Signal transduction through small GTPases—A tale of two GAPs

Cell. Vol. 69, 369-391,

May 1, 1992, Copyright

0 1992 by Cell Press

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Signal Transduction through Small GTPasesA Tale of Two GAPS Alan Hall Chester Beatty Laboratories Institute of Cancer Research London SW3 6JB England

Small GTP-binding proteins related to ras regulate many basic processes in all eukaryotes (Hall, 1990). p21’** is an essential component of receptor-mediated signal transduction pathways regulating growth and differentiation, and although its exact biochemical function is unknown, one potential target protein is rasGAP, a protein that can stimulate the intrinsic GTPase activity of ras. The rho subfamily of small GTPases is functionally distinct from ras. p21”O, for example, controls the organization of polymerized actin in cells. It interacts with a GTPase-activating protein, rhoGAP, which is unrelated in sequence to rasGAP. GAP domains have subsequently been identified in two clinically important genes, NF7 (the neurofibromatosis type 1 susceptibility gene) and bcr (the breakpoint cluster region gene), and the role of GAP proteins in signal transduction pathways has now become a focus of attention. In this issue, the plot thickens still further with the sequence analysis of two proteins that bind to rasGAP-one has homology to proteins involved in mRNA processing, and the other is a multidomain protein that looks like a GTPbinding protein, a transcription factor, and rhoGAP! rasGAP is a 120 kd cytosolic protein found in all mammalian cells. It can stimulate the intrinsic GTPase activity of normal but not oncogenic ras proteins, providing an explanation for why oncogenic ~21”” is hyperactive and constitutively in the GTP-bound form-it is insensitive to down-regulation in vivo. Mutations in ras (between amino acids 30 and 40, the so-called effector domain) block biological activity and GAP-stimulated GTP hydrolysis, suggesting either that the ras target and GAP recognize overlapping sequences, or that GAP is the ras target. Sequence analysis of GAP doesn’t help much in decid-

ing whether it has a signaling function: the C-terminal domain alone can bind to ras and has GAP activity, and although the N-terminal sequence has Src homology (SH) 3 and SH2 domains, it has no other hallmark of an enzyme activity (Figure 1). A direct biochemical demonstration that GAP has a function other than down-regulating ras has come from analyzing the coupling of K’ channels to muscarinic cholinergic receptors in isolated membranes. Addition of recombinant GAP to the membrane inhibits coupling, and since the inhibition can be blocked by neutralizing ras antibodies, both ras and GAP must be acting together. If the N-terminal domain of GAP alone is used, then ras is no longer required, suggesting a model in which the GTP-bound form of ras binding to the catalytic domain of GAP can regulate the interaction between N-terminal SH2 domains and phosphotyrosine on target proteins (Figure 2) (Martin et al., 1992). The ras story took an unexpected turn in 1990, when it was discovered that a domain within neurofibromin, the product of the NF7 gene, has 30% amino acid homology to rasGAP (Figure 1). Expression of this domain reveals that it can act as a rasGTPaseactivating protein. What is the role of neurofibromin? It also appears to bind to the effector domain of ras, leaving open the possibility that neurofibromin could be a down-regulator, a target, or both. Some support for neurofibromin being primarily a down-regulator of ras has been obtained by analyzing malignant Schwannomas from type 1 neurofibromatosis patients (DeClue et al., 1992; Basu et al., 1992). Cell lines derived from these tumors either lack or have reduced levels of neurofibromin protein, and despite almost normal levels of rasGAP, ras is predominantly in the active, GTP-bound form. Furthermore, point mutations that inhibit the GAP activity of neurofibromin have recently been detected in some sporadic tumors (Li et al., 1992). These data suggest. that, in several (maybe all) cell types, NFI is a tumor-suppressor gene, and its product is a down-regulator of ras. Analysis of NF7-like genes in lower organisms leads to the same conclusion. Deletion of IRA 7 and IRA.2 in S. cerevisiae, or gap7 in S. pombe results in increased levels of GTP-bound HAS and gives a phenotype similar to strains containing an activated ras gene (Tanaka et al., 1990; lmai

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et al., 1991). Analogous results are obtained by deleting Drosophila gapl, though its product is unrelated outside its catalytic domain to either rasGAP or neurofibromin (Figure 1) (Gaul et al., 1992). The story could, however, turn out to be more complicated. First, neurofibromin, IRAl, and IRA2 are very large proteins, and only one small domain is required for ras down-regulation. Second, deletion of of lRA7 in S. cerevisiae leads to the mislocalization adenylate cyclase, the targetof RASin thisorganism (Mitts et al., 1991). Neurofibromin may, therefore, be more than just a negative regulator of ras signaling. The identification of rhoGAP, a GAP specific for the rho subfamily of ras-related proteins, has led to some equally intriguing observations. The biological functions of the rho-like small GTPases, rho, ras, and CDC42, are quite distinct from ras. p21mo, for example, is involved in regulating the organization of polymerized actin in cells, while in S. cerevisiae, CDC42 is essential for the assembly of the bud during cell division (Hall, 1992). Like ras, however, rho proteins have a low intrinsic GTPase activity that can be stimulated by a GAP. Originally purified as a 29 kd protein, rhoGAP is found in all cell types but is unrelated in sequence to rasGAP. Although the complete sequence of rhoGAP is not yet available, partial sequence analysis has uncovered a family of related proteins. The first surprise was that a 30 aa peptide derived from rhoGAP has 50% homology to a region close to the C-terminusof bcr (Figure 3; Diekmann et al., 1991) the product of the gene that is rearranged in Philadelphiachromosome (Ph+)-positive, chronic myeloid leukemias (CMLs) and several acute lymphocytic leukemias (ALLs). Expression in E. coli confirms that the C-terminal domain of bcr and of a related protein, n-chimerin, both encode GAPS, but for p21”“, not for p21”o. n-Chimerin appears to be brain specific, and although its function is unknown, it is highly enriched in areas associated with the control of complex learned behaviors (George and Clayton, 1992). The role of bcr in leukemia has been under investigation for several years. In CML, the 9;22 translocation of the Ph+ chromo-

