Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.     

rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif.

Low molecular mass GTP-binding proteins encoded by the mammalian rab genes are found in membranes of the Golgi complex and endosomes, suggesting that they play a role in the movement of exocytic and endocytic vesicles. The basis for the membrane association of these proteins has not been defined. Herein, we demonstrate that terminal cysteine residues in the rab1B, rab2, and rab5 proteins undergo thioether modification by isoprenyl groups when these proteins are translated in vitro in the presence of a radiolabeled isoprenoid precursor, [3H]mevalonate. Results of gel permeation chromatography of the radiolabeled hydrocarbons suggest that these proteins are modified specifically by isoprenyl groups of the 20-carbon diterpene class, rather than the 15-carbon farnesyl class known to be involved in modification of ras proteins. The rab1 and rab2 proteins lack the carboxyl-terminal amino acid motif common to all previously identified isoprenylated proteins, i.e. CXXX, where X is an unspecified amino acid. Analysis of altered translation products generated by site-directed mutagenesis indicates that modification of rab1B protein requires an intact carboxyl-terminal sequence consisting of GGCC. This represents a new amino acid motif for isoprenylation.     

Post-translational modification of low molecular mass GTP-binding proteins by isoprenoid

Several proteins in mammalian cells are modified post-translationally by the isoprenoid, farnesol. Incubation of cultured cells with [3H]mevalonate, an isoprenoid precursor, results in the labeling of multiple polypeptides, the most prominent of which migrate in the range of 21-26 kDa on sodium dodecyl sulfate-polyacrylamide gels. In Rat-6 fibroblasts transformed by H-ras, one of the farnesylated proteins was identified as p21ras by two-dimensional immunoblotting. However, this protein accounted for only a small proportion of the [3H]mevalonate-derived radioactivity incorporated into 21-26-kDa proteins. Murine erythroleukemia cells, which did not express immunodetectable quantities of p21ras, contained several 21-26-kDa farnesylated proteins distributed in both the cytosolic and particulate fractions. At least eight of these proteins were capable of binding [alpha-32P]GTP on nitrocellulose membranes. Pulse-chase studies showed that the isoprenoid modification did not necessarily result in the translocation of the cytosolic proteins to the cell membrane. A prominent group of carboxyl-methylated proteins in murine erythroleukemia cells overlapped with the 21-26-kDa farnesylated proteins on one-dimensional sodium dodecyl sulfate gels. Methylation of this group of proteins was selectively abolished when cells were treated with lovastatin, an inhibitor of isoprenoid synthesis. Addition of exogenous mevalonate to the lovastatin-treated cells fully restored carboxyl methylation. These studies suggest that the 21-26-kDa farnesylated proteins in mammalian cells are members of a recently discovered family of low molecular mass GTP-binding proteins which, although ras-related, appear to be distinct structurally and possibly functionally from the products of the ras genes. The observed isoprenoid-dependent carboxyl methylation of a group of 21-26-kDa proteins suggests that the low molecular mass GTP-binding proteins may undergo a series of post-translational C-terminal cysteine modifications (i.e. farnesylation, carboxyl methylation) analogous to those recently elucidated for p21ras.     

Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets

Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing. To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) [alpha-32P]GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes. The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound [alpha-32P]GTP prominently but was only poorly detected with the GTP gamma S-binding assay. The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu). A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids. In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets. One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein. Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) J. Biol. Chem. 264, 16378-16382). These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds [alpha-32P]GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.     

Posttranslational modification of proteins by isoprenoids in mammalian cells

Isoprenylation is a posttranslational modification that involves the formation of thioether bonds between cysteine and isoprenyl groups derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. Numerous isoprenylated proteins have been detected in mammalian cells. Those identified include K-, N-, and H-p21ras, ras-related GTP-binding proteins such as G25K (Gp), nuclear lamin B and prelamin A, and the gamma subunits of heterotrimeric G proteins. The modified cysteine is located in the fourth position from the carboxyl terminus in every protein where this has been studied. For p21ras, the last three amino acids are subsequently removed and the exposed cysteine is carboxylmethylated. Similar processing events may occur in lamin B and G protein gamma subunits, but the proteolytic cleavage in prelamin A occurs upstream from the modified cysteine. Lamin B and p21ras are modified by C15 farnesyl groups, whereas other proteins such as the G protein gamma subunits are modified by C20 geranylgeranyl chains. Separate enzymes may catalyze these modifications. The structural features that govern the ability of particular proteins to serve as substrates for isoprenylation by C15 or C20 groups are not completely defined, but studies of the p21ras modification using purified farnesyl:protein transferase suggest that the sequence of the carboxyl-terminal tetrapeptide is important. Isoprenylation plays a critical role in promoting the association of p21ras and the lamins with the cell membrane and nuclear envelope, respectively. Future studies of the role of isoprenylation in the localization and function of ras-related GTP-binding proteins and signal-transducing G proteins should provide valuable new insight into the link between isoprenoid biosynthesis and cell growth.     

