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The Ras superfamilies: regulatory proteins and post-translational modifications
The Ras superfamilies: regulatory proteins and post-translational modifications Tony Evans, Matthew Genentech
Inc., South San Francisco,
J. Hart and Richard
California,
and Cornell
The Ras-like CTP-binding proteins comprise that play key roles in a wide variety cell growth, differentiation, secretion, and past few years, it has become clear that regulated by a variety of manners, including of regulatory proteins and post-translational Current
Opinion
As a result of a frenzy of recent activity, both at the biochemical and the molecular genetic level, it is clear that multiple Pas-like GTP-binding proteins are expressed in a wide variety of cells and species. Now that the diversity and scope of the subfamilies (that is, the Ras family and its closest relatives, the Rho family and the Rab family see [l-3] for a more complete deiinition of the subfamily distinctions) are appreciated, the challenge that lies ahead is the dehnition of their biological functions. As the appropriate specilic reagents and experimental systems are generated and identihed, and as the pace of research hastens in this direction away from the mere cataloging of yet more family members, it should become possible to review the regulatory roles of the proteins in speciIic biological processes. That time, however, is not yet at hand and so, in this short commentary, we have decided to focus on the two aspects of the biology of these proteins that we believe represent the most active and exciting areas of current research. One of these is the regulatory proteins that have been identiIied primarily on the basis of their ability to iniluence some aspect of the GTP-bindingGTPa.se cycles of the low-molecularmass GTP-binding proteins. The other is the diversity of post-translational modifications that occur at the ‘CAAX motif and the hypervariable carboxy-terminal region of GTP-binding proteins that are known to be essential for the appropriate localization of the proteins and hence their biological function(s).
Regulatory
proteins
So far, three classes of regulatory proteins for the lowmolecular-mass GTP-binding proteins have been identified; these are the GTPase-activating proteins (GAPS), the
University,
Ithaca,
New York, USA
a large superfamily of proteins of cellular activities, including protein trafficking. During the these CTP-binding proteins are interactions with specific types modification events.
in Cell Biology
Introduction
A. Cerione
1991, 3:185-191
guanine nucleotide exchange proteins (that is, those that facilitate the exchange of GDP for GTP), and the GDPdissociation inhibitors. The GAPS have been by far the most extensively studied, with the Pas-GAP, in particular, having received a great deal of attention particularly because of the possible implication of its ineffective coupling to Pas in the development of certain human cancers. It is, however, reasonable to anticipate that over the next few years exciting new information will become available regarding the molecular features of each of the protein-protein interactions Involved in the regulation of the low-molecular-mass GTP-binding proteins. CTPase-activating
proteins
Since the initial discovery that a distinct protein, GAP, is responsible for stimulating the GTPase activity of Ras, an extensive effort has been directed towards delineating the molecular mechanisms by which this protein acts and how it is regulated. The human Pas-GAP is known to exist in two forms: the type I GAP (molecular mass of approximately 125 kD) appears to be ubiquitous while the type II GAP (molecular mass of approximately 1OOkD) that is generated by alternative splicing has only been found in human placenta (see [4-71 for a much more complete coverage of the structutal properties of the Ras GAP). Although yeast cells do not appear to contain any activity that stimulates the GTPase of mammalian Ras proteins, they do contain two proteins (the products of IRA 2 and IRA2) that show some sequence homology to the carboxy-terminal region of the Pas-GAP and are responsible for stimulating the GTPase activity of the yeast PAS-1 and PAS-2 proteins [8]. Phosphorylation
of Ras-CAP
There have been a number of suggestions regarding the involvement of the Pas-GTP-binding proteins in the sig-
Abbreviations CHO-Chinese
hamster
ovary;
EGkpidermal PDGF-platelet-derived SDS-PACE--sodium
growth
factor; CAP-GTPase-activating growth factor; SDS--sodium dodecyl sulphate polyacrylamide
@ Current
Biology
protein; CDI-CDP-dissociation dodecyl sulphate; gel electrophoresis.
Ltd ISSN 0955+&74
inhibitor;
185
186
Cell regutation
naling pathways of growth factor receptdrs. A clue towads understanding the interplay between growth factor receptors and the Ras proteins has been provided by a series of studies demonstrating the in viva (tyrosine) phosphotylation of the Ras-GAP. Molloy et al [9*], first reported that the addition of both the AA and BB homodimers of platelet-derived growth factor (PDGF) to quiescent fibroblasts elicited a rapid phosphorylation of the Ras-GAP on tyrosine residues. This was demonstrated by immunoprecipita&g phosphoproteins from cell lysates pretreated with PDGF, using antiphosphotyrosine antibodies, and then probing the immunoprecipitates with a specific anti-Ras GAP monoclonal antibody. A potentially important observation from these studies was that in quiescent cells, the Ras-GAPwas found predominantly in the cytosolic fraction, whereas following stimulation of the cells with PDGF, the tyrosine-phosphorylated Ras-GAPS appeared to be most abundant in the membrane. Neither insulin nor the basic fibroblast growth factor were able to elicit the in U~IKJ phosphorylation of the Ras-GAP, while epidermal growth factor (EGF) was able to mimic the effects of PDGF in these cells. A number of subsequent studies conlirmed these findings. For example, Kaplan et al [lo-] also reported that the PDGF receptor stimulated the tyrosine phosphorylation of the Ras-GAP in NIH 3T3 cells and provided evidence from immunoprecipitation experiments that phosphotylated Ras-GAP was capable of associating with a number of other proteins that have been suspected of playing a role in growth factor signaling, including the Raf-1 kinase, phospholipase C-y, and phosphatidylinositol-3 kinase. Kazlauskas et UI! [ 11.1 also reported the formation of complexes between the PDGF receptor and &-GAP in viva and proposed that the phosphoxylation of a specifk tyrosine residue on the PDGF receptor was essential for this association. Overall, the suggestion from these findings is that tyrosine phosphorylation may alter the functional interaction of the GAP protein with Ras and in this way provide the link between growth factor receptor/tyrosine kinases and Ras. One obvious possibility is that the tyrosine phosphorylation of GAP would transiently remove its Ras-attenuating activity and thereby enable Ras-GTP complex to activate mitogenic signaling pathways. So far, however, there has been no indication that the tyrosine phosphorylation of the Ras-GAP has any a&t on its ability to stimulate Ras-GTPase activity. An alternative is that the tyrosine phosphoryiation of GAP causes it to interact with other proteins, rather than with Ras, thereby allowing F&U to remain in an active conformation for a longer period of time. Some support for this notion comes from the findings by Ellis et al. [12] that the Ras-GAP can associate with two other cellular phosphoproteins (molecular mass of approximately 62 kD and 190 kD) following tyrosine phosphorylation. Regulation
of Ras-CAP
upon
T-cell activation
Downward and colleagues [13**] recently published an interesting report suggesting the possible regulation of Ras-GAP activity in T cells by protein kinase C. The initial observation made by these investigators was that while
the Ras protein is almost entirely GDP-bound in untreated T cells, the addition of either the T-cell activating agent, phytohemagglutinin, or a CD3-specilic monoclonal antibody, caused a marked stimulation in the amount of GTP bound to Ras. The investigators were able to show conclusively that the increase in the amount of the GTP-bound Ras species was due to the activation of protein kinase C. Given these observations, two mechanisms were possible. One involved an increase in the rate of GDP-GTP exchange on Ras, while the other involved an inhibition of the Ras-GAP activity. The former possibility was eliminated by the finding that the rate of binding of [c@*P]GTP was rapid and identical in permeabilized cells whether or not these cells were treated with phorbol esters. The Ras-GAP activity, however, was inhibited as much as sixfold in cell lysates derived from phorbol ester-treated cells compared with the GAP activity of untreated cells. Thus, these results suggest that T-cell activation, through the stimulation of protein kinase C, inhibits the Ras-GAP activity and thereby promotes a more persistent activation of the Ras protein. While the simplest mechanism would be a direct phosphorylation of the Ras-GAP by protein kinase C, such a phosphorylation was not detected, leading the investigators to propose that protein kinase C may be phosphorylating (and stimulating) an inhibitor of the Ras-GAP. Relationship between type 7 gene product
Ras-CAP
and the neurofibromatosis
Neurolibromatosis type 1 (NF-1) is an autosomal dominant genetic disease that affects the peripheral nervous system and is characterized by cutaneous pigmental changes and neurofibromas. Recently, three consecutive reports in Cell [ 14**-16**], demonstrated the existence of a structural and functional homology between the Ras-GAP, the yeast IRA proteins, and the neurofibromatosis (NF-I) gene product. Sequence similarities (approximately 30%) exist between a 360.amino-acid region of the NF-1 protein, the catalytic domains of the mammalian GAPS, and the essential domain of the yeast IRA proteins. Significant sequence similarity also appears to exist between NF-1 and IRA-1 in regions that extend beyond the GAP-related domains, thereby suggesting that the NF-1 protein may be more closely related to the IRA-1 protein than it is to the Ras-GAP. The GAPrelated domain of M-1 stimulates the GTPase activity of the normal Ras protein but not the GTPase activity of oncogenic mutants of Ras; this is similar to the case for the Ras-GAP. NF-1, however, appears to bind Ras 20 times more tightly than does Ras-GAP, whereas Ras-GAP has a 30-fold higher specific activity. The observation that the NF-1 protein stimulates Ras-GTPase leads to an obvious suggestion regarding the pathogenesis of neurofibromatosis; specifically, the M-1 protein deactivates Ras molecules in normal cells from which the neurofibromas are derived, but is unable to catalyze Ras deactivation in neurofibromas, thereby accounting for the development of these tumors. It is likely that future lines of investigation will be directed toward the examination of this hypothesis in addition to determining whether NF1 is a phosphosubstrate for tyrosine kinases and if phosphorylation is able to regulate this protein.
