- Email: [email protected]
GAPs for rho-related GTPases
R E V I E W S
Small m-related G'rP-binding proteins (GTPases) are critical for regulating many aspects of cell biology. During the early 1980s, ms oncogenes were first identified in human cancers; since then, the importance of ras in the control of cell growth and differentiation has been fkmly established. More than 50 members of the ms superfamily have now been identified and, on the basis of amino acid sequence similarities, they can be divided into six subfamilies: ms, rho, rab, aft, sat and ran. While the ms subfamily appears to control aspects of cell growth and differentiation, members of the rab, aft, sar and ran subgroups regulate the transport of vesicles and proteins between different intracellular compartmentst. The nine known mammalian rho-like proteins appear to have regulatory roles in the actin cytoskeleton. In Swiss 31"3 cells, rho regulates a signal transduction pathway that links receptors to the assembly of focal adhesion and stress fibres, while rac (a member of the rho subfamily) links receptors to actin pe'/merization at the plasma membrane to produce membrane raffles 2-~. In addition, rac is a regulatory component of the NADPH oxidase system that generates the superoxide free radical in activated phagocytes6. Genetic experiments in Saccharomyces cerevtsiae have revealed that another member of the rho subfamily, Cdc42Sc (the yeast homologue of cdc42Hs/G25K), is required for bud emergence, a process that determines the organization of polymerized actin in this organism. All GTPases act as molecular switches, having an inactive GDP-bound form and an active GTP-bound form. Activation of this conformational switch is catalysed by guanine-nucleotide exchange factors (GEFs). GTPase-activating proteins (GAPs) enhance the low intrinsic GTPase activity of small GTP-binding proteins, which subsequently leads to inactivation. During the past few years, a large number of GEls and GAPs specific for members of the rho subfamily have been identified t. Here, we discuss the GAP proteins
pretam
GAPs for rho-related GTPases NATHAIIELAMARCHEAND AIAN HAIL
.qas.retated6TP-Madlnglz,otel~ (6TPases) of the vbo
subfaml~ pkly importmtl roles in ~
the
orgaMzatiom of the actbtcytoskeletoLA largeumber of
mullifnctfoaal protelas that can stimulate their ttarlm'ic GTPaseactivity have been ideattJ~tL Here, we discuss the uture of such GTPase.ucttvatittgproteOts (GAPs)amf t/mr potent/a//mportaacefor can s/gM///ng. and their potential importance in the rho/rac-mediated signalling pathways. Mammalian rhoGAP-containing proteins Biochemical analysis of cell extracts using recombiham rho led to the identification of a protein with GAP-like activity for the rho subfamily of GTPases. This protein, rhoGAP (29 kDa), was distinct from the only other known mammalian GAP at that time, a rasGAP (120 kDa). Partial amino acid sequence analysis of purified rhoGAP from human spleen tissue and from platelets has led to the isolation of a complete cDNA, which has revealed that the short protein originally found in cell extracts was in fact a proteolytic carboxyterminal product of a larger protein of 50 kDa, p50rhoGAp (Refs 7, 8). The carboxy terminus of p50rhoGAp contains the GAP domain, which extends over approximately 140 amino acids. Sequence analyses have revealed a family of proteins that all contain a closely related rhoGAP domain. To date, nine mammalian genes, the rotund locus of Drosophila raelanogaster, the BEM2 and BEM3 genes of Saccharomyces cerevtstae, and a Caenorbab. dftts elesans gene called CeGap have all been found to encode proteins containing a rhoGAP domain (Table 1). The domains show about 20-40°,6 amino acid identity
oqpalma
m m 0me)
the
me
tdeta
pS0rhoGAP
Mammalltm
Bcr Abr N-¢himaerin ~-chimaerln I>190 p85a P851~ 5BP-1
Mammalian Mammalian Mammalian Mammalian Mammalian Mammalian Mammalian Manunalian
50 145
+ -
÷ +
++ +
7, 8 II
92 54 54 170 85 81 ND
++
÷ + * ,
+ ÷
15, 16 11 18
rotund BEM2 BRM3 CeGAP
Dm~pb~ me/ano~ster Sacchammyc~ ¢ereoMa~ guccbammyce, ~ CaenodmMftts e/~ms
ND 125 155
41
-
-
-
25
ND ND ND + + +
ND ND ND ND ND ÷
ND ND ND
25 26
-
++ +
+, ability, - , inability of the rhoGAP-c~ta~In8 proteins to ~Imulate the GTPase activity of the ¢ o ~ d l n $ ~ I t l c ptmeat, m>, not detennlned, "riG DECEMBER 1994 VOL. 10 NO. 12 o ,994~
~,~.,
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43 6
28
29 29 :30 species-
REVIEWS
and the proteins in which they are contained are generally large and multifunctional (Fig. 1). p5OrhoGAP This ubiquitous protein, which can now be expressed as a recombinant protein in Escherichia coli, can stimulate the GTPase activity of rho, rac and cdc42Hs but, as expected, has no effect on ras and rab proteins7,8. The p50rhoGAP protein appears to have approximately equal affinity for the three rho-related GTPases, but cdc42Hs is its preferred substrate in an in vitro GAP assay7. Surprisingly, p50rhoGAP does not show differential binding affinity for the GDP-bound form and the GTP-bouncl foJm of the GTPases; this is in contrast to rasGAP, which has 100 times greater affinity for the GTP-bound form of ras than for the GDP-bound form9. The only other notable feature of this protein is a proline-rich region upstream from the GAP domain. Such proline-rich sequences are thought to be recognition motifs for proteins containing src homology 3 (SH3) domains. In fact, p50rhoGAP binds in vitro to the SH3 domains of c-src tyrosine kinase and of p850t, the regulatory subunit of phosphatidylinositol 3'-kinases. The possibility that prolinedirected protein interactions with p50rhoGAp exist in vWo is under investigation, p50rhoGAP BCR The physiological role of the large (143 kDa) cytosolic BCR phosphoprotein is still unclear, but it has at least two known enzymatic activities. The amino terminus encodes a novel serine/threonine protein kinase 10, while the carboxy tertninus encodes a GAP domain that stimulates the GTPase activity of rac and cdcq2Hs, but not of rhotL The central segment of BCR has some homology with a GEF called DBL, although no GEF activity has been reported for BCR so far12. BCR was first identified as part of the BCR-ABL fusion oncogene, which is generated by the translocation of sequences encoding the ABL tyrosine kinase on chromosome 9 to BCR sequences on chromosome 22 in human leukaemias positive for the Philadelphia chromosome 13. Two kinds of uanslocation result in the chimaeric proteins p210BCR-ABL and p185BCR-ABL, which are characteristic of chronic myelogenous leukaemia (CML) and acute lymphocytic leukaemia (ALL), respectively. Both proteins lack the GAP domain, and