The prenylation of proteins.

The prenylated proteins represent a newly discovered class of post-translationally modified proteins. The known prenylated proteins include the oncogene product p21ras and other low molecular weight GTP-binding proteins, the nuclear lamins, and the gamma subunit of the heterotrimeric G proteins. The modification involves the covalent attachment of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid moiety in a thioether linkage to carboxyl terminal cysteine. The nature of the attached substituent is dependent on specific sequence information in the carboxyl terminus of the protein. In addition, prenylation entrains other posttranslational modifications forming a reaction pathway. In this article, we review our current understanding of the biochemical reactions involved in prenylation and discuss the possible role of this modification in the control of cellular functions such as protein maturation and cell growth.     

Post-translational processing of rac p21s is important both for their interaction with the GDP/GTP exchange proteins and for their activation of NADPH oxidase.

rac1 and rac2 p21s are ras p21-like small GTP-binding proteins which are implicated in the NADPH oxidase-catalyzed superoxide generation in phagocytes. rac1 and rac2 p21s have a Cys-A-A-Leu (A = aliphatic amino acid) structure in their C-terminal region which may undergo post-translational processing including prenylation, proteolysis, and carboxyl methylation. We studied the function of this post-translational processing of rac p21s in their interaction with the stimulatory and inhibitory GDP/GTP exchange proteins for rac p21s, named smg GDS and rho GDI, and in their NADPH oxidase activation. We produced human recombinant rac1 and rac2 p21s in insect cells and purified them from the membrane and soluble fractions as the post-translationally processed and unprocessed forms, respectively. Post-translationally processed rac1 and rac2 p21s were sensitive to both smg GDS and rho GDI, but post-translationally unprocessed rac1 and rac2 p21s were insensitive to them. The GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-bound form of post-translationally processed rac1 and rac2 p21s stimulated the NADPH oxidase activity, but post-translationally unprocessed rac1 and rac2 p21s were far less effective. These results indicate that both rac1 and rac2 p21s stimulate the NADPH oxidase activity and that their post-translational processing is important not only for their interaction with smg GDS and rho GDI but also for their NADPH oxidase activation.     

The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.

The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.     

Sos-mediated activation of rac1 by p66shc

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1-eps8-e3b1 tricomplex. The NH(2)-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1-eps8-e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.     

Interaction of mastoparan with the low molecular mass GTP-binding proteins rho/rac

Mastoparan, which has been shown to active G proteins, inhibits the ADP-ribosylation of 20 kDa human platelet membrane proteins catalyzed by Clostridium botulinum exoenzyme C3 half-maximally and maximally (90%) at 20 and 100 microM concentrations, respectively. Inhibition of ADP-ribosylation was enhanced by GTP-gamma S. Mastoparan increased GTP hydrolysis by porcine brain rho protein and stimulated GTP binding in a concentration dependent manner. The data suggest that mastoparan not only interacts with heterotrimeric G proteins but also with low molecular mass GTP-binding proteins of the rho/rac family.     

Heterogeneity of Entamoeba histolytica rac genes encoding p21rac homologues

Entamoeba histolytica (Eh), the parasite that causes amebic dysentery, is the only protozoan that phagocytoses bacteria, epithelial cells and red blood cells. Numerous low-molecular-weight GTP-binding proteins, called p21^rac1, are implicated in signal transduction and actin polymerization during phagocytosis by macrophages and Dictyostelium discoideum (Dd). Here, molecular cloning techniques were used to obtain four Eh rac genes that encoded putative p21^rac1, as well as segments of two Eh rac pseudogenes. The predicted Eh p21^rac1, which share 55–81% amino acid (aa) identities with each other, include one that closely resembles the p21^rac1 of man, Dd, Drosophila melanogaster and Caenorhabditis elegans; two that resemble the p21^rac1 of Dd; and one that is unique. An alignment of the Eh rac ORF with other rac family proteins reveals multiple as that distinguish p21^rac1p21^rac1 and p21^rac1. We conclude that the Eh genes encoding amebic p21^rac1, which are the first identified from a protozoan parasite, are numerous and heterogeneous.     