The Ras superfamilies Other
CAPS
It is now felt that the Ras-GAP, NF-1, and the IRAs are members of what may be a large superfamily of GTPaseactivating proteins. GAPS for the Rap-1A and Smg p21 (Rap-1B) proteins have been purilied [17,18] and a partially puriiied preparation of a GAP activity for the Rho protein has also been described [19]. It is interesting that the Rho-GAP appears to have a molecular massof approximately 30 kD. This low-molecular-mass GAP may be the prototype of a distinct subgroup of the GAP superfamily, as we have partially purilied a low-molecularmass (approximately 25 kD) GAP activity for the human platelet CDC42Hs (Gp/G25K) protein. In the future, it will be interesting to determine whether the high and low molecular mass GAPScan be subgrouped on the basis of common functional properties. Exchange proteins for Ras Role of tyrosine kinases in GDP-UP
exchange Given the key role that Ras appears to play in mitogenie signaling pathways, a common belief has been that growth factor receptors are able to elicit the activation of Ras molecules by catalyziig GDPGTP exchange. Three reports appeared during the past year which supported such a suggestion. Firstly, Satoh et al. [20] used a Swiss 3T3 cell line that overexpresses c-Ha-?-us to show that more GTP-bound Ras complexes were detected in normal cells relative to quiescent cells. When the quiescent cells were stimulated with fetal bovine serum to promote DNA synthesis, the amount of Ras-GTP was increased twofold. This effect could be fully mimicked by the addition of PDGF, whereas the addition of bombesin plus insulin, which also induce DNA synthesis, did not elicit any changes in the amount of GTP-bound Ras. In a subsequent study performed by the same group [21], the EGF receptor tyrosine kinase and the e&B2/neu and V-S-C gene products were also shown to increase the amount of GTP-bound Ras, in a manner essentially identical to the stimulation by PDGF. Gibbs and colleagues [ 221 have also reported very similar results in NIH 3T3 cells transformed with either the V-PC or the v-ubl oncogenes. At this point, the key question is how do these different tyrosine kinases, which presumably exist at different points within a mitogenic signaling pathway, or perhaps even within diIferent (parallel) pathways, stimulate an increase in the amount of Ras-GTP species. On the basis of the discussion on regulatory proteins, it is tempting to speculate that tyrosine phosphorylation of GAP is involved in these effects; that is, by directly or indirectly inhibiting GAP action on Ras, the amount of Ras-GTP is increased. It is also possible, however, that some or all of these tyrosine kinases are able to influence directly the protein(s) responsible for eliciting GDPGTP exchange. Characterization from Ras
of factors that stimulate
GDP dissociation
Three different research groups have now reported the identification of an activity that stimulates the dissociation of GDP from Ras. Wolfman and Macara [23**] first reported linding an apparent guanine nucleotide exchange activity for Ras in brain cytosol. This exchange activity
Evans, Hart, Cerione
was shown to be sensitive to heat and to require the presence of phosphatase inhibitors. The activity was observed with v-Ha-Ras as well as with c-Ha-Ras, but it was not able to elicit the dissociation of GDP from another low-molecular-mass GTP-binding protein, Rab-3A The dose-response curve for the cytosolic exchange activity was sharply sigmoidal with a Hill coefficient of 3.2, lndleating that the interaction between Ras and the exchange factor was highly co-operative. Gel filtration experiments suggested that the exchange factor had a molecular size of loo-16okD. Downward, Weinberg and colleagues also reported the identillcation of a putative exchange activity for Ras [ 24.1, in this case, from human placenta. These investigators partially purified the activity through a series of chromatographic steps and found that the activity eluted through the gel filtration step with an apparent molecular mass of 60 kD. A number of mutant forms of Ras were examined for their ability to interact with the exchange factor. In most cases, the mutants were also responsive to the exchange activity, although two mutants (where amino acid substitutions occurred at positions 59 or 61) showed greatly reduced sensitivity to the exchanger. Most recently, Huang, Kung, and Kamata [25**] reported the purification of an exchange factor to near homogeneity. Unlike the activities described above, this exchange activity was identised in brain membranes rather than in the cytosol. The purified exchange factor migrated on sodium dodecyl sulphate polyacrylamlde gels with an apparent molecular mass of 35 kD; however, during an earlier gel llltration step, it eluted with an apparent mass of 100 kD. This latter result was similar to that reported by Wolfman and Macara [23**] and suggests that the exchange factor may be associated with other cellular proteins. At present, the relationship between the different exchange factors that have been described for the Ras protein is unknown. Given that the exchange factor purified by Huang and colleagues [25”] was able to stirnulate the GDP-GTP exchange activity of a number of low-molecular-mass GTP-binding proteins in addition to c-Ha-Ras, namely, the R&s, Rho, Rap-14 and Rab-1B proteins, suggests that it may represent a distinct protein from the factor identified by Wolf&n and Macara [23**] (which did not work with Rab3B). Some lmportam questions for the future include whether there is a family of exchange proteins similar to the family of GAP, which show different degrees of speclhcity for the various low-molecular-mass GTP-binding proteins and/or whether the localization of a specific exchange protein, or its interaction with other cellular proteins, a&cts its functional properties? It will be of interest to determine whether phosphorylation or other types of post-translational modifications have key roles ln the regulation of exchange activities, as well as to determine the mechanisms by which the exchange activities are initially activated by external stimuli. GDP-dissociation
inhibitory
proteins
Recently, Takai and his colleagues [26@*,27-291 have identiiied another potential class of regulatory proteins
187
188
Cell regujation
for low-molecular-mass GTP-binding pr&eins; speciiitally, proteins that inhibit the dissociation of GDP, i.e. GDP-dissociation inhibitors (GDIs)). Sasaki et al [26-l first reported the puriiication of a GDI for smg p25A &b&4), a GTP-binding protein from bovine brain cyto&l. The smg p25A protein is present in high abundance in brain where it is mainly localized in presynaptic plasma membranes and vesicles, possibly playing a role in secretory processes. The purified GDI, which has an apparent molecular mass of approximately 60 kD, appears speciIically to block the dissociation of [3H]GDP from smg p25A, while having no effect on the dissociation of other guanine nucleotides such as [%]GTPyS. Takai and his colleagues [ 27.1 have cloned the cDNA for this GDI from a bovine brain cDNA library and showed that it codes for a protein of molecular mass of approximately 55kD. Interestingly, the mRNA for this protein was found in a number of tissues that do not contain the message for smg p25A, suggesting that this GDI may Interact with other GTP-binding proteins. Thus far, it has been shown that the put-&d GDI will not inhibit the dissociation of GDP from c-Ras, Rho-12, or the Rap-1B proteins; however, these investigators have found that a 25 kD GTP-binding protein from human platelets, which is not smg p25A, will interact with the smg p25A-GDI. An obvious related question is whether other types of GDIs exist. It is likely that there are other types, and in fact Ueda, Takai and coworkers [28] have recently identilled a second GDI protein that interacts with a liver cytosol 24kD GTP-binding protein, and which appears to be structurally similar to, although distinct from, the smg p25A-GDI. These investigators have also very recently puriiied and cloned a GDI protein that appears to be speciiic for the Rho GTP-binding proteins [29]. It is interesting that the apparent molecular mass of the Rho-GDI protein (approximately 25 kD) is similar to, although slightly lower than, the apparent molecular mass of the Rho-GAP protein. A question of some interest is what specific roles the GDI proteins play in the functions of low-molecular-mass GTP-binding proteins? Takai and his colleagues have already started to answer this question for the smg p25A protein [30*] on the basis of the results of localization studies. It appears that the smg p25A-GDI protein forms a 1:l complex with the GDP-bound smg p25A and will not bind to either the GTP-bound protein nor to a guanine nucleotide-depleted protein. This GDI-GTP-binding protein complex appears to inhibit the association of the GTP-binding protein with the membrane. This makes it tempting to speculate that the GDI protein interacts with the GTP-binding protein at its carboxyl terminus (i.e. at the fourth cysteine from the carboxyl terminus), where fatty acid modifications have been suggested to be essential for the association of these GTP-binding proteins with the membrane. The competition by the GDI protein for the carboxy-terminal site may result in the dissociation of the GTP-biding protein from the membrane surface and its ultimate localization in the cytosol. Some type of regulatory signal could then be involved in releasing the GDI protein from the GTP-binding protein and enabling the latter to reassociate with the membrane.