some results in the formation of ~21Obc’-*~‘, a fusion protein between N-terminal bcr sequences and the abl oncoprotein (Figure 3), and it is thought that a phosphoserine residue from bcr interacts with an SH2 domain in abl to activate its tyrosine kinase activity (Pendergast et al., 1991). However, bcr is turning out to be a much more complicated multidomain protein than initially thought (Figure 3). In addition to interacting with abl’s SH2 domain, the N-terminus encodes a novel serine kinase unrelated in sequence to other cellular kinases (Maru and Witte, 1991). Downstream of this kinase domain is a region with homology to the product of the dbl oncogene, recently shown to be a guanine nucleotide exchange factor for the CDC42 GTP-binding protein (Hart et al., 1991), and the C-terminus of bcr encodes a GAP for rat. The biological implications of these recent observations have not yet been addressed. For example, in addition to the deregulated abl tyrosine kinase, ~21Obc”*~ found in CML contains the bcr serine kinase and the putative nucleotide exchange factor domain related to dbl. On the other hand, p1856”‘-aMfound in ALL (Figure 3) which appears to be a more potent transforming protein, does not contain the dbl domain. Also, it is not clear whether the abl-bcr products of the reciprocal translocation, which would contain the GAP domain, are expressed. Another protein of considerable interest in growth control has a domain related to rhoGAP-the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase (Otsu et al., 1991). Although it has not yet been shown to have any GAP activity, the homology points to an interaction between p85 and one or more of the rho-like proteins. The organization of p85 is somewhat reminiscent of rasGAP, having SH2 and SH3 domains linked to a GAP-like domain (Figure 3). Perhaps an interaction between p85 and a rho-like protein allows its SH2 and SH3 domains to interact with target proteins. The most intriguing revelation to date concerning GAPrelated proteins involves the association of rasGAP with tyrosine kinase receptors and two phosphotyrosinecontaining proteins, p62 and ~190, upon stimulation of cells with growth factors. Settleman et al. (1992) and Wong et al. (1992) have obtained by immunoprecipitation sufficient ~190 and p62 amino acid sequence to allow the isolation of cDNA clones. p82 shows extensive sequence similarity to an hnRNP protein, GRP33, and recombinant p62 binds nucleic acid. It is hard to speculate what the role of the rasGAP-p62 complex might be, but it could be involved in some aspect of mRNA processing. Analysis of the ~190 clone reveals that its C-terminus looks like rhoGAP! With over 40% amino acid homology to rhoGAP/ bcrGAP/chimerin, it will be surprising if it does not show GAP activity toward one or more rho-related proteins. The open reading frame of ~190 has more surprises in store. Its N-terminus has three sequence motifs indicative of a GTP-binding protein, and although ~190 has not yet been shown to bind GTP, it’s a good bet that it will. The most dramatic discovery, however, must be that the central region of ~190 encodes a transcriptional repressor of the glucocorticoid receptor gene. Is this the long sought after link between the plasma membrane and the nucleus?

It’s certainly the best candidate so far-a transcription factor linked directly to regulatory proteins found at the plasma membrane. It could be that rasGAP regulates the activity of a transcription factor by sequestering it at the plasma membrane or by modifying it in some way; it’s too early to tell. At the very least, it effectively rules out a simple down-regulatory role for rasGAP in receptormediated signal transduction. What is not yet clear is whether ras is required for the formation of the rasGAP~190 complex, and if it is, whether that is its only function. In conclusion, the identification of rasGAP and rhoGAP, two prototypes for the ras and rho subfamily of small GTPases, has led to some remarkable discoveries over the last couple of years. The identification of related proteins is perhaps in itself not so surprising, but their complex multidomain character is intriguing (Figure 3). From the perspective of the rhoGAP domain, this could mean that, in addition to their other functions, ~85, bcr, or ~190 might be upstream regulators of rho-like GTP-binding proteins. If the proposed interaction between ras and rasGAP is any kind of a paradigm, however, a downstream model might be favored, in which rho-like GTP-binding proteins regulate the activities of ~85, bcr, and/or p190 by interacting with their GAP-like domains. We now know something about the cellular function of rho-like proteins: they regulate the formation of multimolecular actin-associated complexes at the plasma membrane (Hall, 1992); perhaps these include ~85, bcr, and pl90. Interestingly, ~85 contains an SH3 domain, which is found in a subset of proteins that can associate with cortical actin. bcr does not have an SH3 domain but can apparently activate the SH3 domain of abl, allowing it to associate with the cytoskeleton. Similarly, ~190 does not have an SH3 domain, but its partner rasGAP does. There is clearly a long way to go yet in understanding the complex interactions between small GTP-binding proteins and their GAPS. rasGAP- and rhoGAP-related proteins are going to fuel the imagination for some time to come.

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