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.     

ADP-ribosylation of the rho/rac gene products by botulinum ADP-ribosyltransferase: identity of the enzyme and effects on protein and cell functions

1. Botulinum C1 toxin and C3 exoenzyme were purified from the culture filtrate of type C Clostridium botulinum strain 003-9, and specific antibodies were raised against each protein. Immunochemical analysis using these antibodies revealed the presence of minute amount of a C3-like molecule in C1 toxin preparation which tightly binds to the toxin component(s). This enzyme complex was separated from the major neurotoxin. Thus, the ADP-ribosyltransferases in C1 and D toxins and C3 exoenzyme appear to come from the same origin, and should be called together botulinum C3 enzyme. 2. Botulinum C3 enzyme ADP-ribosylates the rho and rac gene products, a family of small molecular weight GTP-binding proteins homologous to ras p21s. This ADP-ribosylation occurs at Asn41 of the rho products which is located in their putative effector domain, suggesting that it interferes interaction of these GTP binding proteins with their effector molecules. 3. When incubated with PC-12 cells, the enzyme inhibits cell growth and induces neurites and acetylcholine esterase. Several lines of evidence suggest that the ADP-ribosylation of the rho/rac proteins is responsible for these changes.     

Isoprenoid modification of G25K (Gp), a low molecular mass GTP-binding protein distinct from p21ras

Cultured murine erythroleukemia (MEL) cells synthesize a number of low molecular mass GTP-binding proteins that undergo post-translational modification by isoprenoids. We used two-dimensional electrophoresis and immunoblotting to show that a 23-24-kDa protein labeled by the isoprenoid precursor [3H]mevalonate was specifically recognized by an antibody to G25K (Gp), a low molecular mass GTP-binding protein originally purified from placental, platelet, and brain membranes. Several isoelectric variants of G25K were detected in MEL cells, and all were radiolabeled with [3H]mevalonte. The G25K-immunoreactive protein did not cross-react with pan-ras antibody. Although mature p21ras is known to be localized in the cell membrane, most of the isoprenylated G25K was found in the 100,000 x g supernatant fraction when cells were lysed in buffer without detergent. Blocking isoprenoid synthesis by incubation of MEL cells with lovastatin resulted in a decrease in the concentration of G25K in the particulate fraction and a corresponding increase in immunodetectable protein in the soluble fraction. Lovastatin treatment also produced shifts in the electrophoretic mobilities of the G25K isoforms on two-dimensional gels. These observations are consistent with the idea that isoprenylation plays a permissive role in the association of G25K with the cell membrane or other organelles. However, the high proportion of soluble isoprenylated G25K in MEL cells under normal culture conditions suggests that the role of the isoprenoid modification may be more complex than simply serving as a structural anchor for stable insertion of proteins into the lipid bilayer.     

Post-translational modifications of p21rho proteins.

Post-translational modifications of the ras proteins, which are required for plasma membrane localization and biological function of the proteins, have been shown to include prenylation and carboxymethylation at the carboxyl terminal cysteine residue of the cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid (CAAX) box. In addition, p21Ha-ras and p21N-ras, but not p21K-ras (B), are palmitoylated. The three mammalian rho proteins (A, B, and C) are also members of the ras superfamily but have distinct biological activities and different intracellular distributions from p21ras. Analysis showed all three rho proteins are modified by a COOH-terminal carboxymethylation similar to p21ras, whereas p21rhoC labeled with [3H]mevalonic acid in vivo revealed the presence of a C20 prenoid, similar to that already described for p21rhoA. However, in vivo and in vitro studies of p21rhoB showed this protein to be modified by both C15 and C20 prenoids. Mutation of C193 in the CAAX box abolished prenylation, whereas mutation of the adjacent C192 resulted in a significant reduction in the amount of the C20, but not C15 prenoid, recovered from p21rhoB. In vivo labeling studies with [3H]palmitic acid and mutational analysis showed that both cysteine residues at 189 and 192 upstream of the CAAX box in p21rhoB are sites for palmitoylation. We conclude that there are different populations of post-translationally modified p21rhoB in the cell and that the sequence specificity for geranylgeranyl- and farnesyltransferases may be more complicated than previously proposed.     

Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.     

The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases

Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho family of GTPases.