Post-translational modifications motif and in the hypervariable
at the CAAX region
To date, the majority of the studies that have addressed the nature and importance of the modiiications that occur post-translationally at the carboxyl terminus of this superfamily of proteins have focused on the Ras proteins. Clearly, the valuable information derived from such studies will be directly applicable to the other family members, but careful investigations will have to verify this as being the case. The carboxy-terminal CAAX motif of Ras proteins undergoes a triplet of post-translational modifications (step 1 processing) that are required for membrane association. The cysteine residue (Cys186) is alkylated by a polyisoprenoid (for Ras, this probably represents the addition of a Cysl5 farnesyl moiety but for the other family members this may not be the case- see later), the AAX amino acids are removed by proteolysis, and methyl ester&cation takes place probably at the or-carboxyl group of the new carboxy-terminal cysteine. These modiiications convert the primary translation product (pro-p21) into an intermediate form, c-p21 (cytosolic processed ~21). The latter form is signiiicantly more hydrophobic, associates weakly with cell membranes, but is found predominantly in the SlOO fraction. Step 2 processing comprises palmitoylation of cysteine residues within the hypervariable region of these proteins. Palmitoylation signiiicantly increases both the extent and avidity of membrane binding. Hancock and coworkers [31,32] have shown that both step 1 and step 2 modifications are required for the correct targeting of ~21 H&s to the plasma membrane. They have also shown that in ~21 K&s(B), which is not palmitoylated because there is no cysteine residue within the hypervariable domain, a polybasic domain (six consecutive lysine residues-positions 175-180) is essential for the plasma membrane targeting of this form of Ras. A similar positioning of groups of basic residues occurs in a number of the Ras-related proteins. So what, over the past year, has made us focus on these modifications in this raiew? Firstly, the involvement of metabolites of the mevalonate pathway (for reviews see [33,34,35**]) has sparked interest in the possibility that manipulation of this regulatory system may be of use in the treatment of certain forms of cancer. This fact has kindled great interest in the enzyme(s) responsible for the modiiications. Reiss et al. [36*] have reported the identification, purification and characterization of a famesylprotein transferase from rat brain, which transfers the farnesyl moiety from famesyl pyrophosphate to a cysteine in p21 Ras proteins. The enzyme was puriiled approximately 60 000-fold from rat brain cytosol through the use of a chromatography step based on the ability of the enzymes to bind to a hexapeptide containing the consensus sequence (CAAX) for famesylation. The puriIied enzyme migrated on gel iilnation chromatography with an apparent molecular mass of 7&100 kD. SDS-PAGEshowed two closely spaced (approximately 5OkD) protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as four residues, which contained the CAAX mo-
The Ras superfamilies
tif, and the same peptides acted as alternative substrates that competed with p21 Ras for famesyiation. Schaber et al [37*] have shown, using [sH]famesyl-PPi as a precursor and Escbericb~ coli-expressed Ras, that forms of Ras having the CAAX sequence are radiolabeled upon incubation with the cytosolic fraction of bovine brain. Forms of Ras having a deletion of the CAAX sequence or a Cys-to-Ser substitution in this sequence were not substrates. The enzyme responsible for this activity had a molecular mass of approximately 19OkD as estimated by gel liltration and required a divalent cation for activity. Manne et al [38*] have described similar enzyme activities capable of catalyzing the famesylation of unprocessed ~21 Ras proteins in vitro at the correct residue (Cys186). This activity was reported to be present in all mammalian cell lines and tissues which were tested. Gel filtration analysis of a partially purified preparation revealed two peaks of activity at 250-300 kD and SO-130 kD. Given these initial reports of purified and partially purified activities, we can clearly look forward in the coming year to the microsequencing of the proteins and cloning of the genes that encode these enzymes. The availability of these recombinant molecules should greatly assist in the screening for potential inhibitors of Ras oncogene function. Again, biochemical as well as genetical approaches are proving to be complementary. Schafer et al. [35**] have shown that in the yeast Succburomyces cerevt&e, the product of the DPRl-RAMI gene encodes an essential and limiting component of a protein prenyltransferase. As the yeast protein could also prenylate human Ha-Ras ~21, they speculate that the human DPRlRAM1 analogue may be a useful target for anticancer chemotherapy. Goodman et al. [39**] have further expanded upon these observations in that they have shown that another mutant, RAM2 was also defective in the transferase activity, hence demonstrating that at least two genes, DPRZ-RAMI and RAM2, are required for the farnesykransferase activity in yeast. Secondly, the molecular identification of the form of the modifying prenyl group has demonstrated that certain proteins are famesylated whereas others are geranylgeranylated. Farnsworth ef al [40**] identified the novel isoprenoid modification by labeling HeLa cells with [3H]mevalonic acid and analysing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analysed by liquid chromatography and mass spectrometry and the approach revealed an all frunsgeranylgeranyl group as a major isoprenoid modification. Rilling et al. [41**] demonstrated similar findings from the mevalonate-derived portion of prenylated proteins from Chinese hamster ovary (CHO) cells. Rilling and colleagues [42] went on to demonstrate that approximately 10% of the radioactive products released from labeled extracts of CHO cells showed the same chromatographic properties as geranylgeranylcysteine. Kawata et al [43*] have provided evidence that the smg p21B (Rap-1B) protein purified from human platelets is modified by geranylgeranyIation. Similarly, Yamane et al [44*] have shown that the membrane-bind-
Evans, Hart, Cerione
ing domain of the CDC42H, (Gp/G25K) protein contains an all fruns geranylgeranyl cysteinyl methyl ester at its carboxyl terminus. The biological significance of these different prenyl modifications may be determined in he coming year(s). The structural information that governs whether a protein is famesylated or geranylgemnylated should be forthcoming as increased numbers of studies identify the modifying prenyi group of specifk proteins. To go hand in hand with this, we anticipate that the enzyme(s) responsible for such modifications will be purified and cloned. Clearly, we have much to discover about the enzymology and the biological meaning of the intricate ways in which the cell chooses to modify this important superfamily of proteins. Conclusions What do we have to look forward to in the coming year? It seems likely that we will see the publication of studies identifying, purifying, and cloning novel regulatory proteins that will further extend our knowledge of what regulates the activities of the Ras superfamily of proteins. It is also likely that the enzymes responsible for the posttranslational modiEcations that are crucial for the biological function will be more fully understood. We can also hope to see great strides in our understanding of the biological processes in which the various members of the superfamily participate. References
and recommended
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1.
HALL A: The Cellular Functions of Small GTP-Binding teins. Nature 1930, 24963-O.
2.
BOURNEHR, SANDER DA MCCORMICKF: The GTPase Superfaimly: a Conserved Switch for Diverse Cell Functions. Nalure 1990, 348:12%132.
3.
CHARDtNP, SANDERC, WlTllNGHOFERA: The Ras h-OteitI Family: Evolutionary Tree and the Role of Conserved Amino Acids. Bimbemishy 1991, in press.
4.
MCCORMICKF: Ras GTPase Activating Protein: Sii mitter and Signal Terminator. Cell 1989, 565-8.
5.
HAU k Ras and GAP 1990, 61:921-923.
6.
MCCORMICKF: The World According to GAP. Oncogene 1990, 5:1281-1283.
7.
WIGUR MH: GAPs in Understanding 346:696-697.
8.
TANAKA K, ~VAKAFUKUM, SATOH T, M&~Au MS, GIBBS JB, MATSUMOTOK, KMRO Y, TOH-E A; S. cetwtsiae Genes IRA1 and IRA2 Encode Proteins that may be Functionally Equivalent to Mammalian Ras GTPase Activating Protein. C&1990, 60:8oHKI7.
Who’s Controlling
Pro-
Trans-
Whom? Cefl
Ras. Nature
1990,
MOKOY CJ, BOT’JARODP, FLEhit~c TP, MARSHALMS, Gmss JB, AARONSONSA PDGF Induction of Tyrosine Phosphorylation of GTPase Activating Protein. Nature 1989, 342:711-714. This represents the Erst report that the I&-GAP is phosphotylated in vi00 in response to growth factors (that is PDGF and EGF). 9. l
189
190
Cell regulation Saw DR, MORRISONDK, WONG G, MCCO&K F, W~llu~s IT: PDGF &Receptor Stimulates Tyroslne Phosphorylatlon of GAP and Association of GAP with a Siig Complex. Cd 1998, 61:125-133. This study ptesents the tirst convincing demonstration that the EasGAP is dhectly coupled to, and phosphotylated by, the PDGF receptor in vrvh Moreover, it proposes the existence of growth-factor receptorslgnahg complexes, which include the Ras-GAP, the Eaf-1 kinase. the phosphatidylinositol-3 kinase, and phosphollpase C-y. 10.
18.
Pouws PG, RUB[NFEU)B, EVANST, MCCORMICKF: PurlScation of a Plasma Membrane-Associated GTPase Activating Protein (GAP) Specilic for rapl/krev-1 from HL60 Cells. Proc Nat1 Acud Sci USA 1991, 88~239243.
19.
Gm MD, SERFAJ, 0~a.s CV, H~tt A: Identification of Diitinct Cytoplasmic Targets for Ras/R-Ras and Rho Regulatory Proteins. J Bid C%em 1989, 264:10-13.
..
20.
KUMJSKG A, Etus C, PAWSONT, CooPER JA Siding of GAP to Acthated PI&F Receptors. Science 1990, 247:1578-1581. kis paper describes the formation of a complex between the PDGF receptor and the Ras-GAP. The important lindings here include the demonstration that kinase-inactive PDGF-receptor mutants do not assodate with I&-GAP and that the mutation of either (or both) of the receptor autophosphotylation sites (Tyr751 and Tyr857) reduced the amount of associated GAP by 88%.
SATOH T, ENDO M, NAKAFUKU M, NAKAMuRAS, K,UIRO Y: Platelet-Derived Growth Factor Stimulates Formation of Active p2 lnrs. GTP complex in Swiss 3T3 Cells. Proc Nat1 Acad Sci USA 1990, 87~59935997.
21.
SATOH T, Ervno M, NAUFUKU M, AK~YAMAT, Ywcrro T, Kr\zr~o Y: Accumulation of ~21~. GTP ln Response to Stimulation with Epidermal Growth Factor and Oncogene Products with Tposine Kinase Activity. Proc Nat1 Acud Sci USA 1990, 87:79267929.
12.
22.
GIBBS JB, hrlw~~tt MS, SCO~N~CKEM, DEVON RF, VOGEL US: Modulation of Guanine Nucleotides Bound to Ras in NlH 3T3 Cells by Oncogenes, Growth Factors, and the GTPase Activating Protein (GAP). J Bid &em 1990, 265:20437-20442.
11.
Eun C, Mom M, MCCORMICKF, PAWSONT: Phosphotylation of GAP and GAP-Associated Proteins by Transforming the Mitogenic ‘Qrosine Kinases. N&u-e 1990, 343:377-381.
DO-m J, Gruvtis JD, Wm PH, EAYrER S, CANlREu DA: Stimulation of p21m upon T-Cell Activation. Nature 1990, 346:71!&723. This paper is of outstanding interest as it describes the direct involvement of the Ras protein ln T-cell activation. ln addition, the data indicate that the increase in the activation state of Eas, which accompanies T-cell activation, is the outcome of a reduction in the 8as GTPase activity. This reduction in GTPase activity appears to be the result of a protein kinase C-mediated phosphorylation of a regulatory component of the l&GAP.
WOEMAN A, MAW IG: A Cytosolic Proteins Catalyzes the 23. .. Release of GDP from ~21~. Science 1990, 247:67-69. Presents the first report of the identiiication of a cytosolic factor that catalyzes the dissociation of GDP from Eas. Such a factor could play an extremely important role in the regulation of Eas action since it presumably would be responsible for catalyzing the GDP-GTP exchange reaction and eliciting the actnation of Eas. Gel liltration indicated that this cytosolic factor has a molecular mass of 100-160 kD.
14. ..
24. .
13. ..
XU G, Lm B, TARA~~AK, DUNN D, WC0D D, GES~ELANDR, WHITE R, WEISS R, TAMANOI F: The Catalytic Domain of the Neurolibromatc&s ‘Qpe 1 Gene Product Stimulates Ras GTPsse and Complements ira Mutants of S. cereuisfue. Cell 1990, 63835841. This paper, and the two that follow it in the issue [15**,16**] report the spectacular findings that the f&s-GAP shares both structural and functional homology with the neurofibromatosis gene (NF- I) product. This study teports that the NFl product stimulates Ras GTPase activity and that the expression of the HF.1 domain that is homologous to Eas-GAP also complements ira- 1 and it-u.2 mutations in yeast m GA VISKOCHIUD, BOUAG G, MCCABEPC, CROS~!ZR WJ, 15. .. HAUBRUCKH, CONROY I, CLARK R, O’CONNEU P, CAVI?HON RM, Inns MA MCCORMICKF: The GAP-Related Domain of the Neurofibromatosis Type 1 Gene Product Interacts with Ras ~21. &I1 1990, 6384-9. The second publication in the series of three (see [14**,16**]) de scribing the functional homology between the l&s-GAP and the NF.1 gene product This report demonstrated that the NF-1 product had a 20-fold h&her alfinity for Eas than did the &s-GAP protein, while the specific activity for the NF I-stimulated Ras-GTPaseactivity was approximately 30 times lower than the Eas-GAP-stimulated activity. 16. ..