Bcr
p185BCR-ABL also lacks the DBL-related domain. It has recently been shown that 70°6 of CML patients analysed express the reciprocal chimaeric transcript ABL-BCR (Ref. 14). It is tempting to speculate that expression of a chimaeric ABL-BCR protein leads to deregulated GAP activity with important biological effects, although such a protein has not yet been found in leukaemic cells. ABR The ABR gene, which was originally identified by its close relatedness to BCR, encodes a protein with 68% amino acid identity to BCR. ABR contains DBL-related sequences at its amino terminus and a rhoGAP domain at the carboxy terminus. However, it lacks the serine/threonine kinase domain found in BCR. It has been reported that ABR has GAP activity on rac and cdc42Hs (Refs 15, 16). N- and ~8-cbimaerin N-chimaerin and 13-chimaerin, which are expressed in the brain and testes, respectively, have significant homology with the rhoGAP domain at their carboxyl terminuslTa8 and have been shown to be GAPs for rac, but not for rho or cdc42Hs (Refs 11. 18). Unlike
-I
I
P
meal
Abr N-chimaerin 13-chimaerin p190 p85 3BP-1 rotund BEM2
BEM3
I
CeGAP I
P
P
[ rhoGAP dbl
~
GTPase
I
SH3 SH2 ] Cysteine-dch
Ser/Thrkinase Pleckstdn
~ P
Pleckstrin
1FIGIJIIE1. The multifunctionalcharacter of GTPase-activatingproteins (GAPs) for the dlo subfamilyof GTPases. The homologous domains are identifiedat the Ixmomof tile figure. The full-lengthsequences of the cDNAsencoding 3BP-1 and BEM2have not yet been published.
TIG DECEMBER1994 VOL. 10 No. 12 437
REVIEWS
although it is as yet unknown whether 3BP-1 has GAP activity.
pS0rhoGAP, N-chimaerin has been found to interact preferentially with the GTP-bound form of rac (Ref. 19). In addition to their GAP domains, chimaerins also contain cysteine-rich sequences similar to those found in the regulatory domain of protein kinase C. It appears that lipids can bind to this domain and affect the racGAP activity of N-c"htmaerin, but whether this is physiologically significant is unclear. Two spliced variants of N- and ~-chimaerin have each been reported to contain an additional src homology 2 (SH2) domain z°,21. Tbep l gOprotein The plg0 protein is of particular interest because it was f'ast identified as a tyrosine-phosphorylated, rasGAPassociated protein in cells transformed by tyrosine kinase, as well as in fibroblasts that had been stimulated with growth factors. In addition to having a carboxyterminal rhoGAP domain, p190 has a putative aminoterminal GTP-binding domain. It was originally thought that the central domain encodes a transcriptional repressor of the gene encoding the glucocorticoid receptor, but this is currently under debate 2z. Settleman and his collaborators showed that p190 has GAP activity on rho, rac and cdc42Hs in vitro, which strongly suggests that the interaction of pl20rasGAP with p190 could serve to couple the ms and rho signalling pathways23. Some evidence obtained recently by Pawson et al. supports this hypothesis 24. They observed that when the amino terminus of rasGAP is expressed in fibroblasts, it is constitutively associated with p190. Reduction of serum levels to 0.5°/6 results in disruption of the actin cytoskeleton and cell adhesion in a similar manner to that observed in Swiss 3T3 cells after downregulation of rho (Ref. 3). It is possible that the binding of the amino terminus of rasGAP to plg0 stimulates its rhoGAP activity, thereby downregulating rho. p85a and "B The genes p85a and "B encode regulatory subuntts of phosphatidyltnositol 3'.kinase2S. They are similar in overall structure, containing an amino-terminal SH3 domain, two carboxy-terminal SH2 domains, and an intervening rhoGAP domain (Fig. 1). The p850t protein does not appear to exert GAP activity on rho, rac or cdc42Hs in vitro. It is possible that p850t stimulates the GTPase activity of an unknown rho-like GTP-binding protein; a!tematively, although p850t lacks GAP activity it might still interact with rho-related GTPases. p850t is constitutively complexed with pll0, the catalytic subunit of phosphatidylinositol 3'-kinase, and at least part of its function is to relocalize the activity of the kinase to membrane receptors in response to stimulation of cells with growth factors. The role of the GAP domain is unclear. 3BP-1 In a search for proteins that bind with high specificity to the SH3 domain of the protooncogene ABL, a partial cDNA clone, 3BP-1, was obtained26. The SH3 binding site on 3BP-1 was subsequently localized to a stretch of ten amino acids very rich in proline residues, and it is thought that this is a common recognition motif of all SH3 binding sites27. Interestingly, 3BP-1 also contains sequences homologous with rhoGAP,
Nonmammalian rhoGAP-containing proteins The product of the rotund locus in Drosophila is involved in the morphogenesis of the adult appendages, and includes a sequence with 30-50°/6 similarity to the rhoGAP domain. It also contains a cysteine-rich region similar to that found in protein kinase C and in chimaerins ~. In yeast, the products of the BEM2 and BEM3 genes, along with the GTPase Cdc42Sc, play a role in bud emergence, a process involving cell-cycle-regulated reorganization of the cortical cytoskeleton. Domains within the BEM2 and BEM3 proteins have striking homology with the rhoGAP domain; BEM3 stimulates the GTPase activity of yeast Rhol and Cdc42Sc in vitro, while BEM2 acts as a GAP only for the yeast Rhol protein zg. In addition to the GAP domain, BEM3 contains a pleckstrin domain, which is a motif found in a number of proteins implicated in signalling processes. A protein containing a pleckstrin domain and exhibiting homologous sequences to the rhoGAP domain has also been found in C. elegans3O. CeGAP stimulates the GTPase activity of the three C. elegans proteins CeRhoA, CeRacl and Cdc42Ce, and also of human RHOA. Surprisingly, it also has a weak effect on the C. elegans ras protein, Let-60, and on the human ras and rab3A proteins. CeGAP is the first rhoGAP-like protein discovered to have an effect on ras, but the significance of this is presendy unclear.
