Jul 31, 1995 - ras and rho subfamilies are, in particular, involvedin regulating signal transduction pathways. Ras is crucial for controlling cell growth and ...
The EMBO Journal vol.14 no.21 pp.5297-5305, 1995
Rac GTPase interacts with GAPs and target proteins through multiple effector sites
Dagmar Diekmann, Catherine D.Nobes, Peter D.Burbelo, Arie Abol and Alan Hall2 CRC Oncogene and Signal Transduction Group, MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, UK and 'Onyx Pharmaceuticals, 3031 Research Drive, Building A, Richmond, CA 94806, USA 2Corresponding author
Rac, a small GTPase in the ras superfamily, regulates at least two biological processes in animal cells: (i) the polymerization of actin and the assembly of integrin complexes to produce lamellipodia and ruffles; and (ii) the activity of an NADPH oxidase in phagocytic cells. NADPH oxidase activation is mediated through a rac effector protein, p67phox, and using chimeras made between rac and the closely related GTPase, rho, we have identified two distinct effector sites in rac, one Nterminal and one C-terminal, both of which are required for activation of p67phoxr The same two effector sites are essential for rac-induced actin polymerization in fibroblasts. p65PAK, a ubiquitous serine/ threonine kinase, interacts with rac at both the N- and C-terminal effector sites, but the GTPase-activating protein, ber interacts with rac at a different region. This makes p65PAK, but not bcr, a candidate effector of rac-induced lamellipodium formation. Keywords: actin/bcr/NADPH oxidase/p65PAK kinase/rac/ rho
Introduction Over 50 members of the ras superfamily of small GTPbinding proteins have been identified in mammalian cells (Hall, 1990). They act as molecular switches to control a wide range of biological processes, but members of the ras and rho subfamilies are, in particular, involved in regulating signal transduction pathways. Ras is crucial for controlling cell growth and differentiation and its function is to localize the cytoplasmic serine/threonine kinase c-raf 1 to the plasma membrane where it is then able to stimulate the MAP (ERKI, 2) kinase cascade (Leevers et al., 1994; Stokoe et al., 1994) The mammalian rho-subfamily of small GTPases consists of five distinct proteins: rho, rac, cdc42, rhoG and TC1O (Hall, 1994). In Swiss 3T3 cells, rho regulates a signal transduction pathway linking growth factor receptors to the assembly of focal adhesions and actin stress fibres (Ridley and Hall, 1992). Rac, on the other hand, controls the polymerization of actin and the assembly of associated focal complexes at the plasma membrane to produce lamellipodia and membrane ruffles in response to growth factor stimulation (Ridley et al., 1992; Nobes © Oxford University Press
and Hall, 1995). The third important member of this subfamily, cdc42, triggers the formation of filopodia and associated focal complexes when injected into subconfluent Swiss 3T3 cells (Nobes and Hall, 1995). A distinct biological role for rac has been described in phagocytic cells, where it is required for activation of the NADPH oxidase enzyme complex. The complex consists of two membrane-bound cytochrome b558 subunits (p22 and gp9l) and two proteins recruited from the cytosol, P47phox and P67phox (Segal and Abo, 1993). In addition to these four essential components, the activity of the complex is dependent on rac (Abo et al., 1991). It has been possible to reconstitute NADPH oxidase activity in a cellfree system using purified cytochrome b, P47phoxg P67phox and rac in the GTP-form (Abo et al., 1992). Finally, it has recently been reported that constitutively activated rac is capable of transforming ratl fibroblast cells to a malignant phenotype, suggesting that rac and perhaps other rho-related GTPases can signal to the nucleus and affect gene transcription (Qiu et al., 1995). To understand the molecular basis of the diverse biological activities associated with the rho family of GTPases, it is important to identify their downstream target molecules, or effectors. We have recently identified p67phox as the rac target in the NADPH oxidase complex and suggested that the enzyme complex assembles independently of rac, but that its activity is dependent on the subsequent interaction of rac.GTP with p67phox (Diekmann et al., 1994). A biochemical target for rac in fibroblasts has not yet been identified. One candidate, the serine/ threonine kinase p65 PAK, is stimulated by rac, but not rho, in a GTP-dependent manner (Manser et al., 1994). p65PAK is related to STE20, a Saccharomyces cerevisiae kinase required for pheromone activation of the MAP kinase pathway in this organism and this provides further support for the idea that rac might signal to the nucleus in mammalian cells (Leberer et al., 1992; Ramer and Davis, 1993). Other potential downstream targets of the rho family members are GTPase-activating proteins or GAPs. At least five mammalian GAPs have so far been identified that are specific for the rho family: rhoGAP, p190 and myrS stimulate the GTPase activity of rho, rac and cdc42, while bcr, the product of the breakpoint cluster gene, and n-chimerin are active on rac and cdc42, but not rho (Diekmann et al., 1991; Settleman et al., 1992; Lancaster et al., 1994; Reinhard et al., 1995). To identify the sites on rac that interact with target molecules, we have compared the biochemical and biological activities of rac/rho chimeras. Rac has an Nterminal effector site, surrounding amino acids 30-40, that is essential for activation of NADPH oxidase and for induction of actin polymerization leading to lamellipodia. Although this site is necessary for both these activities, it
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is not sufficient and a second, C-terminal, effector site at amino acids 143-175 is also required. The same two effector sites are needed for p65PAK activation, but a distinct region of rac, amino acids 74-90, is required for interaction with bcr. These results raise the possibility that p65PAK, but not bcr, is a candidate effector of lamellipodium formation.