BAL~E~~ERR, MA~CHUK D, Boousio M, SAULINOA, ETCHER R, WIG~ERM, Cow F: The m-1 Locus Encodes a Protein Functionally Related to Mamma&an GAP and Yeast IRA Proteins. Cell 1990, 63851859. The third paper ln a series (see [14.*,15**]) repotting that the NF.1 gene codes for a protein structurally and functionally related to the RasGAP. Thls paper demonstrates that the NFI product shares extensive homology with the IRA.1 and IRA2 gene products of S. cereuisiue and that a segment of the NFl cDNA gene, when expressed in yeast, will complement the loss of IRA function and will inhibit both the wild-type and the mutant-activated human Ha-ras genes following their expression ln yeast. 17.
KBCUCHlA, Small T, ARAKl S, HATA Y, T,ucu Y: PurlScation and Characterization from Bovine Brain Cytosol of Two GTPaseAcththg Roteins Speciiic for smg ~21, a GTPBinding Rotein Having the Same Rffector Domain as c-Ras ~21s. J Biol C&m 1989, 264:913H136.
D~WMW~ARD J, R~HL E, WV L WEINBERGPA Identification of a Nucleotide Exchange-Promoting Activity for p21N. Prm Nat1 Acud Sci USA 1990, 87:5998&02. This report describes the identiiication of a nucleotide exchange-promoting activity for Ras in cytoplasmic extracts of human placenta. This activity was partially purified through a series of chromatographic steps, and gel titration indicated that it had a molecular mass of 68 kD. 25. ..
HUANG YK, KUNG H.F. KAMATA T: Purification of a Factor Capable of Stimulating the Guanine Nucleotide Exchange Reaction of Ras Proteins and its Effect on Ras-Related Small Molecular Mass G Rotelns. Prx NatI Acud Sci USA 1990, 878008-8012. This represents the third report of a factor capable of stimulating the guanine nucleotide exchange reaction of Eas. This is the only report, however, describing the complete pur&cation of such a factor, and in this case, the factor was isolated from membranes rather than from cytosol. On the basis of SDS-PAGE, the factor appeared to have a molecular mass of 35 kD. The ability of this factor to catalyze the dissociation of GDP from a number of 8as-related GTP-binding proteins suggests that it may represent a common regulatory component for these different proteins. 26. ..
Shyw T. K~KUCHIA, ARAKl S, HATA Y, BOMURA M, KURODA S, TM Y: Purification and Characterization from Bovine Brain Cytosol of a Rotein that Inhibits the Dissociation of GDP from the Subsequent Siding of GTP to smg p25A. a Ras p21-like GTP-Biding Protein. J Biol C!wm 1990, 265:23332337. The importance of this study stems from the fact that it introduces, for the first time, a third class of regulatory components for low-molecularmass GTP-binding proteins, namely the GDls. This report describes the purllication and characterization from brain cytosol of an activity that inhibits the dissociation of GDP from the smg p25A (Eab.3) protein. 27. .
Mmm Y, KIKUCHI A, Artuo S, HATA Y, KONDONJ, Txtu.rvtsm Y. Tm Y: Molecular Cloning and Characterization of a Novel Type of Regulatory Protein (GDI) for smg p25A, a Ras pal-Like GTP-Binding Protein. Mol and Cell Bid 1990, 10:411’+4122. This report describes the lirst molecular cloning of a GDI protein. The cDNA for this GDI (which works on the smg p25A, or Rab3, protein) encodes a protein of molecular weight of approximately 50 000 kD. The
The Ras superfamilies mRNA for this GDI was found In alI tissues examined (even in tissues that do not contain smg p25A), which suggests that this GDI protein may regulate other members of the low molecular mass GTP-biding protein famiIy. 28.
UEDA T, T~iozv,wi Y, Omotu T, OHVANAGI H, SA~T~HY, TAKA~ Y: Purification and Characterization from Rat liver Cytosol of a GDP Dissociation Inhibitor (GDI) for Liver 24 k G, a Ras pll-L&e GTP-Binding Protein, with Proper&s Siiar to Those of smg p25A GDI. Biochem~ 1991, 30:909-917.
29.
F~KUMOTOY, KAIBucttt K, Hots Y, Fu~lolcr H, Art~to S, UEDA 4 TAKA~ Y: Molecular Cloning and Characterization of a Novel Type of Regulatory Protein (GDI) for the Rho Proteins, Ras p21-like SmaR GTP-Binding Proteins. T, mUCHI
Chacqene
1990,
51321-1328.
30. .
Arwa S, Kmum A, ETA Y, MMURA M, TM Y: Regulation of Reversible Biiding of smg p25A, a Ras p21-l&e GTP-BiidIng Protein, to Synaptic Plasma Membranes and Vesicles by its Specific ReguIatoty Protein, GDP Dissociation Inhibitor. J Bill them 1990, 265:13007-13015. This paper describes Important and new Information regarding the role of the GDI protein for smg p25A SpecificalIy, this paper shows that both the GTP-bound and GDP-bound forms of smg p25A bind reversibIy to synaptic membranes and that the GDI binds SelectiveIy to the GDP-bound fonn of the protein and causes it to dissociate from the membrane surface. JF, MAGEE AJ, CH~LOSJE, MARSHAU. CJ: AR Ras Proteins are PoIyisoprenyIated but only Some are PaImitoyhued. Cell 1989, 5731167-1177.
31.
Wcoc~
32.
HANCOCK JF, PATERSON H, MhRswu JF: A Polybasic Domain or PaImItoyIation is Required in Addition to the CMX Motif to LocaRze p2lRa.s to the Plasma Membrane. Cell 1990, 63:133-139.
33.
GOWIN JL, BROWN MS: Regulation of the MevaIonate Pathway. Nature 1998, 343425-430.
34.
SCHAFER WR, Km II, STERNE R, THORNER J, KIM SH,
RWE J: Ge-
netic and PhannacologicaI Suppression of Oncogenic Mutations in RAS Genes of Yeast and Humans. Science 1989, 245:37!&385. 35. ..
SCHAFERWE, TRUEBLOODCE, YANG C-C. MAYER MP, ROSENBERG S, POULTERCD, K1M S-H, Rw J: Enzymatic Coupling of Cholesterol Intermediates to a Mating Pheromone Precursor and to the Ras Protein. Science 1990, 249~11331139. Demonstrates that mutations in the yeast DPRl-l+LMl gene causes a defect in the prenylation reaction. Implies that as the yeast protein could ako prenyiate human Ha&s p21 precursor, the human DPRl-RAM1 analogue may be a usefuI target for anticancer chemotherapy. 36. .
REM Y, Gomm JI SEABE MC, CA%% PJ, BROWNMS: Inhibition of Puritied p2 1Ras FamesyI: Protein Transferase by Cys-MX Tetrapeptides. Cell 1990, 62:8188. IdentiIicatIon, purikation, and characterization of a famesyl : protein transferax from rat brain cytosol. Competitive inhibition of enzyme activity by peptides as short as four residues that contained the Cys-AAX motif.
37. .
SCHABERMD, O’HA~A MB, JD, Mocwi SL, MARsw JB: Polyisoprenylation of Transferaw. / Biol &em Identification and pattial putication
from
bovine
brain
cytosol.
G.utw VM, MOUSERSD, BERGSTROM MS, Frunti PA, DIXON RAF, Gmas Ras In Vfrro by a Famesyl-Protein
1990, 265:14701-14704. of a famesyl transfemse Demonstration that deletion of
activity
the CAAX
Evans, Hart, Cerione
motif or a Cys-to Ser-mutation rendered the E cokxpressed tein, not a substrate.
Ras pro-
MANNEV, ROBERTSD, TOB~NA, O’ROURKEEO, DE VRG~O M, MEyEas C, AHMED N, Kuaz B, Rest M, KUNG H-F, B-to M: Identification and PreRminary Characterization of ProteinCystelne Farnesyltransferase. Pnx Nat1 Acud Sci USA 1990, 877541-7545. Description of enzyme activities capable of catahzing the farnesylation of unprocessed Ras p21 proteins in &r-o at the correct residue (Cyslf36). Implies that the in oftm assay will be useful in screening for potential inhibitors of the rus oncogene function. GOODMANLE, JUDD SR, F,uwsworo?t CC, POWERSS, GE~B MI-I, 39. .. Gmwxr JA TAMANOI F: Mutants of Sac&aromy#s cerw vfdae Defective in the Famesylation of Ras Proteins. Proc Nat1 Acud Sci USA 1990, 87966W669. Identification in crude soluble extracts of yeast ceUs of an acdvity that cataIyzes the transfer of a fame@ moiety from famesyl pyrophosphate to the yeast Ras-2 protein. A yeast mutant, DPRl-RAMl, Is shown to be defective in famesyhransfemse activity. AddItionaUy, another mutant, RAM-2, is shown to be defective in the transferax activity. Implies that at least two genes are required for the farnesyItransferase activity in yeast. 38. .
40. ..
F~twsworrm CC, GELB MI-I, GLOMSETJA: Identlftcation of GeranyIgeranyl-Mod&d Proteins in HeLa CeIIs. Science 1990,
247:32&322.
Identification of a novel Isoprenoid modikation, aII frans geranyIgeranyi, by labeling HeIa ceUswith [ 3H] mewIonIc acid and anaiysii proteotytic digests of total ceU protein. 41. ..
WG HC, BREUNGERE, Efi?-snr WW, Cruw PF: PrenyIated Proteins: the Structure of the Isoprenoid Group. Science 1990, 247:318320. IdentiIication of the mevalonate-derived portion of a prenybted protein from CHO cells as diterpenoid (C20). linkage of the group to a carboxy-terminal cysteine as a thioether. 42.
E~%-!?nuWW, LEVER DC, RUNG HC: RenyIated Proteins: Synthesis of GeranyIgeranylcystelne and Identltlcation of this Thioetber Amino Acid as a Component of Roteins in CHO Cells. Proc Nat1 Acad Sci USA 1990, 87:7352-7354.
KAWATA M, FARNSWORTH CC, YOSHIDAY, GELB MI-I, G~~MSET JA, TAKA~Y: Post-TransIationalIy Processed Structure of the Human Platelet Protein smg ~218: Evidence for GeranyIgeranykttion and CarboxyI MethyIation of the C-Terminal Cysteine. Pnx Nat1 Acud Sci USA 1990, 87:8960+X%. Results indicating that the smg p21B (RaplB) protein puriIied from human platelet membranes is post-transIationaIIy mod&d by geranyi geranyiation of Cys181. First demonstration of such a modification of one of the Ras superfamiIy members. 43. .
44. .
YAMANE HK, F~swOtt’rtt CC, m H, EVANS T, HoW~tn W, GEIB MH, Gt.o~ssr JA, CUW
T Evans, Department of CeII Biology, Genentech Inc., South San Francisco, California 94080, USA MJ Hart and RA Cerione, Departments of Biochemistry, CeII and Mokular Biology, and Department of Pharmacology, Schurman HaII, Cornell University, Ithaca, New York 14853, USA.