Whyso many rhoGAPs? An intriguing characteristic of proteins containing the rhoGAP domain is their diversity and number. This is in contrast to the rasGAP domain, which has been described in only two mammalian proteins: pl20rasGAP and neuroflbromin, the product of the neurofibromatosis gene3t,32. As discussed above, the different rhoGAP-containing proteins are not uniquely specific for individual members of the rho subfamily, at least in vitro. This Is also the case for rasGAP proteins: pl20rasGAP and neurofibromin will also stimulate the GTPase activity of a closely related protein, R-ms (Ref. 33), and pl20rasGAP interacts with raplA, although it cannot stimulate its GTPase activity. In fact, a specific rapGAP has been identified for raplA and rap2 that displays no homology with any other known GAP protein34. Why should cells produce such a wide range of proteins with rhoGAp activity? Except for the tissuespecific chimaerins, all these rhoGAP.-containing proteins appear to be ubiquitously expressed; cleady, they must be highly regulated in the cell so that the rho-like GTPases are not always turned off. The multifunctional character of the rhoGAP-containing proteins suggests that the GAP function is regulated by specific protein-protein interactions, either directly or by affecting its subcellular site of action. The identification of proteins interacting with rhoGAPs is the next step in clarifying their role. l~motons of r h ~ The ability of GAP proteins to downregulate small GTPases is well established. Microinjection experiments
"riG DECEMBER1994 VOL. 10 No. 12
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REVIEWS
in Swiss 3T3 cells have shown that Tm p190, Bcr and p50rhoGAP interfere with rho/rac signalling pathways35. Interestingly, p190 preferentially dm6AP-eonta~ lamein(sn~ bindinSmoti0 inhibits the formation of stress fibres mediated by rho but is unable p50rhoGAP(227-236) A P K P M P P R P P to inhibit rac-induced membrane Bcr(772-781) A V P N I P L V P D ruffles. Bcr stimulates the GTPase plg0 (!036-1045) N K V P P P V K P K activity of rac but not rho in vitro, p190 (1446-1455) G A A L A P L Q P w L P V A P G and so it is not surprising that p85a(91-100) P P R P P K P P microinjection of Bcr specifically p85a(303.-312) P A P A L P L P A R P R blocks rac-induced membrane p8515(92-101) G P R P p85~, (294-303) A P P A L P P K P P ruffles but not the rho signalling 3BP-1 (267-276) P T M P P P L P P V pathway, p50rhoGAP, which has a rotund (153-162) A P R V A P M V P A broad specificity for the rho-like BEM3 (364-373) E S L P P P P A P P proteins in vitro, with a marked BEM3 (568-577) P D L P L P T L P D preference for cdc42Hs, inhibits the SH3 binding motif X p a P p X P formation of stress fibres but not consensus42 the induction of membrane ruffles. These experiments suggest that a, hydrophobic residue; p, praline preferred; P, conserved praline; SH3, Src there may be, in fact, more specihomology' 3; X, nonconserved residues. ficity associated with different rhoGAP-containing proteins in viva than would be expected from the GAP assays in vitro. This suggests that these domains may be involved in The possibility that GAPs could in principle also act controllh,,g the interactions of signalling proteins with downstream of GTPases as effector proteins was first the actin c3,toskeleton; for example, phospholipase C'y suggested for pl20rasGAP. This protein interacts with and GRB-2 appear to interact with microf'daments and residues 24--46 of ras, the effector site essential for the membrane raffles through their SH3 domains41. Perbiological activity of ras (Ref. 36). More recently, it has haps specific SH3-containing proteins are responsible been shown that c-raf, a mitogen-activated protein for the recruitment of rhoGAPs via a praline-rich region • kinase kinase kinase and a ras effector target, has a to complexes that are associated with the actin cytoweak GAP effect on ras (Ref. 37). Interestingly, phos- skeleton at the plasma membrane. In conclusion, the biochemical functions of rho, rac pholipase CI5 and the ~/subunit of phosphodiesterase, the effectors of the heterotrimeric G proteins Gq and and other members of the rho subfamily of ras-related transducin, respectively, are also GAPs. It would be of proteins are still unknown but it has been suggested great interest if rhoGAP-containing proteins were found that, in general, they regulate the formation of to act downstream in rho/rac-mediated signalling path- multimolecular complexes that are associated with ways, but so far there is no evidence for this. In fact, p67phox, a protein that has recently been shown to be the effector of rac in the NADPH oxidase system, does not have GAP activity towards rac (Ref. 