines within its basic cluster reduces the electrostatic attraction, producing translocation of the protein from the membrane to the cytosol by a simple 'electrostatic switch' mechanism.
The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.
To understand better the potential functional importance of the dual-lipid modifications found in a number of intracellular proteins of eukaryotes, we have examined how "tenaciously" various doubly-lipid-modified peptides, with sequences and lipid modifications reflecting those found in intracellular proteins, are anchored to lipid bilayer membranes. Fluorescent-labeled peptides bearing dual-lipid modifications were incorporated into large unilamellar egg phosphatidylcholine/phosphatidylglycerol vesicles, and the kinetics of spontaneous intervesicle transfer of the lipopeptides were monitored by a fluorescence-dequenching assay. Lipopeptides incorporating the stable "dual-anchor" motif -C(geranylgeranyl)XC(geranylgeranyl)-OMe found in several rab and homologous proteins exhibit very slow rates of interbilayer transfer (t1/2 > 50 h), as do lipopeptides incorporating myristoyl-GC(palmitoyl)X- and -C(palmitoyl)XC(farnesyl)-OMe motifs found in various src-related intracellular tyrosine kinases and G-protein alpha-subunits and in p21H-ras, respectively. Lipopeptides terminating in an unmethylated -C(geranylgeranyl)C(geranylgeranyl)-OH motif show somewhat greater but still very slow rates of spontaneous interbilayer transfer (t1/2 = ca. 10 h). Extrapolating from these results, we estimate that the rate of spontaneous desorption of the corresponding doubly-anchored proteins from membranes should be much slower than that of regulated, protein-mediated release (effected by binding to an "escort" protein or by de-S-acylation). As a result the intracellular distributions of these species (and particularly their targeting to specific intracellular membranes) are likely to be governed (and regulated) primarily by kinetic rather than thermodynamic factors.(ABSTRACT TRUNCATED AT 250 WORDS)
We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.
Prenylation is a class of lipid modification involving covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. Known prenylated proteins include fungal mating factors, nuclear lamins, Ras and Ras-related GTP-binding proteins (G proteins), the subunits of trimeric G proteins, protein kinases, and at least one viral protein. Prenylation promotes membrane interactions of most of these proteins, which is not surprising given the hydrophobicity of the lipids involved. In addition, however, prenylation appears to play a major role in several protein-protein interactions involving these species. The emphasis in this review is on the enzymology of prenyl protein processing and the functional significance of prenylation in cellular events. Several other recent reviews provide more detailed coverage of aspects of prenylation that receive limited attention here owing to length restrictions (1-4).
Prenylated proteins contain a covalently linked cholesterol intermediate near their carboxyl-termini. Maturation of most prenylated
proteins involves proteolytic removal of the last three amino acids. Two genes in Saccharomyces cerevisiae, RCE1 and AFC1, were identified that appear to be responsible for this processing. The Afc1 protein is a zinc protease that participates
in the processing of yeast a-factor mating pheromone. The Rce1 protein contributes to the processing of both Ras protein and a-factor. Deletion of both AFC1 and RCE1 resulted in the loss of proteolytic processing of prenylated proteins. Disruption of RCE1 led to defects in Ras localization and signaling and suppressed the activated phenotype associated with the allele RAS2val19.
Several members of the Src family of protein tyrosine kinases have a N-terminal dual acylation motif which specifies their myristoylation and S-acylation. These lipid modifications are necessary for correct intracellular localisation to the plasma membrane and to detergent-resistant glycolipid-enriched membrane domains (GEMs). Using chimaeras of the Lck dual acylation motif with two normally cytosolic proteins (chloramphenicol acetyl transferase and galectin-3), we show here that this motif is sufficient to encode correct lipid modification and to target these chimaeras to the plasma membrane, as demonstrated by subcellular fractionation and confocal immunofluorescence microscopy of transiently transfected COS cells. In addition, the chimaeras are resistant to extraction with cold non-ionic detergent, indicating targeting to GEM subdomains in the plasma membrane. The dual acylation motif has potential for targeting proteins to specific plasma membrane subdomains involved in signalling.