191
Inc., South San Francisco,
J. Hart and Richard
California,
and Cornell
The Ras-like CTP-binding proteins comprise that play key roles in a wide variety cell growth, differentiation, secretion, and past few years, it has become clear that regulated by a variety of manners, including of regulatory proteins and post-translational Current
Opinion
As a result of a frenzy of recent activity, both at the biochemical and the molecular genetic level, it is clear that multiple Pas-like GTP-binding proteins are expressed in a wide variety of cells and species. Now that the diversity and scope of the subfamilies (that is, the Ras family and its closest relatives, the Rho family and the Rab family see [l-3] for a more complete deiinition of the subfamily distinctions) are appreciated, the challenge that lies ahead is the dehnition of their biological functions. As the appropriate specilic reagents and experimental systems are generated and identihed, and as the pace of research hastens in this direction away from the mere cataloging of yet more family members, it should become possible to review the regulatory roles of the proteins in speciIic biological processes. That time, however, is not yet at hand and so, in this short commentary, we have decided to focus on the two aspects of the biology of these proteins that we believe represent the most active and exciting areas of current research. One of these is the regulatory proteins that have been identiIied primarily on the basis of their ability to iniluence some aspect of the GTP-bindingGTPa.se cycles of the low-molecularmass GTP-binding proteins. The other is the diversity of post-translational modifications that occur at the ‘CAAX motif and the hypervariable carboxy-terminal region of GTP-binding proteins that are known to be essential for the appropriate localization of the proteins and hence their biological function(s).
Regulatory
proteins
So far, three classes of regulatory proteins for the lowmolecular-mass GTP-binding proteins have been identified; these are the GTPase-activating proteins (GAPS), the
University,
Ithaca,
New York, USA
a large superfamily of proteins of cellular activities, including protein trafficking. During the these CTP-binding proteins are interactions with specific types modification events.
in Cell Biology
Introduction
A. Cerione
1991, 3:185-191
guanine nucleotide exchange proteins (that is, those that facilitate the exchange of GDP for GTP), and the GDPdissociation inhibitors. The GAPS have been by far the most extensively studied, with the Pas-GAP, in particular, having received a great deal of attention particularly because of the possible implication of its ineffective coupling to Pas in the development of certain human cancers. It is, however, reasonable to anticipate that over the next few years exciting new information will become available regarding the molecular features of each of the protein-protein interactions Involved in the regulation of the low-molecular-mass GTP-binding proteins. CTPase-activating
proteins
Since the initial discovery that a distinct protein, GAP, is responsible for stimulating the GTPase activity of Ras, an extensive effort has been directed towards delineating the molecular mechanisms by which this protein acts and how it is regulated. The human Pas-GAP is known to exist in two forms: the type I GAP (molecular mass of approximately 125 kD) appears to be ubiquitous while the type II GAP (molecular mass of approximately 1OOkD) that is generated by alternative splicing has only been found in human placenta (see [4-71 for a much more complete coverage of the structutal properties of the Ras GAP). Although yeast cells do not appear to contain any activity that stimulates the GTPase of mammalian Ras proteins, they do contain two proteins (the products of IRA 2 and IRA2) that show some sequence homology to the carboxy-terminal region of the Pas-GAP and are responsible for stimulating the GTPase activity of the yeast PAS-1 and PAS-2 proteins [8]. Phosphorylation
of Ras-CAP
There have been a number of suggestions regarding the involvement of the Pas-GTP-binding proteins in the sig-
Abbreviations CHO-Chinese
hamster
ovary;
EGkpidermal PDGF-platelet-derived SDS-PACE--sodium
growth
factor; CAP-GTPase-activating growth factor; SDS--sodium dodecyl sulphate polyacrylamide
@ Current
Biology
protein; CDI-CDP-dissociation dodecyl sulphate; gel electrophoresis.
Ltd ISSN 0955+&74
inhibitor;
185
186
Cell regutation
naling pathways of growth factor receptdrs. A clue towads understanding the interplay between growth factor receptors and the Ras proteins has been provided by a series of studies demonstrating the in viva (tyrosine) phosphotylation of the Ras-GAP. Molloy et al [9*], first reported that the addition of both the AA and BB homodimers of platelet-derived growth factor (PDGF) to quiescent fibroblasts elicited a rapid phosphorylation of the Ras-GAP on tyrosine residues. This was demonstrated by immunoprecipita&g phosphoproteins from cell lysates pretreated with PDGF, using antiphosphotyrosine antibodies, and then probing the immunoprecipitates with a specific anti-Ras GAP monoclonal antibody. A potentially important observation from these studies was that in quiescent cells, the Ras-GAPwas found predominantly in the cytosolic fraction, whereas following stimulation of the cells with PDGF, the tyrosine-phosphorylated Ras-GAPS appeared to be most abundant in the membrane. Neither insulin nor the basic fibroblast growth factor were able to elicit the in U~IKJ phosphorylation of the Ras-GAP, while epidermal growth factor (EGF) was able to mimic the effects of PDGF in these cells. A number of subsequent studies conlirmed these findings. For example, Kaplan et al [lo-] also reported that the PDGF receptor stimulated the tyrosine phosphorylation of the Ras-GAP in NIH 3T3 cells and provided evidence from immunoprecipitation experiments that phosphotylated Ras-GAP was capable of associating with a number of other proteins that have been suspected of playing a role in growth factor signaling, including the Raf-1 kinase, phospholipase C-y, and phosphatidylinositol-3 kinase. Kazlauskas et UI! [ 11.1 also reported the formation of complexes between the PDGF receptor and &-GAP in viva and proposed that the phosphoxylation of a specifk tyrosine residue on the PDGF receptor was essential for this association. Overall, the suggestion from these findings is that tyrosine phosphorylation may alter the functional interaction of the GAP protein with Ras and in this way provide the link between growth factor receptor/tyrosine kinases and Ras. One obvious possibility is that the tyrosine phosphorylation of GAP would transiently remove its Ras-attenuating activity and thereby enable Ras-GTP complex to activate mitogenic signaling pathways. So far, however, there has been no indication that the tyrosine phosphorylation of the Ras-GAP has any a&t on its ability to stimulate Ras-GTPase activity. An alternative is that the tyrosine phosphoryiation of GAP causes it to interact with other proteins, rather than with Ras, thereby allowing F&U to remain in an active conformation for a longer period of time. Some support for this notion comes from the findings by Ellis et al. [12] that the Ras-GAP can associate with two other cellular phosphoproteins (molecular mass of approximately 62 kD and 190 kD) following tyrosine phosphorylation. Regulation
of Ras-CAP
upon
T-cell activation
Downward and colleagues [13**] recently published an interesting report suggesting the possible regulation of Ras-GAP activity in T cells by protein kinase C. The initial observation made by these investigators was that while
the Ras protein is almost entirely GDP-bound in untreated T cells, the addition of either the T-cell activating agent, phytohemagglutinin, or a CD3-specilic monoclonal antibody, caused a marked stimulation in the amount of GTP bound to Ras. The investigators were able to show conclusively that the increase in the amount of the GTP-bound Ras species was due to the activation of protein kinase C. Given these observations, two mechanisms were possible. One involved an increase in the rate of GDP-GTP exchange on Ras, while the other involved an inhibition of the Ras-GAP activity. The former possibility was eliminated by the finding that the rate of binding of [c@*P]GTP was rapid and identical in permeabilized cells whether or not these cells were treated with phorbol esters. The Ras-GAP activity, however, was inhibited as much as sixfold in cell lysates derived from phorbol ester-treated cells compared with the GAP activity of untreated cells. Thus, these results suggest that T-cell activation, through the stimulation of protein kinase C, inhibits the Ras-GAP activity and thereby promotes a more persistent activation of the Ras protein. While the simplest mechanism would be a direct phosphorylation of the Ras-GAP by protein kinase C, such a phosphorylation was not detected, leading the investigators to propose that protein kinase C may be phosphorylating (and stimulating) an inhibitor of the Ras-GAP. Relationship between type 7 gene product
Ras-CAP
and the neurofibromatosis
Neurolibromatosis type 1 (NF-1) is an autosomal dominant genetic disease that affects the peripheral nervous system and is characterized by cutaneous pigmental changes and neurofibromas. Recently, three consecutive reports in Cell [ 14**-16**], demonstrated the existence of a structural and functional homology between the Ras-GAP, the yeast IRA proteins, and the neurofibromatosis (NF-I) gene product. Sequence similarities (approximately 30%) exist between a 360.amino-acid region of the NF-1 protein, the catalytic domains of the mammalian GAPS, and the essential domain of the yeast IRA proteins. Significant sequence similarity also appears to exist between NF-1 and IRA-1 in regions that extend beyond the GAP-related domains, thereby suggesting that the NF-1 protein may be more closely related to the IRA-1 protein than it is to the Ras-GAP. The GAPrelated domain of M-1 stimulates the GTPase activity of the normal Ras protein but not the GTPase activity of oncogenic mutants of Ras; this is similar to the case for the Ras-GAP. NF-1, however, appears to bind Ras 20 times more tightly than does Ras-GAP, whereas Ras-GAP has a 30-fold higher specific activity. The observation that the NF-1 protein stimulates Ras-GTPase leads to an obvious suggestion regarding the pathogenesis of neurofibromatosis; specifically, the M-1 protein deactivates Ras molecules in normal cells from which the neurofibromas are derived, but is unable to catalyze Ras deactivation in neurofibromas, thereby accounting for the development of these tumors. It is likely that future lines of investigation will be directed toward the examination of this hypothesis in addition to determining whether NF1 is a phosphosubstrate for tyrosine kinases and if phosphorylation is able to regulate this protein.