38). Furthermore, preliminary analysis suggests that p50rhoGAP does not interact at the effector site of rho and does not distinguish between the GTP-bound and the GDPbound forms of the protein39; both results would be unexpected if pS0rhoGAP is a target of the rho-like proteins. It seems likely that the activities of the different rhoGAPs are restricted to discrete sites within the cell, although so far there is no direct evidence for this. A closer analysis of the rhoGAP-containing proteins reveals that praline-rich sequences are found in six of the mammalian rhoGAps, in the rotund protein and in the yeast BEM3 protein (Table 2). As mentioned above, FtGt~ 2. Proposed model for the role of GTPase-activating p50rhoGAP binds to the SH3 domains of c-src tyrosine proteins (GAPs)for the rho subfamilyof GTPases. In the cytosol, kinase and p850t in vitro a, while p850t interacts via the GDP-bound form of rho is associated with rhoGDl, a praline-rich sequences with SH3 domains of the non- guanine-nucleotidedissociation inhibitorthat can inhibitthe exchange between GDP and GTP on all rho-like proteins43. receptor src tyrosine kinase proteins lyn, fyn and abl 't°. Dissociationof rho from rhoGDl and its conversionto the It is possible. ;.hat the interaction of rhoGAPs with SH3- GTP-bound form promotes the formationof multimolecular containing proteins may be a common mechanism for complexes at a plasma membrane target5 (filledbox). We either targeting rhoGAPs to specific protein complexes propose that a rhoGAP is recruited to this membranecomplex in the cell, or for regulating their GAP activity, or both. along with other cytosolicproteins (X, Y), where it acts to It is noteworthy that SH3 domains are found in several downregulate further rho activity,it is not known whether cytoskeletal proteins such as spectrin and myosin 1. it has any additionalrole in the complex.
I~o-QDPI [rhoGDI] ~
'FIG DECEMBER 1994 VOL. 10 No. 12
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REVIEWS
polymerized actin at the plasma membrane 5. It is possible that rhoGAP-containing proteins, recruited to these complexes through their interaction with SH3containing proteins, act in a localized fashion to downregulate rho-like GTPases at these sites (Fig. 2). Whether these proteins also contribute in other ways to the rho/rac signalling pathways is unclear; the identification of specific proteins interacting with members of the rhoGAP family should help clarify the role of this type of GAP.
Aclmowledgem~ts
We thank the CRC (UK) and the Human Frontier Science Program for their support. N.L. holds a fellowship from Fonds de La Recherche en Sant6 du Qu6bec.
References
! Boguski, M.S. and McCormick, F. (1993) Nature 366, 643-654 2 Nobes, C. and Hall, A. (1994) Curt. Opin. Genet. Dev. 4, 77-81 3 Ridley, A.J. and Hail, A. (1992) CellTO, 389-399 4 Ridley, A.J. etal. (1992) Ceil70, 401-410 5 Hall, A. (1994) Annu. Rev. Cell Biol. 10, 31-54 6 Bokoch, G.M. (1994) Curr. Opfn. CellBiol. 6, 212-218 7 Lancaster, C.A. etal. (1994)./. Btol. Chem. 269, 1137-1142 8 Baffod, E.T. etal. (1993)J. Btol. Chem. 268, 26059-26062 9 Schaber, M.D. etaL (1989) Proteins6, 306-315 lO Mare, Y. and Wine, O.N. (1991) Ce1167, 459--468 11 Die!~mann,D. etai. (1991) Nature351, 400-402 12 Adams,J.M. et al. (1992) Oncogene7, 611.-618 13 Heisterkamp, N. et al. (1985) Nature 315, 758-761 14 Melo,J., Gordon, D.E., Cross, N.C.P. and Goldman, J.M. (1993) Blood81, 158-165 15 Tan, E,C,, Leung, T,, Manser, E, and Lim, L, (1993)./. Biol. Chem, 268, 27291-27298 16 Heisterkamp, N. et al. (1993)./. Biol. Chem, 268, 16903-16906 17 Hall, C, etal. (1990) J, Mot, Biol, 211, 11-16 18 Leung, T,, How, B-E,, Manser, E, and Lira, L, (1993) J, Btoi. Chem. 268, 3815--3816 19 Ahmed, S, et al. (1994) J. Biol. Chem, 269, 17642-17648 20 Hail, C, et at. (1993) Moi, Cell, Biol. 13, 4986-4998
Comlqs
21 Leung, T., How, B.E., Manser, E. and Lim, L. (1994)./. Biol. Chem. 269, 12888-12892 22 Settleman, J., Narasimhan, V., Foster, L.C. and Weinberg, R.A. (1992) Cell69, 539-549 23 Settleman, J., Albright, C.F., Foster, L.C. and Weinberg, R.A. (1992) Nature 359, 153--154 24 McGlade, J. et al. (1993) EMBOJ. 12, 3073-3081 25 Otsu, M. et al. (1991) Ce!!65, 91-104 26 Cicchetti, P., Mzyer, B.J., Thiei, G. and Baltimore, D. (1992) Science 257, 803--806 27 Ren, R., Mayer, B.J., Cicchetti, P. and Baltimore, D. (1.993) Science 259, 1157-1161 28 Agnel, M., Roder, L., Vola, C. and Griffin-Shea, R. (1992) Mol. Cell. Biol. 12, 5111-5122 29 Zheng, Y., Cerione, R. and Bender, A. (1994)J. Biol. Chem. 269, 2369-2372 30 Chen, W., Blanc, J. and Lim, L. (1994) J. Biol. Chem. 269, 820--823 31 Trahey, M. and McCormick, F. (1987) Science238, 542-545 32 Martin, G.A. et al. (1990) Cell63, 843--849 33 Rey, I., Taylor-Harris, P., van Erp, H. and Hall, A. (1994) Oncogene 9, 685--692 34 Rubinfeld, B. et al. (1992) Mol. Cell. Biol. 12, 4634--4642 35 Ridley, A.J. et al. (1993) EMBOJ. 12, 5151--5160 36 Cal~s, C., Hancock, J.F., Marshall, C.J. and Hall, A. (1988) Nature 332, 548-551 37 Wame, P.H., Viciana, P.R. and Downward, J. (1993) Nature 364, 352-355 38 Diekmann, D. et al. (1994) Science 265, 531-533 39 Self, A.J., Paterson, H.F. and Hall, A. (1993) Oncogeneg, 655--661 40 Kapeller, R. etal. (1994)J. Biol. Chem. 269, 1927-1933 41 Bar-Sagi, D. etal. (1993) Cell74, 83-91 42 Yu, H. et al. (1994) Cell76, 933-945 43 Hiraoka, K, et al, (1992) Blocbem. Btophys. ICes.Commun. 182, 921--930