Many signaling proteins, including the proto-oncogene Ras, require the covalent addition of a lipid chain for proper function. This prenylated protein is then earmarked for further proteolysis and methylation. In his Perspective, Gelb discusses two reports in this issue that illuminate how the lipid is initially attached to the protein [Park et al . ([p. 1800][1])] and how the later proteolytic modifications are accomplished [Boyartchuk et al . ([p. 1796][2])]
[1]: /lookup/doi/10.1126/science.275.5307.1800
[2]: /lookup/doi/10.1126/science.275.5307.1796
We have used a series of fluorescent lipid-modified peptides, based on the farnesylated C-terminal sequence of mature N-ras [-GCMGLPC(farnesyl)-OCH3], to investigate the membrane-anchoring properties of this region of the protein and its reversible modification by S-acylation in cultured mammalian fibroblasts. The farnesylated peptide associates with lipid bilayers (large unilamellar phospholipid vesicles) with high affinity but in a rapidly reversible manner. Additional S-palmitoylation of the peptide suppresses its ability to desorb from, and hence to diffuse between, lipid bilayers on physiologically significant time scales. NBD-labeled derivatives of the farnesylated N-ras C-terminal heptapeptide, when incubated with CV-1 cells in culture, are taken up by the cells and reversibly S-acylated in a manner similar to that observed previously for the parent protein. The S-acylation process is highly specific for modification of a cysteine rather than a serine residue but tolerates replacement of the peptide-linked farnesyl moiety by other hydrophobic groups. Fluorescence microscopy reveals that in CV-1 cells the S-acylated form of the peptide is localized preferentially to the plasma membrane, as has been observed for N-ras itself. This plasma membrane localization is unaffected by either reduced temperature (15 degrees C) or exposure to brefeldin A, treatments which inhibit various trafficking steps within the secretory pathway. These results suggest that in mammalian cells the plasma membrane localization of mature N-ras is maintained by a 'kinetic trapping' mechanism based on S-acylation of the protein at the level of the plasma membrane itself.
RhoA/B/C and CDC42/Rac, which form two subgroups of the rho guanosine triphosphatase (GTPase) family, regulate various aspects of actin cytoskeleton organisation. In cytosol, guanosine diphosphate (GDP) dissociation inhibitor (GDI) interacts with and maintains rho GTPases in their inactive GDP-bound form. RhoGDI is a ubiquitously expressed GDI, whereas D4/LyGDI is hematopoietic cell-specific and 10-fold less potent than RhoGDI in binding to and regulating rho GTPases. We have combined microanalytical liquid chromatography with the use of specific antibodies in order to separate D4/LyGDI and RhoDGI-complexes from the cytosol of U937 cells and to demonstrate that the two GDIs associate with different rho protein partners. RhoGDI can form a complex with CDC42Hs, RhoA, Rac1 and Rac2, while none of these GTPases was found to interact with D4/LyGDI. In addition, we found that stimulation of U937 cells with phorbol ester leads to phosphorylation of D4/LyGDI. Our results suggest that LyGDI forms complexes with specific rho GTPases expressed in hematopoietic cells where it may regulate specific pathways.
We have examined the association with lipid vesicles of fluorescent lipidated peptides based on the farnesylated, polybasic carboxy-terminal region of mature K-ras4B, which functions physiologically as an autonomous plasma membrane-targeting motif. While the peptides bind to neutral lipid (phosphatidylcholine/phosphatidylethanolamine) vesicles with relatively low affinity, the vesicle-binding affinity increases exponentially as increasing amounts of anionic lipids are incorporated into the vesicle bilayers. Competitive vesicle-binding experiments reveal that the K-ras4B carboxy-terminal sequence accordingly discriminates strongly between lipid surfaces of differing surface charge, such that two lipid bilayers differing in anionic lipid content by 10 mol % will show a 45-fold preferential accumulation of the lipidated peptide in the more negatively charged surface. At the same time, the carboxyl-terminal region of K-ras4B exhibits no preferential binding to particular anionic lipids, including the polyanionic species phosphatidylinositol-4'-phosphate and phosphatidylinositol-4',5'-bisphosphate, beyond that predicted on the basis of surface-charge effects. The K-ras4B carboxyl-terminal sequence dissociates rapidly (with half-times of seconds or less) from lipid bilayers containing up to 40 mol % anionic lipid. These results suggest that the targeting of the mature K-ras4B carboxy-terminus to the plasma membrane, if it is based on interactions with plasma membrane lipids, is not mediated by a kinetic-trapping mechanism or by specific binding to particular anionic lipids but may rest on the sensitive surface potential-sensing function of this region of the protein.
Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry 34, 12543-12552]. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25(Mm285), was 4.6 nM, i.e., a 500-fold lower affinity than the Ras.GDP interaction. The affinities defining the ternary complex Ras. nucleotide.Cdc25(Mm285) are several orders of magnitude lower. The maximum acceleration by Cdc25(Mm285) of the GDP dissociation from Ras was more than 10(5)-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras. Cdc25(Mm285) show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras. GDP.Cdc25(Mm285) is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, we demonstrate that the disruption of the Mg2+-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras.