The Ras superfamilies Other
CAPS
It is now felt that the Ras-GAP, NF-1, and the IRAs are members of what may be a large superfamily of GTPaseactivating proteins. GAPS for the Rap-1A and Smg p21 (Rap-1B) proteins have been purilied [17,18] and a partially puriiied preparation of a GAP activity for the Rho protein has also been described [19]. It is interesting that the Rho-GAP appears to have a molecular massof approximately 30 kD. This low-molecular-mass GAP may be the prototype of a distinct subgroup of the GAP superfamily, as we have partially purilied a low-molecularmass (approximately 25 kD) GAP activity for the human platelet CDC42Hs (Gp/G25K) protein. In the future, it will be interesting to determine whether the high and low molecular mass GAPScan be subgrouped on the basis of common functional properties. Exchange proteins for Ras Role of tyrosine kinases in GDP-UP
exchange Given the key role that Ras appears to play in mitogenie signaling pathways, a common belief has been that growth factor receptors are able to elicit the activation of Ras molecules by catalyziig GDPGTP exchange. Three reports appeared during the past year which supported such a suggestion. Firstly, Satoh et al. [20] used a Swiss 3T3 cell line that overexpresses c-Ha-?-us to show that more GTP-bound Ras complexes were detected in normal cells relative to quiescent cells. When the quiescent cells were stimulated with fetal bovine serum to promote DNA synthesis, the amount of Ras-GTP was increased twofold. This effect could be fully mimicked by the addition of PDGF, whereas the addition of bombesin plus insulin, which also induce DNA synthesis, did not elicit any changes in the amount of GTP-bound Ras. In a subsequent study performed by the same group [21], the EGF receptor tyrosine kinase and the e&B2/neu and V-S-C gene products were also shown to increase the amount of GTP-bound Ras, in a manner essentially identical to the stimulation by PDGF. Gibbs and colleagues [ 221 have also reported very similar results in NIH 3T3 cells transformed with either the V-PC or the v-ubl oncogenes. At this point, the key question is how do these different tyrosine kinases, which presumably exist at different points within a mitogenic signaling pathway, or perhaps even within diIferent (parallel) pathways, stimulate an increase in the amount of Ras-GTP species. On the basis of the discussion on regulatory proteins, it is tempting to speculate that tyrosine phosphorylation of GAP is involved in these effects; that is, by directly or indirectly inhibiting GAP action on Ras, the amount of Ras-GTP is increased. It is also possible, however, that some or all of these tyrosine kinases are able to influence directly the protein(s) responsible for eliciting GDPGTP exchange. Characterization from Ras
of factors that stimulate
GDP dissociation
Three different research groups have now reported the identification of an activity that stimulates the dissociation of GDP from Ras. Wolfman and Macara [23**] first reported linding an apparent guanine nucleotide exchange activity for Ras in brain cytosol. This exchange activity
Evans, Hart, Cerione
was shown to be sensitive to heat and to require the presence of phosphatase inhibitors. The activity was observed with v-Ha-Ras as well as with c-Ha-Ras, but it was not able to elicit the dissociation of GDP from another low-molecular-mass GTP-binding protein, Rab-3A The dose-response curve for the cytosolic exchange activity was sharply sigmoidal with a Hill coefficient of 3.2, lndleating that the interaction between Ras and the exchange factor was highly co-operative. Gel filtration experiments suggested that the exchange factor had a molecular size of loo-16okD. Downward, Weinberg and colleagues also reported the identillcation of a putative exchange activity for Ras [ 24.1, in this case, from human placenta. These investigators partially purified the activity through a series of chromatographic steps and found that the activity eluted through the gel filtration step with an apparent molecular mass of 60 kD. A number of mutant forms of Ras were examined for their ability to interact with the exchange factor. In most cases, the mutants were also responsive to the exchange activity, although two mutants (where amino acid substitutions occurred at positions 59 or 61) showed greatly reduced sensitivity to the exchanger. Most recently, Huang, Kung, and Kamata [25**] reported the purification of an exchange factor to near homogeneity. Unlike the activities described above, this exchange activity was identised in brain membranes rather than in the cytosol. The purified exchange factor migrated on sodium dodecyl sulphate polyacrylamlde gels with an apparent molecular mass of 35 kD; however, during an earlier gel llltration step, it eluted with an apparent mass of 100 kD. This latter result was similar to that reported by Wolfman and Macara [23**] and suggests that the exchange factor may be associated with other cellular proteins. At present, the relationship between the different exchange factors that have been described for the Ras protein is unknown. Given that the exchange factor purified by Huang and colleagues [25”] was able to stirnulate the GDP-GTP exchange activity of a number of low-molecular-mass GTP-binding proteins in addition to c-Ha-Ras, namely, the R&s, Rho, Rap-14 and Rab-1B proteins, suggests that it may represent a distinct protein from the factor identified by Wolf&n and Macara [23**] (which did not work with Rab3B). Some lmportam questions for the future include whether there is a family of exchange proteins similar to the family of GAP, which show different degrees of speclhcity for the various low-molecular-mass GTP-binding proteins and/or whether the localization of a specific exchange protein, or its interaction with other cellular proteins, a&cts its functional properties? It will be of interest to determine whether phosphorylation or other types of post-translational modifications have key roles ln the regulation of exchange activities, as well as to determine the mechanisms by which the exchange activities are initially activated by external stimuli. GDP-dissociation
inhibitory
proteins
Recently, Takai and his colleagues [26@*,27-291 have identiiied another potential class of regulatory proteins
187
188
Cell regujation
for low-molecular-mass GTP-binding pr&eins; speciiitally, proteins that inhibit the dissociation of GDP, i.e. GDP-dissociation inhibitors (GDIs)). Sasaki et al [26-l first reported the puriiication of a GDI for smg p25A &b&4), a GTP-binding protein from bovine brain cyto&l. The smg p25A protein is present in high abundance in brain where it is mainly localized in presynaptic plasma membranes and vesicles, possibly playing a role in secretory processes. The purified GDI, which has an apparent molecular mass of approximately 60 kD, appears speciIically to block the dissociation of [3H]GDP from smg p25A, while having no effect on the dissociation of other guanine nucleotides such as [%]GTPyS. Takai and his colleagues [ 27.1 have cloned the cDNA for this GDI from a bovine brain cDNA library and showed that it codes for a protein of molecular mass of approximately 55kD. Interestingly, the mRNA for this protein was found in a number of tissues that do not contain the message for smg p25A, suggesting that this GDI may Interact with other GTP-binding proteins. Thus far, it has been shown that the put-&d GDI will not inhibit the dissociation of GDP from c-Ras, Rho-12, or the Rap-1B proteins; however, these investigators have found that a 25 kD GTP-binding protein from human platelets, which is not smg p25A, will interact with the smg p25A-GDI. An obvious related question is whether other types of GDIs exist. It is likely that there are other types, and in fact Ueda, Takai and coworkers [28] have recently identilled a second GDI protein that interacts with a liver cytosol 24kD GTP-binding protein, and which appears to be structurally similar to, although distinct from, the smg p25A-GDI. These investigators have also very recently puriiied and cloned a GDI protein that appears to be speciiic for the Rho GTP-binding proteins [29]. It is interesting that the apparent molecular mass of the Rho-GDI protein (approximately 25 kD) is similar to, although slightly lower than, the apparent molecular mass of the Rho-GAP protein. A question of some interest is what specific roles the GDI proteins play in the functions of low-molecular-mass GTP-binding proteins? Takai and his colleagues have already started to answer this question for the smg p25A protein [30*] on the basis of the results of localization studies. It appears that the smg p25A-GDI protein forms a 1:l complex with the GDP-bound smg p25A and will not bind to either the GTP-bound protein nor to a guanine nucleotide-depleted protein. This GDI-GTP-binding protein complex appears to inhibit the association of the GTP-binding protein with the membrane. This makes it tempting to speculate that the GDI protein interacts with the GTP-binding protein at its carboxyl terminus (i.e. at the fourth cysteine from the carboxyl terminus), where fatty acid modifications have been suggested to be essential for the association of these GTP-binding proteins with the membrane. The competition by the GDI protein for the carboxy-terminal site may result in the dissociation of the GTP-biding protein from the membrane surface and its ultimate localization in the cytosol. Some type of regulatory signal could then be involved in releasing the GDI protein from the GTP-binding protein and enabling the latter to reassociate with the membrane.
Post-translational modifications motif and in the hypervariable
at the CAAX region
To date, the majority of the studies that have addressed the nature and importance of the modiiications that occur post-translationally at the carboxyl terminus of this superfamily of proteins have focused on the Ras proteins. Clearly, the valuable information derived from such studies will be directly applicable to the other family members, but careful investigations will have to verify this as being the case. The carboxy-terminal CAAX motif of Ras proteins undergoes a triplet of post-translational modifications (step 1 processing) that are required for membrane association. The cysteine residue (Cys186) is alkylated by a polyisoprenoid (for Ras, this probably represents the addition of a Cysl5 farnesyl moiety but for the other family members this may not be the case- see later), the AAX amino acids are removed by proteolysis, and methyl ester&cation takes place probably at the or-carboxyl group of the new carboxy-terminal cysteine. These modiiications convert the primary translation product (pro-p21) into an intermediate form, c-p21 (cytosolic processed ~21). The latter form is signiiicantly more hydrophobic, associates weakly with cell membranes, but is found predominantly in the SlOO fraction. Step 2 processing comprises palmitoylation of cysteine residues within the hypervariable region of these proteins. Palmitoylation signiiicantly increases both the extent and avidity of membrane binding. Hancock and coworkers [31,32] have shown that both step 1 and step 2 modifications are required for the correct targeting of ~21 H&s to the plasma membrane. They have also shown that in ~21 K&s(B), which is not palmitoylated because there is no cysteine residue within the hypervariable domain, a polybasic domain (six consecutive lysine residues-positions 175-180) is essential for the plasma membrane targeting of this form of Ras. A similar positioning of groups of basic residues occurs in a number of the Ras-related proteins. So what, over the past year, has made us focus on these modifications in this raiew? Firstly, the involvement of metabolites of the mevalonate pathway (for reviews see [33,34,35**]) has sparked interest in the possibility that manipulation of this regulatory system may be of use in the treatment of certain forms of cancer. This fact has kindled great interest in the enzyme(s) responsible for the modiiications. Reiss et al. [36*] have reported the identification, purification and characterization of a famesylprotein transferase from rat brain, which transfers the farnesyl moiety from famesyl pyrophosphate to a cysteine in p21 Ras proteins. The enzyme was puriiled approximately 60 000-fold from rat brain cytosol through the use of a chromatography step based on the ability of the enzymes to bind to a hexapeptide containing the consensus sequence (CAAX) for famesylation. The puriIied enzyme migrated on gel iilnation chromatography with an apparent molecular mass of 7&100 kD. SDS-PAGEshowed two closely spaced (approximately 5OkD) protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as four residues, which contained the CAAX mo-
The Ras superfamilies
tif, and the same peptides acted as alternative substrates that competed with p21 Ras for famesyiation. Schaber et al [37*] have shown, using [sH]famesyl-PPi as a precursor and Escbericb~ coli-expressed Ras, that forms of Ras having the CAAX sequence are radiolabeled upon incubation with the cytosolic fraction of bovine brain. Forms of Ras having a deletion of the CAAX sequence or a Cys-to-Ser substitution in this sequence were not substrates. The enzyme responsible for this activity had a molecular mass of approximately 19OkD as estimated by gel liltration and required a divalent cation for activity. Manne et al [38*] have described similar enzyme activities capable of catalyzing the famesylation of unprocessed ~21 Ras proteins in vitro at the correct residue (Cys186). This activity was reported to be present in all mammalian cell lines and tissues which were tested. Gel filtration analysis of a partially purified preparation revealed two peaks of activity at 250-300 kD and SO-130 kD. Given these initial reports of purified and partially purified activities, we can clearly look forward in the coming year to the microsequencing of the proteins and cloning of the genes that encode these enzymes. The availability of these recombinant molecules should greatly assist in the screening for potential inhibitors of Ras oncogene function. Again, biochemical as well as genetical approaches are proving to be complementary. Schafer et al. [35**] have shown that in the yeast Succburomyces cerevt&e, the product of the DPRl-RAMI gene encodes an essential and limiting component of a protein prenyltransferase. As the yeast protein could also prenylate human Ha-Ras ~21, they speculate that the human DPRlRAM1 analogue may be a useful target for anticancer chemotherapy. Goodman et al. [39**] have further expanded upon these observations in that they have shown that another mutant, RAM2 was also defective in the transferase activity, hence demonstrating that at least two genes, DPRZ-RAMI and RAM2, are required for the farnesykransferase activity in yeast. Secondly, the molecular identification of the form of the modifying prenyl group has demonstrated that certain proteins are famesylated whereas others are geranylgeranylated. Farnsworth ef al [40**] identified the novel isoprenoid modification by labeling HeLa cells with [3H]mevalonic acid and analysing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analysed by liquid chromatography and mass spectrometry and the approach revealed an all frunsgeranylgeranyl group as a major isoprenoid modification. Rilling et al. [41**] demonstrated similar findings from the mevalonate-derived portion of prenylated proteins from Chinese hamster ovary (CHO) cells. Rilling and colleagues [42] went on to demonstrate that approximately 10% of the radioactive products released from labeled extracts of CHO cells showed the same chromatographic properties as geranylgeranylcysteine. Kawata et al [43*] have provided evidence that the smg p21B (Rap-1B) protein purified from human platelets is modified by geranylgeranyIation. Similarly, Yamane et al [44*] have shown that the membrane-bind-
Evans, Hart, Cerione
ing domain of the CDC42H, (Gp/G25K) protein contains an all fruns geranylgeranyl cysteinyl methyl ester at its carboxyl terminus. The biological significance of these different prenyl modifications may be determined in he coming year(s). The structural information that governs whether a protein is famesylated or geranylgemnylated should be forthcoming as increased numbers of studies identify the modifying prenyi group of specifk proteins. To go hand in hand with this, we anticipate that the enzyme(s) responsible for such modifications will be purified and cloned. Clearly, we have much to discover about the enzymology and the biological meaning of the intricate ways in which the cell chooses to modify this important superfamily of proteins. Conclusions What do we have to look forward to in the coming year? It seems likely that we will see the publication of studies identifying, purifying, and cloning novel regulatory proteins that will further extend our knowledge of what regulates the activities of the Ras superfamily of proteins. It is also likely that the enzymes responsible for the posttranslational modiEcations that are crucial for the biological function will be more fully understood. We can also hope to see great strides in our understanding of the biological processes in which the various members of the superfamily participate. References
and recommended
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1.