Results An N-terminal effector region is essential in rac In ras, a single N-terminal effector site spanning residues 22-46 is both necessary and sufficient for interacting with the target protein c-rafl and for inducing downstream biological effects (Zhang et al., 1990; Self et al., 1993). A corresponding region has also been shown to be essential for the biological activity of rho, since amino acid substitutions at codons 34 and 36 (corresponding to 32 and 34 in ras and rac) block induction of actin stress fibres (Self et al., 1993). We reported recently that rac with substitutions in the 30-40 region was unable to interact with p67phox and was inactive in an in vitro NADPH oxidase assay (Diekmann et al., 1994). To determine whether this N-terminal region of rac is required to induce lamellipodia in fibroblasts, we have microinjected sub-confluent Swiss 3T3 fibroblasts with recombinant, constitutively activated (V12rac) rac proteins containing additional amino acid substitutions at codon 35 (threonine-alanine), 38 (aspartic acid->alanine) or 40 (tyrosine-4lysine). None of these three proteins is able to induce actin polymerization or focal complex assembly associated with lamellipodia (data not shown). We conclude that an N-terminal effector site surrounding amino acids 30-40 is essential for all the biological effects so far described for the rho and rac proteins. An additional region is required for bcrGAP sensitivity of rac It has been shown that the N-terminal effector region of ras is the interaction site for the two GTPase-activating proteins rasGAP and NFI (Adari et al., 1988; Cales et al., 1988). In contrast, mutational analysis of the N-terminal 60 amino acids of rho suggests that this is not the site of interaction with rhoGAP (Self et al., 1993); the binding site for rhoGAP on rho and rac is currently unknown. However, since rho is effectively insensitive to bcrGAP we have made use of rac/rho chimeras to look for the site on rac conferring sensitivity. Figure lA shows that a rac73rho chimera (N-terminal 73 residues from rac, the remainder from rho) is 30-fold less sensitive to bcrGAP stimulation than rac. The chimera has unaltered sensitivity towards rhoGAP (data not shown) confirming that the conformational integrity of the GTPase is not compromised. By screening additional chimeras, we have localized a small region of rac between residues 74 and 90, which when replaced with sequences from rho (rac73rho9Orac) results in the same 30-fold reduction in bcrGAP activity (Figure lA). Confirmation that this region confers sensitivity to bcrGAP was obtained by introducing residues 74-90 of rac into rho. This chimera (rho73rac90rho) is 200-fold more sensitive than rho to bcrGAP. We refer to the region 74-90 as the 'bcrGAP site'. Figure lA reveals that the rac73rho chimera is neverthe-
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less more sensitive to bcrGAP than is rho. We conclude that full sensitivity to bcrGAP requires two sites on rac: the N-terminal effector site and the GAP site. In agreement with this a rac' '7rho chimera is as sensitive as rac to bcrGAP (Figure IA). Interestingly, the Asp38-4Ala and Tyr4O-+Lys N-terminal effector site substitutions, previously shown to block the biological activity of rac, have no significant effect on bcrGAP-stimulated GTPase activity (Figure iB). A Thr35-Ala change abolished sensitivity to both bcrGAP and rhoGAP as expected, since this amino acid is required for the correct coordination of Mg2+ in the p21 structure (Pai et al., 1989). To determine whether residues 74-90 of rac are also required for interaction with bcrGAP as well as for its sensitivity to GTPase stimulation we have used a dot blot assay. Activated (L61) rac loaded with [y-32P]GTP interacts strongly with bcrGAP protein spotted onto nitrocellulose filters, but under these conditions neither L63rho nor L61rac73rho90rac show any detectable interaction (Figure IC). We conclude that residues 74-90 in rac are required not only for bcrGAP-stimulated GTPase activity but also for physical interaction between the two proteins.
Two sites in rac are required for p67phox binding and NADPH oxidase activation The affinity of p67phox for rac is dramatically reduced by the N-terminal effector site substitutions Ala38rac, Ala35rac, or Lys40rac (Figure 2 and Diekmann et al., 1994). These three substitutions also block activation of NADPH oxidase by rac, confirming that p67phox is the rac target in the complex (Figure 2 and Diekmann et al., 1994). To determine if the N-terminal effector site is sufficient for p67phox interaction, recombinant p67phox protein was spotted onto nitrocellulose filters and probed with [y-32P]GTP-labelled rac/rho chimeras. Figure 2 shows that, as expected, rho73rac does not interact with p67phox under these conditions (