Protein farnesyltransferase and protein geranylgeranyltransferase-I catalyze the prenylation of a cysteinyl group located four residues upstream of the carboxyl terminus. The identity of the carboxyterminal residue plays a significant role in determining the ability of compounds to bind to each enzyme and to serve as substrate. We compared the binding and substrate specificities of peptides with carboxyterminal substitutions to determine which residues promote selectivity and which residues promote recognition by both enzymes. Using tetrapeptide inhibitors with the general structure l-penicillamine-valine-isoleucine-X and substrates with the structure Lys-Lys-Ser-Ser-Cys-Val-Ile-X, we measured their respective Ki, Km, and kcat values for both recombinant rat protein farnesyltransferase and recombinant rat protein geranylgeranyltransferase-I. We studied the roles of carboxyterminal branched residues (leucine, isoleucine, valine, and penicillamine) and linear residues (methionine, cysteine, homocysteine, alanine, aminobutyrate, and aminohexanoate) in promoting interaction with the enzymes. For protein geranylgeranyltransferase-I, peptide substrates with carboxyterminal branched or linear residues had Km values that were 5- to 15-fold greater than the Ki values of the corresponding peptide inhibitors. For protein farnesyltransferase, peptide substrates with carboxyterminal branched residues, proline, or homoserine had Km values that were 7- to 200-fold greater than the Ki values of the corresponding peptide inhibitors. For protein farnesyltransferase the Km and Ki values for peptides ending with linear residues were in general agreement. Our studies indicate that the substrate and inhibitor binding specificities of protein geranylgeranyltransferase was much more restricted than those of protein farnesyltransferase.
Covalent lipid modifications anchor numerous signalling proteins to the cytoplasmic face of the plasma membrane. These modifications mediate protein-membrane and protein-protein interactions and are often essential for function. Protein palmitoylation, due to its reversible nature, may be particularly important for modulating protein function during cycles of activation and deactivation. Despite intense investigation, the exact functions of protein palmitoylation are not well understood. However, it is clear that palmitoylation can affect a protein's affinity for membranes, subcellular localization, and interactions with other proteins. In this review, recent advances in understanding the functions and mechanisms of protein palmitoylation are discussed, with particular emphasis on how this lipid affects the biochemistry and cell biology of signalling proteins.
Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduction and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions. Here we present a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells.
The RhoGDI proteins serve as key multifunctional regulators of Rho family GTP-binding proteins. The 2.6 A X-ray crystallographic structure of the Cdc42/RhoGDI complex reveals two important sites of interaction between GDI and Cdc42. First, the amino-terminal regulatory arm of the GDI binds to the switch I and II domains of Cdc42 leading to the inhibition of both GDP dissociation and GTP hydrolysis. Second, the geranylgeranyl moiety of Cdc42 inserts into a hydrophobic pocket within the immunoglobulin-like domain of the GDI molecule leading to membrane release. The structural data demonstrate how GDIs serve as negative regulators of small GTP-binding proteins and how the isoprenoid moiety is utilized in this critical regulatory interaction.
Although its precise function has not yet been established, galectin-1 seems to play a role in tumor progression. In this study, we investigated galectin-1 mRNA expression in human glioma specimens and glioma cell lines. Northern blot analysis showed higher galectin-1 mRNA levels in glioma tissues. The 0.7-kb galectin-1 mRNA transcript was detected, and the expression level correlated with the malignant state, from low-grade astrocytoma to glioblastoma. In several human glioma specimens, immunohistochemical examination with antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the protein showed high levels of galectin-1 expression. To clarify the correlation between the expression of galectin-1 and the malignancy of gliomas, we examined whether expression of antisense galectin-1 would suppress tumor growth in rat 9L cells that express high levels of galectin-1. The cells were transfected with a plasmid DNA that produces antisense galectin-1 mRNA under the control of the metallothionein promoter, and stable clones expressing low levels of galectin-1 protein in comparison with control clones were isolated. Cells with low levels of galectin-1 displayed dramatic phenotypic changes in their morphology and growth properties compared with vector-transfected control 9L cells. Our data suggest that decreased expression of galectin-1 may arrest the growth of rat 9L cells.
Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rab family by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated to what extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp) and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoids indicated that in all cases association is rapid, with rate constants in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak binding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K(d) of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate constant of 0.082 s(-1) which is very similar to that of GGpp. The implications of the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.