N. l a o ~ o t B AND A. HAU m~ tS rum MR¢ ldmo~ffomr FOR Mo~cet.~g CSU BtOtOG¥ aNO DBPARFMENF OF BIOCHEMISTRg UNIVBRSHY COUEG~ LONOON, GOWBa $ ~ LONDON,UK WC'IE 6B~.
soon ~TIG...
Insights into lymphocyte development from X-linked immune deficiences byJ. Belmont
Flavonold genes in plants
- a
model network for quantitative
genetics?
by a. B o w e n
Finding the hairpin in the haystack - searching for RNA motifs by Z D a n d e k a r a n d M, W. H e n t z e
Tram-splicing and polycistronlc transcription in C, elegans by Z B l u m e n t b a l
JAKs and STATsin signalling by the cytoidne receptor supeffamtly byJ.N. Ible aridlY. Kerr
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Small m-related G'rP-binding proteins (GTPases) are critical for regulating many aspects of cell biology. During the early 1980s, ms oncogenes were first identified in human cancers; since then, the importance of ras in the control of cell growth and differentiation has been fkmly established. More than 50 members of the ms superfamily have now been identified and, on the basis of amino acid sequence similarities, they can be divided into six subfamilies: ms, rho, rab, aft, sat and ran. While the ms subfamily appears to control aspects of cell growth and differentiation, members of the rab, aft, sar and ran subgroups regulate the transport of vesicles and proteins between different intracellular compartmentst. The nine known mammalian rho-like proteins appear to have regulatory roles in the actin cytoskeleton. In Swiss 31"3 cells, rho regulates a signal transduction pathway that links receptors to the assembly of focal adhesion and stress fibres, while rac (a member of the rho subfamily) links receptors to actin pe'/merization at the plasma membrane to produce membrane raffles 2-~. In addition, rac is a regulatory component of the NADPH oxidase system that generates the superoxide free radical in activated phagocytes6. Genetic experiments in Saccharomyces cerevtsiae have revealed that another member of the rho subfamily, Cdc42Sc (the yeast homologue of cdc42Hs/G25K), is required for bud emergence, a process that determines the organization of polymerized actin in this organism. All GTPases act as molecular switches, having an inactive GDP-bound form and an active GTP-bound form. Activation of this conformational switch is catalysed by guanine-nucleotide exchange factors (GEFs). GTPase-activating proteins (GAPs) enhance the low intrinsic GTPase activity of small GTP-binding proteins, which subsequently leads to inactivation. During the past few years, a large number of GEls and GAPs specific for members of the rho subfamily have been identified t. Here, we discuss the GAP proteins
pretam
GAPs for rho-related GTPases NATHAIIELAMARCHEAND AIAN HAIL
.qas.retated6TP-Madlnglz,otel~ (6TPases) of the vbo
subfaml~ pkly importmtl roles in ~
the
orgaMzatiom of the actbtcytoskeletoLA largeumber of
mullifnctfoaal protelas that can stimulate their ttarlm'ic GTPaseactivity have been ideattJ~tL Here, we discuss the uture of such GTPase.ucttvatittgproteOts (GAPs)amf t/mr potent/a//mportaacefor can s/gM///ng. and their potential importance in the rho/rac-mediated signalling pathways. Mammalian rhoGAP-containing proteins Biochemical analysis of cell extracts using recombiham rho led to the identification of a protein with GAP-like activity for the rho subfamily of GTPases. This protein, rhoGAP (29 kDa), was distinct from the only other known mammalian GAP at that time, a rasGAP (120 kDa). Partial amino acid sequence analysis of purified rhoGAP from human spleen tissue and from platelets has led to the isolation of a complete cDNA, which has revealed that the short protein originally found in cell extracts was in fact a proteolytic carboxyterminal product of a larger protein of 50 kDa, p50rhoGAp (Refs 7, 8). The carboxy terminus of p50rhoGAp contains the GAP domain, which extends over approximately 140 amino acids. Sequence analyses have revealed a family of proteins that all contain a closely related rhoGAP domain. To date, nine mammalian genes, the rotund locus of Drosophila raelanogaster, the BEM2 and BEM3 genes of Saccharomyces cerevtstae, and a Caenorbab. dftts elesans gene called CeGap have all been found to encode proteins containing a rhoGAP domain (Table 1). The domains show about 20-40°,6 amino acid identity