HALL A: The Cellular Functions of Small GTP-Binding teins. Nature 1930, 24963-O.
2.
BOURNEHR, SANDER DA MCCORMICKF: The GTPase Superfaimly: a Conserved Switch for Diverse Cell Functions. Nalure 1990, 348:12%132.
3.
CHARDtNP, SANDERC, WlTllNGHOFERA: The Ras h-OteitI Family: Evolutionary Tree and the Role of Conserved Amino Acids. Bimbemishy 1991, in press.
4.
MCCORMICKF: Ras GTPase Activating Protein: Sii mitter and Signal Terminator. Cell 1989, 565-8.
5.
HAU k Ras and GAP 1990, 61:921-923.
6.
MCCORMICKF: The World According to GAP. Oncogene 1990, 5:1281-1283.
7.
WIGUR MH: GAPs in Understanding 346:696-697.
8.
TANAKA K, ~VAKAFUKUM, SATOH T, M&~Au MS, GIBBS JB, MATSUMOTOK, KMRO Y, TOH-E A; S. cetwtsiae Genes IRA1 and IRA2 Encode Proteins that may be Functionally Equivalent to Mammalian Ras GTPase Activating Protein. C&1990, 60:8oHKI7.
Who’s Controlling
Pro-
Trans-
Whom? Cefl
Ras. Nature
1990,
MOKOY CJ, BOT’JARODP, FLEhit~c TP, MARSHALMS, Gmss JB, AARONSONSA PDGF Induction of Tyrosine Phosphorylation of GTPase Activating Protein. Nature 1989, 342:711-714. This represents the Erst report that the I&-GAP is phosphotylated in vi00 in response to growth factors (that is PDGF and EGF). 9. l
189
190
Cell regulation Saw DR, MORRISONDK, WONG G, MCCO&K F, W~llu~s IT: PDGF &Receptor Stimulates Tyroslne Phosphorylatlon of GAP and Association of GAP with a Siig Complex. Cd 1998, 61:125-133. This study ptesents the tirst convincing demonstration that the EasGAP is dhectly coupled to, and phosphotylated by, the PDGF receptor in vrvh Moreover, it proposes the existence of growth-factor receptorslgnahg complexes, which include the Ras-GAP, the Eaf-1 kinase. the phosphatidylinositol-3 kinase, and phosphollpase C-y. 10.
18.
Pouws PG, RUB[NFEU)B, EVANST, MCCORMICKF: PurlScation of a Plasma Membrane-Associated GTPase Activating Protein (GAP) Specilic for rapl/krev-1 from HL60 Cells. Proc Nat1 Acud Sci USA 1991, 88~239243.
19.
Gm MD, SERFAJ, 0~a.s CV, H~tt A: Identification of Diitinct Cytoplasmic Targets for Ras/R-Ras and Rho Regulatory Proteins. J Bid C%em 1989, 264:10-13.
..
20.
KUMJSKG A, Etus C, PAWSONT, CooPER JA Siding of GAP to Acthated PI&F Receptors. Science 1990, 247:1578-1581. kis paper describes the formation of a complex between the PDGF receptor and the Ras-GAP. The important lindings here include the demonstration that kinase-inactive PDGF-receptor mutants do not assodate with I&-GAP and that the mutation of either (or both) of the receptor autophosphotylation sites (Tyr751 and Tyr857) reduced the amount of associated GAP by 88%.
SATOH T, ENDO M, NAKAFUKU M, NAKAMuRAS, K,UIRO Y: Platelet-Derived Growth Factor Stimulates Formation of Active p2 lnrs. GTP complex in Swiss 3T3 Cells. Proc Nat1 Acad Sci USA 1990, 87~59935997.
21.
SATOH T, Ervno M, NAUFUKU M, AK~YAMAT, Ywcrro T, Kr\zr~o Y: Accumulation of ~21~. GTP ln Response to Stimulation with Epidermal Growth Factor and Oncogene Products with Tposine Kinase Activity. Proc Nat1 Acud Sci USA 1990, 87:79267929.
12.
22.
GIBBS JB, hrlw~~tt MS, SCO~N~CKEM, DEVON RF, VOGEL US: Modulation of Guanine Nucleotides Bound to Ras in NlH 3T3 Cells by Oncogenes, Growth Factors, and the GTPase Activating Protein (GAP). J Bid &em 1990, 265:20437-20442.
11.
Eun C, Mom M, MCCORMICKF, PAWSONT: Phosphotylation of GAP and GAP-Associated Proteins by Transforming the Mitogenic ‘Qrosine Kinases. N&u-e 1990, 343:377-381.
DO-m J, Gruvtis JD, Wm PH, EAYrER S, CANlREu DA: Stimulation of p21m upon T-Cell Activation. Nature 1990, 346:71!&723. This paper is of outstanding interest as it describes the direct involvement of the Ras protein ln T-cell activation. ln addition, the data indicate that the increase in the activation state of Eas, which accompanies T-cell activation, is the outcome of a reduction in the 8as GTPase activity. This reduction in GTPase activity appears to be the result of a protein kinase C-mediated phosphorylation of a regulatory component of the l&GAP.
WOEMAN A, MAW IG: A Cytosolic Proteins Catalyzes the 23. .. Release of GDP from ~21~. Science 1990, 247:67-69. Presents the first report of the identiiication of a cytosolic factor that catalyzes the dissociation of GDP from Eas. Such a factor could play an extremely important role in the regulation of Eas action since it presumably would be responsible for catalyzing the GDP-GTP exchange reaction and eliciting the actnation of Eas. Gel liltration indicated that this cytosolic factor has a molecular mass of 100-160 kD.
14. ..
24. .
13. ..
XU G, Lm B, TARA~~AK, DUNN D, WC0D D, GES~ELANDR, WHITE R, WEISS R, TAMANOI F: The Catalytic Domain of the Neurolibromatc&s ‘Qpe 1 Gene Product Stimulates Ras GTPsse and Complements ira Mutants of S. cereuisfue. Cell 1990, 63835841. This paper, and the two that follow it in the issue [15**,16**] report the spectacular findings that the f&s-GAP shares both structural and functional homology with the neurofibromatosis gene (NF- I) product. This study teports that the NFl product stimulates Ras GTPase activity and that the expression of the HF.1 domain that is homologous to Eas-GAP also complements ira- 1 and it-u.2 mutations in yeast m GA VISKOCHIUD, BOUAG G, MCCABEPC, CROS~!ZR WJ, 15. .. HAUBRUCKH, CONROY I, CLARK R, O’CONNEU P, CAVI?HON RM, Inns MA MCCORMICKF: The GAP-Related Domain of the Neurofibromatosis Type 1 Gene Product Interacts with Ras ~21. &I1 1990, 6384-9. The second publication in the series of three (see [14**,16**]) de scribing the functional homology between the l&s-GAP and the NF.1 gene product This report demonstrated that the NF-1 product had a 20-fold h&her alfinity for Eas than did the &s-GAP protein, while the specific activity for the NF I-stimulated Ras-GTPaseactivity was approximately 30 times lower than the Eas-GAP-stimulated activity. 16. ..
BAL~E~~ERR, MA~CHUK D, Boousio M, SAULINOA, ETCHER R, WIG~ERM, Cow F: The m-1 Locus Encodes a Protein Functionally Related to Mamma&an GAP and Yeast IRA Proteins. Cell 1990, 63851859. The third paper ln a series (see [14.*,15**]) repotting that the NF.1 gene codes for a protein structurally and functionally related to the RasGAP. Thls paper demonstrates that the NFI product shares extensive homology with the IRA.1 and IRA2 gene products of S. cereuisiue and that a segment of the NFl cDNA gene, when expressed in yeast, will complement the loss of IRA function and will inhibit both the wild-type and the mutant-activated human Ha-ras genes following their expression ln yeast. 17.