oqpalma
m m 0me)
the
me
tdeta
pS0rhoGAP
Mammalltm
Bcr Abr N-¢himaerin ~-chimaerln I>190 p85a P851~ 5BP-1
Mammalian Mammalian Mammalian Mammalian Mammalian Mammalian Mammalian Manunalian
50 145
+ -
÷ +
++ +
7, 8 II
92 54 54 170 85 81 ND
++
÷ + * ,
+ ÷
15, 16 11 18
rotund BEM2 BRM3 CeGAP
Dm~pb~ me/ano~ster Sacchammyc~ ¢ereoMa~ guccbammyce, ~ CaenodmMftts e/~ms
ND 125 155
41
-
-
-
25
ND ND ND + + +
ND ND ND ND ND ÷
ND ND ND
25 26
-
++ +
+, ability, - , inability of the rhoGAP-c~ta~In8 proteins to ~Imulate the GTPase activity of the ¢ o ~ d l n $ ~ I t l c ptmeat, m>, not detennlned, "riG DECEMBER 1994 VOL. 10 NO. 12 o ,994~
~,~.,
o , ~ 9 s ~ , 9 ~ 7 oo
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28
29 29 :30 species-
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and the proteins in which they are contained are generally large and multifunctional (Fig. 1). p5OrhoGAP This ubiquitous protein, which can now be expressed as a recombinant protein in Escherichia coli, can stimulate the GTPase activity of rho, rac and cdc42Hs but, as expected, has no effect on ras and rab proteins7,8. The p50rhoGAP protein appears to have approximately equal affinity for the three rho-related GTPases, but cdc42Hs is its preferred substrate in an in vitro GAP assay7. Surprisingly, p50rhoGAP does not show differential binding affinity for the GDP-bound form and the GTP-bouncl foJm of the GTPases; this is in contrast to rasGAP, which has 100 times greater affinity for the GTP-bound form of ras than for the GDP-bound form9. The only other notable feature of this protein is a proline-rich region upstream from the GAP domain. Such proline-rich sequences are thought to be recognition motifs for proteins containing src homology 3 (SH3) domains. In fact, p50rhoGAP binds in vitro to the SH3 domains of c-src tyrosine kinase and of p850t, the regulatory subunit of phosphatidylinositol 3'-kinases. The possibility that prolinedirected protein interactions with p50rhoGAp exist in vWo is under investigation, p50rhoGAP BCR The physiological role of the large (143 kDa) cytosolic BCR phosphoprotein is still unclear, but it has at least two known enzymatic activities. The amino terminus encodes a novel serine/threonine protein kinase 10, while the carboxy tertninus encodes a GAP domain that stimulates the GTPase activity of rac and cdcq2Hs, but not of rhotL The central segment of BCR has some homology with a GEF called DBL, although no GEF activity has been reported for BCR so far12. BCR was first identified as part of the BCR-ABL fusion oncogene, which is generated by the translocation of sequences encoding the ABL tyrosine kinase on chromosome 9 to BCR sequences on chromosome 22 in human leukaemias positive for the Philadelphia chromosome 13. Two kinds of uanslocation result in the chimaeric proteins p210BCR-ABL and p185BCR-ABL, which are characteristic of chronic myelogenous leukaemia (CML) and acute lymphocytic leukaemia (ALL), respectively. Both proteins lack the GAP domain, and
Bcr
p185BCR-ABL also lacks the DBL-related domain. It has recently been shown that 70°6 of CML patients analysed express the reciprocal chimaeric transcript ABL-BCR (Ref. 14). It is tempting to speculate that expression of a chimaeric ABL-BCR protein leads to deregulated GAP activity with important biological effects, although such a protein has not yet been found in leukaemic cells. ABR The ABR gene, which was originally identified by its close relatedness to BCR, encodes a protein with 68% amino acid identity to BCR. ABR contains DBL-related sequences at its amino terminus and a rhoGAP domain at the carboxy terminus. However, it lacks the serine/threonine kinase domain found in BCR. It has been reported that ABR has GAP activity on rac and cdc42Hs (Refs 15, 16). N- and ~8-cbimaerin N-chimaerin and 13-chimaerin, which are expressed in the brain and testes, respectively, have significant homology with the rhoGAP domain at their carboxyl terminuslTa8 and have been shown to be GAPs for rac, but not for rho or cdc42Hs (Refs 11. 18). Unlike
-I
I
P
meal
Abr N-chimaerin 13-chimaerin p190 p85 3BP-1 rotund BEM2
BEM3
I
CeGAP I
P
P
[ rhoGAP dbl
~
GTPase
I
SH3 SH2 ] Cysteine-dch
Ser/Thrkinase Pleckstdn
~ P
Pleckstrin
1FIGIJIIE1. The multifunctionalcharacter of GTPase-activatingproteins (GAPs) for the dlo subfamilyof GTPases. The homologous domains are identifiedat the Ixmomof tile figure. The full-lengthsequences of the cDNAsencoding 3BP-1 and BEM2have not yet been published.