KBCUCHlA, Small T, ARAKl S, HATA Y, T,ucu Y: PurlScation and Characterization from Bovine Brain Cytosol of Two GTPaseAcththg Roteins Speciiic for smg ~21, a GTPBinding Rotein Having the Same Rffector Domain as c-Ras ~21s. J Biol C&m 1989, 264:913H136.
D~WMW~ARD J, R~HL E, WV L WEINBERGPA Identification of a Nucleotide Exchange-Promoting Activity for p21N. Prm Nat1 Acud Sci USA 1990, 87:5998&02. This report describes the identiiication of a nucleotide exchange-promoting activity for Ras in cytoplasmic extracts of human placenta. This activity was partially purified through a series of chromatographic steps, and gel titration indicated that it had a molecular mass of 68 kD. 25. ..
HUANG YK, KUNG H.F. KAMATA T: Purification of a Factor Capable of Stimulating the Guanine Nucleotide Exchange Reaction of Ras Proteins and its Effect on Ras-Related Small Molecular Mass G Rotelns. Prx NatI Acud Sci USA 1990, 878008-8012. This represents the third report of a factor capable of stimulating the guanine nucleotide exchange reaction of Eas. This is the only report, however, describing the complete pur&cation of such a factor, and in this case, the factor was isolated from membranes rather than from cytosol. On the basis of SDS-PAGE, the factor appeared to have a molecular mass of 35 kD. The ability of this factor to catalyze the dissociation of GDP from a number of 8as-related GTP-binding proteins suggests that it may represent a common regulatory component for these different proteins. 26. ..
Shyw T. K~KUCHIA, ARAKl S, HATA Y, BOMURA M, KURODA S, TM Y: Purification and Characterization from Bovine Brain Cytosol of a Rotein that Inhibits the Dissociation of GDP from the Subsequent Siding of GTP to smg p25A. a Ras p21-like GTP-Biding Protein. J Biol C!wm 1990, 265:23332337. The importance of this study stems from the fact that it introduces, for the first time, a third class of regulatory components for low-molecularmass GTP-binding proteins, namely the GDls. This report describes the purllication and characterization from brain cytosol of an activity that inhibits the dissociation of GDP from the smg p25A (Eab.3) protein. 27. .
Mmm Y, KIKUCHI A, Artuo S, HATA Y, KONDONJ, Txtu.rvtsm Y. Tm Y: Molecular Cloning and Characterization of a Novel Type of Regulatory Protein (GDI) for smg p25A, a Ras pal-Like GTP-Binding Protein. Mol and Cell Bid 1990, 10:411’+4122. This report describes the lirst molecular cloning of a GDI protein. The cDNA for this GDI (which works on the smg p25A, or Rab3, protein) encodes a protein of molecular weight of approximately 50 000 kD. The
The Ras superfamilies mRNA for this GDI was found In alI tissues examined (even in tissues that do not contain smg p25A), which suggests that this GDI protein may regulate other members of the low molecular mass GTP-biding protein famiIy. 28.
UEDA T, T~iozv,wi Y, Omotu T, OHVANAGI H, SA~T~HY, TAKA~ Y: Purification and Characterization from Rat liver Cytosol of a GDP Dissociation Inhibitor (GDI) for Liver 24 k G, a Ras pll-L&e GTP-Binding Protein, with Proper&s Siiar to Those of smg p25A GDI. Biochem~ 1991, 30:909-917.
29.
F~KUMOTOY, KAIBucttt K, Hots Y, Fu~lolcr H, Art~to S, UEDA 4 TAKA~ Y: Molecular Cloning and Characterization of a Novel Type of Regulatory Protein (GDI) for the Rho Proteins, Ras p21-like SmaR GTP-Binding Proteins. T, mUCHI
Chacqene
1990,
51321-1328.
30. .
Arwa S, Kmum A, ETA Y, MMURA M, TM Y: Regulation of Reversible Biiding of smg p25A, a Ras p21-l&e GTP-BiidIng Protein, to Synaptic Plasma Membranes and Vesicles by its Specific ReguIatoty Protein, GDP Dissociation Inhibitor. J Bill them 1990, 265:13007-13015. This paper describes Important and new Information regarding the role of the GDI protein for smg p25A SpecificalIy, this paper shows that both the GTP-bound and GDP-bound forms of smg p25A bind reversibIy to synaptic membranes and that the GDI binds SelectiveIy to the GDP-bound fonn of the protein and causes it to dissociate from the membrane surface. JF, MAGEE AJ, CH~LOSJE, MARSHAU. CJ: AR Ras Proteins are PoIyisoprenyIated but only Some are PaImitoyhued. Cell 1989, 5731167-1177.
31.
Wcoc~
32.
HANCOCK JF, PATERSON H, MhRswu JF: A Polybasic Domain or PaImItoyIation is Required in Addition to the CMX Motif to LocaRze p2lRa.s to the Plasma Membrane. Cell 1990, 63:133-139.
33.
GOWIN JL, BROWN MS: Regulation of the MevaIonate Pathway. Nature 1998, 343425-430.
34.
SCHAFER WR, Km II, STERNE R, THORNER J, KIM SH,
RWE J: Ge-
netic and PhannacologicaI Suppression of Oncogenic Mutations in RAS Genes of Yeast and Humans. Science 1989, 245:37!&385. 35. ..
SCHAFERWE, TRUEBLOODCE, YANG C-C. MAYER MP, ROSENBERG S, POULTERCD, K1M S-H, Rw J: Enzymatic Coupling of Cholesterol Intermediates to a Mating Pheromone Precursor and to the Ras Protein. Science 1990, 249~11331139. Demonstrates that mutations in the yeast DPRl-l+LMl gene causes a defect in the prenylation reaction. Implies that as the yeast protein could ako prenyiate human Ha&s p21 precursor, the human DPRl-RAM1 analogue may be a usefuI target for anticancer chemotherapy. 36. .
REM Y, Gomm JI SEABE MC, CA%% PJ, BROWNMS: Inhibition of Puritied p2 1Ras FamesyI: Protein Transferase by Cys-MX Tetrapeptides. Cell 1990, 62:8188. IdentiIicatIon, purikation, and characterization of a famesyl : protein transferax from rat brain cytosol. Competitive inhibition of enzyme activity by peptides as short as four residues that contained the Cys-AAX motif.
37. .
SCHABERMD, O’HA~A MB, JD, Mocwi SL, MARsw JB: Polyisoprenylation of Transferaw. / Biol &em Identification and pattial putication
from
bovine
brain
cytosol.
G.utw VM, MOUSERSD, BERGSTROM MS, Frunti PA, DIXON RAF, Gmas Ras In Vfrro by a Famesyl-Protein
1990, 265:14701-14704. of a famesyl transfemse Demonstration that deletion of
activity
the CAAX
Evans, Hart, Cerione
motif or a Cys-to Ser-mutation rendered the E cokxpressed tein, not a substrate.
Ras pro-
MANNEV, ROBERTSD, TOB~NA, O’ROURKEEO, DE VRG~O M, MEyEas C, AHMED N, Kuaz B, Rest M, KUNG H-F, B-to M: Identification and PreRminary Characterization of ProteinCystelne Farnesyltransferase. Pnx Nat1 Acud Sci USA 1990, 877541-7545. Description of enzyme activities capable of catahzing the farnesylation of unprocessed Ras p21 proteins in &r-o at the correct residue (Cyslf36). Implies that the in oftm assay will be useful in screening for potential inhibitors of the rus oncogene function. GOODMANLE, JUDD SR, F,uwsworo?t CC, POWERSS, GE~B MI-I, 39. .. Gmwxr JA TAMANOI F: Mutants of Sac&aromy#s cerw vfdae Defective in the Famesylation of Ras Proteins. Proc Nat1 Acud Sci USA 1990, 87966W669. Identification in crude soluble extracts of yeast ceUs of an acdvity that cataIyzes the transfer of a fame@ moiety from famesyl pyrophosphate to the yeast Ras-2 protein. A yeast mutant, DPRl-RAMl, Is shown to be defective in famesyhransfemse activity. AddItionaUy, another mutant, RAM-2, is shown to be defective in the transferax activity. Implies that at least two genes are required for the farnesyItransferase activity in yeast. 38. .
40. ..
F~twsworrm CC, GELB MI-I, GLOMSETJA: Identlftcation of GeranyIgeranyl-Mod&d Proteins in HeLa CeIIs. Science 1990,
247:32&322.
Identification of a novel Isoprenoid modikation, aII frans geranyIgeranyi, by labeling HeIa ceUswith [ 3H] mewIonIc acid and anaiysii proteotytic digests of total ceU protein. 41. ..
WG HC, BREUNGERE, Efi?-snr WW, Cruw PF: PrenyIated Proteins: the Structure of the Isoprenoid Group. Science 1990, 247:318320. IdentiIication of the mevalonate-derived portion of a prenybted protein from CHO cells as diterpenoid (C20). linkage of the group to a carboxy-terminal cysteine as a thioether. 42.
E~%-!?nuWW, LEVER DC, RUNG HC: RenyIated Proteins: Synthesis of GeranyIgeranylcystelne and Identltlcation of this Thioetber Amino Acid as a Component of Roteins in CHO Cells. Proc Nat1 Acad Sci USA 1990, 87:7352-7354.
KAWATA M, FARNSWORTH CC, YOSHIDAY, GELB MI-I, G~~MSET JA, TAKA~Y: Post-TransIationalIy Processed Structure of the Human Platelet Protein smg ~218: Evidence for GeranyIgeranykttion and CarboxyI MethyIation of the C-Terminal Cysteine. Pnx Nat1 Acud Sci USA 1990, 87:8960+X%. Results indicating that the smg p21B (RaplB) protein puriIied from human platelet membranes is post-transIationaIIy mod&d by geranyi geranyiation of Cys181. First demonstration of such a modification of one of the Ras superfamiIy members. 43. .
44. .
YAMANE HK, F~swOtt’rtt CC, m H, EVANS T, HoW~tn W, GEIB MH, Gt.o~ssr JA, CUW
T Evans, Department of CeII Biology, Genentech Inc., South San Francisco, California 94080, USA MJ Hart and RA Cerione, Departments of Biochemistry, CeII and Mokular Biology, and Department of Pharmacology, Schurman HaII, Cornell University, Ithaca, New York 14853, USA.
191