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although it is as yet unknown whether 3BP-1 has GAP activity.
pS0rhoGAP, N-chimaerin has been found to interact preferentially with the GTP-bound form of rac (Ref. 19). In addition to their GAP domains, chimaerins also contain cysteine-rich sequences similar to those found in the regulatory domain of protein kinase C. It appears that lipids can bind to this domain and affect the racGAP activity of N-c"htmaerin, but whether this is physiologically significant is unclear. Two spliced variants of N- and ~-chimaerin have each been reported to contain an additional src homology 2 (SH2) domain z°,21. Tbep l gOprotein The plg0 protein is of particular interest because it was f'ast identified as a tyrosine-phosphorylated, rasGAPassociated protein in cells transformed by tyrosine kinase, as well as in fibroblasts that had been stimulated with growth factors. In addition to having a carboxyterminal rhoGAP domain, p190 has a putative aminoterminal GTP-binding domain. It was originally thought that the central domain encodes a transcriptional repressor of the gene encoding the glucocorticoid receptor, but this is currently under debate 2z. Settleman and his collaborators showed that p190 has GAP activity on rho, rac and cdc42Hs in vitro, which strongly suggests that the interaction of pl20rasGAP with p190 could serve to couple the ms and rho signalling pathways23. Some evidence obtained recently by Pawson et al. supports this hypothesis 24. They observed that when the amino terminus of rasGAP is expressed in fibroblasts, it is constitutively associated with p190. Reduction of serum levels to 0.5°/6 results in disruption of the actin cytoskeleton and cell adhesion in a similar manner to that observed in Swiss 3T3 cells after downregulation of rho (Ref. 3). It is possible that the binding of the amino terminus of rasGAP to plg0 stimulates its rhoGAP activity, thereby downregulating rho. p85a and "B The genes p85a and "B encode regulatory subuntts of phosphatidyltnositol 3'.kinase2S. They are similar in overall structure, containing an amino-terminal SH3 domain, two carboxy-terminal SH2 domains, and an intervening rhoGAP domain (Fig. 1). The p850t protein does not appear to exert GAP activity on rho, rac or cdc42Hs in vitro. It is possible that p850t stimulates the GTPase activity of an unknown rho-like GTP-binding protein; a!tematively, although p850t lacks GAP activity it might still interact with rho-related GTPases. p850t is constitutively complexed with pll0, the catalytic subunit of phosphatidylinositol 3'-kinase, and at least part of its function is to relocalize the activity of the kinase to membrane receptors in response to stimulation of cells with growth factors. The role of the GAP domain is unclear. 3BP-1 In a search for proteins that bind with high specificity to the SH3 domain of the protooncogene ABL, a partial cDNA clone, 3BP-1, was obtained26. The SH3 binding site on 3BP-1 was subsequently localized to a stretch of ten amino acids very rich in proline residues, and it is thought that this is a common recognition motif of all SH3 binding sites27. Interestingly, 3BP-1 also contains sequences homologous with rhoGAP,
Nonmammalian rhoGAP-containing proteins The product of the rotund locus in Drosophila is involved in the morphogenesis of the adult appendages, and includes a sequence with 30-50°/6 similarity to the rhoGAP domain. It also contains a cysteine-rich region similar to that found in protein kinase C and in chimaerins ~. In yeast, the products of the BEM2 and BEM3 genes, along with the GTPase Cdc42Sc, play a role in bud emergence, a process involving cell-cycle-regulated reorganization of the cortical cytoskeleton. Domains within the BEM2 and BEM3 proteins have striking homology with the rhoGAP domain; BEM3 stimulates the GTPase activity of yeast Rhol and Cdc42Sc in vitro, while BEM2 acts as a GAP only for the yeast Rhol protein zg. In addition to the GAP domain, BEM3 contains a pleckstrin domain, which is a motif found in a number of proteins implicated in signalling processes. A protein containing a pleckstrin domain and exhibiting homologous sequences to the rhoGAP domain has also been found in C. elegans3O. CeGAP stimulates the GTPase activity of the three C. elegans proteins CeRhoA, CeRacl and Cdc42Ce, and also of human RHOA. Surprisingly, it also has a weak effect on the C. elegans ras protein, Let-60, and on the human ras and rab3A proteins. CeGAP is the first rhoGAP-like protein discovered to have an effect on ras, but the significance of this is presendy unclear.
Whyso many rhoGAPs? An intriguing characteristic of proteins containing the rhoGAP domain is their diversity and number. This is in contrast to the rasGAP domain, which has been described in only two mammalian proteins: pl20rasGAP and neuroflbromin, the product of the neurofibromatosis gene3t,32. As discussed above, the different rhoGAP-containing proteins are not uniquely specific for individual members of the rho subfamily, at least in vitro. This Is also the case for rasGAP proteins: pl20rasGAP and neurofibromin will also stimulate the GTPase activity of a closely related protein, R-ms (Ref. 33), and pl20rasGAP interacts with raplA, although it cannot stimulate its GTPase activity. In fact, a specific rapGAP has been identified for raplA and rap2 that displays no homology with any other known GAP protein34. Why should cells produce such a wide range of proteins with rhoGAp activity? Except for the tissuespecific chimaerins, all these rhoGAP.-containing proteins appear to be ubiquitously expressed; cleady, they must be highly regulated in the cell so that the rho-like GTPases are not always turned off. The multifunctional character of the rhoGAP-containing proteins suggests that the GAP function is regulated by specific protein-protein interactions, either directly or by affecting its subcellular site of action. The identification of proteins interacting with rhoGAPs is the next step in clarifying their role. l~motons of r h ~ The ability of GAP proteins to downregulate small GTPases is well established. Microinjection experiments
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in Swiss 3T3 cells have shown that Tm p190, Bcr and p50rhoGAP interfere with rho/rac signalling pathways35. Interestingly, p190 preferentially dm6AP-eonta~ lamein(sn~ bindinSmoti0 inhibits the formation of stress fibres mediated by rho but is unable p50rhoGAP(227-236) A P K P M P P R P P to inhibit rac-induced membrane Bcr(772-781) A V P N I P L V P D ruffles. Bcr stimulates the GTPase plg0 (!036-1045) N K V P P P V K P K activity of rac but not rho in vitro, p190 (1446-1455) G A A L A P L Q P w L P V A P G and so it is not surprising that p85a(91-100) P P R P P K P P microinjection of Bcr specifically p85a(303.-312) P A P A L P L P A R P R blocks rac-induced membrane p8515(92-101) G P R P p85~, (294-303) A P P A L P P K P P ruffles but not the rho signalling 3BP-1 (267-276) P T M P P P L P P V pathway, p50rhoGAP, which has a rotund (153-162) A P R V A P M V P A broad specificity for the rho-like BEM3 (364-373) E S L P P P P A P P proteins in vitro, with a marked BEM3 (568-577) P D L P L P T L P D preference for cdc42Hs, inhibits the SH3 binding motif X p a P p X P formation of stress fibres but not consensus42 the induction of membrane ruffles. These experiments suggest that a, hydrophobic residue; p, praline preferred; P, conserved praline; SH3, Src there may be, in fact, more specihomology' 3; X, nonconserved residues. ficity associated with different rhoGAP-containing proteins in viva than would be expected from the GAP assays in vitro. This suggests that these domains may be involved in The possibility that GAPs could in principle also act controllh,,g the interactions of signalling proteins with downstream of GTPases as effector proteins was first the actin c3,toskeleton; for example, phospholipase C'y suggested for pl20rasGAP. This protein interacts with and GRB-2 appear to interact with microf'daments and residues 24--46 of ras, the effector site essential for the membrane raffles through their SH3 domains41. Perbiological activity of ras (Ref. 36). More recently, it has haps specific SH3-containing proteins are responsible been shown that c-raf, a mitogen-activated protein for the recruitment of rhoGAPs via a praline-rich region • kinase kinase kinase and a ras effector target, has a to complexes that are associated with the actin cytoweak GAP effect on ras (Ref. 37). Interestingly, phos- skeleton at the plasma membrane. In conclusion, the biochemical functions of rho, rac pholipase CI5 and the ~/subunit of phosphodiesterase, the effectors of the heterotrimeric G proteins Gq and and other members of the rho subfamily of ras-related transducin, respectively, are also GAPs. It would be of proteins are still unknown but it has been suggested great interest if rhoGAP-containing proteins were found that, in general, they regulate the formation of to act downstream in rho/rac-mediated signalling path- multimolecular complexes that are associated with ways, but so far there is no evidence for this. In fact, p67phox, a protein that has recently been shown to be the effector of rac in the NADPH oxidase system, does not have GAP activity towards rac (Ref. 38). Furthermore, preliminary analysis suggests that p50rhoGAP does not interact at the effector site of rho and does not distinguish between the GTP-bound and the GDPbound forms of the protein39; both results would be unexpected if pS0rhoGAP is a target of the rho-like proteins. It seems likely that the activities of the different rhoGAPs are restricted to discrete sites within the cell, although so far there is no direct evidence for this. A closer analysis of the rhoGAP-containing proteins reveals that praline-rich sequences are found in six of the mammalian rhoGAps, in the rotund protein and in the yeast BEM3 protein (Table 2). As mentioned above, FtGt~ 2. Proposed model for the role of GTPase-activating p50rhoGAP binds to the SH3 domains of c-src tyrosine proteins (GAPs)for the rho subfamilyof GTPases. In the cytosol, kinase and p850t in vitro a, while p850t interacts via the GDP-bound form of rho is associated with rhoGDl, a praline-rich sequences with SH3 domains of the non- guanine-nucleotidedissociation inhibitorthat can inhibitthe exchange between GDP and GTP on all rho-like proteins43. receptor src tyrosine kinase proteins lyn, fyn and abl 't°. Dissociationof rho from rhoGDl and its conversionto the It is possible. ;.hat the interaction of rhoGAPs with SH3- GTP-bound form promotes the formationof multimolecular containing proteins may be a common mechanism for complexes at a plasma membrane target5 (filledbox). We either targeting rhoGAPs to specific protein complexes propose that a rhoGAP is recruited to this membranecomplex in the cell, or for regulating their GAP activity, or both. along with other cytosolicproteins (X, Y), where it acts to It is noteworthy that SH3 domains are found in several downregulate further rho activity,it is not known whether cytoskeletal proteins such as spectrin and myosin 1. it has any additionalrole in the complex.
I~o-QDPI [rhoGDI] ~
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polymerized actin at the plasma membrane 5. It is possible that rhoGAP-containing proteins, recruited to these complexes through their interaction with SH3containing proteins, act in a localized fashion to downregulate rho-like GTPases at these sites (Fig. 2). Whether these proteins also contribute in other ways to the rho/rac signalling pathways is unclear; the identification of specific proteins interacting with members of the rhoGAP family should help clarify the role of this type of GAP.
Aclmowledgem~ts
We thank the CRC (UK) and the Human Frontier Science Program for their support. N.L. holds a fellowship from Fonds de La Recherche en Sant6 du Qu6bec.
References
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N. l a o ~ o t B AND A. HAU m~ tS rum MR¢ ldmo~ffomr FOR Mo~cet.~g CSU BtOtOG¥ aNO DBPARFMENF OF BIOCHEMISTRg UNIVBRSHY COUEG~ LONOON, GOWBa $ ~ LONDON,UK WC'IE 6B~.
soon ~TIG...
Insights into lymphocyte development from X-linked immune deficiences byJ. Belmont
Flavonold genes in plants
- a
model network for quantitative
genetics?
by a. B o w e n
Finding the hairpin in the haystack - searching for RNA motifs by Z D a n d e k a r a n d M, W. H e n t z e
Tram-splicing and polycistronlc transcription in C, elegans by Z B l u m e n t b a l
JAKs and STATsin signalling by the cytoidne receptor supeffamtly byJ.N. Ible aridlY. Kerr
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