The PAK family of kinases are regulated through interaction with the small GTPases Cdc42 and Rac1, but little is known of the signaling components immediately upstream or downstream of these proteins. We have purified and cloned a new class of Rho-p21 guanine nucleotide exchange factor binding tightly through its N-terminal SH3 domain to a conserved proline-rich PAK sequence with a Kd of 24 nM. This PAK-interacting exchange factor (PIX), which is widely expressed and enriched in Cdc42- and Rac1-driven focal complexes, is required for PAK recruitment to these sites. PIX can induce membrane ruffling, with an associated activation of Rac1. Our results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex.

The p21-activated kinase PAK is targeted to focal complexes (FCs) through interactions with the SH3 domains of the PAK-interacting exchange factor PIX and Nck. PIX is a Rac GTP exchange factor that also binds the G-protein-coupled receptor kinase-interacting protein known as GIT1. Overexpression of GIT1 in fibroblasts or epithelial cells causes a loss of paxillin from FCs and stimulates cell motility. This is due to the direct interaction of a C-terminal 125-residue domain of GIT1 with paxillin, under the regulation of PIX. In its activated state, GIT1 can promote FC disassembly independent of actin-myosin contractile events. Additionally, GIT directly couples to a key component of FCs, focal adhesion kinase (FAK), via a conserved Spa2 homology domain. We propose that GIT1 and FAK cooperate to promote motility both by directly regulating focal complex dynamics and by the activation of Rac.PAKs are a family of kinases activated on interacting with Cdc42 and Rac GTPases (3,21). PAK associates in Drosophila with phosphotyrosine-rich cytoskeletal structures (10) and in cultured cells with focal complexes (FCs) which represent sites of attachment with the substratum (20). PAK association with FCs is independent of the Cdc42/Rac1-binding domain. Constitutively active PAK promotes FC disassembly (20) which can be blocked by the inhibitory PAK 83-149 fragment (41). Cells expressing kinase-deficient PAK exhibit more stable FCs and increased formation of Rac-dependent lamellipodia (26). Rac1 activation can occur through the ubiquitous PAK-interacting exchange factor PIX, which specifically binds PAKs (1, 22). PAK plays an essential role in axonal guidance in Drosophila, achieved through its binding via the second SH3 domain of Nck (13). PAK binding to PIX through a unique SH3 interaction plays an important role in coordinating the activation of Cdc42 and Rac1 in a phosphatidylinositol 3-kinase (PI-3-(kinase)-dependent manner (40). It is an association with PIX that allows PAK to localize to FCs (22), although this interaction is negatively regulated by PAK activation and autophosphorylation (42).Recently PIX has been shown to interact with a family of proteins first designated G-protein-coupled receptor kinase (GRK)-interacting targets (GIT [28]); these proteins contain at their N termini a domain that can function as an Arf GTPase-activating protein (GAP). This family of tyrosinephosphorylated ϳ90-kDa proteins can also bind paxillin (2, 37), indicating that PAK might be linked to FCs through this interaction. Interestingly, a close link between Arfs and FCs is indicated by the observation that the related Arf GAP ASAP (29) and PAG3 (16) both localize to FCs and can promote their turnover.FCs are integrin-dependent sites linking the extracellular matrix (ECM) to the actin-rich cytoskeleton. Their formation is influenced particularly by RhoA, Cdc42, and Rac1 (24). RhoA induces the formation of steady-state FCs (or focal adhesions) which allow cultured cells to remain attached to the ECM. RhoA-and Cdc42-induced FCs require ...

No abstract

The Rho GTPases are a family of molecular switches that are critical regulators of signal transduction pathways in eukaryotic cells. They are known principally for their role in regulating the cytoskeleton, and do so by recruiting a variety of downstream effector proteins. Kinases form an important class of Rho effector, and part of the biological complexity brought about by switching on a single GTPase results from downstream phosphorylation cascades. Here we focus on our current understanding of the way in which different Rho-associated serine/threonine kinases, denoted PAK (p21-activated kinase), MLK (mixed-lineage kinase), ROK (Rho-kinase), MRCK (myotonin-related Cdc42-binding kinase), CRIK (citron kinase) and PKN (protein kinase novel), interact with and are regulated by their partner GTPases. All of these kinases have in common an ability to dimerize, and in most cases interact with a variety of other proteins that are important for their function. A diversity of known structures underpin the Rho GTPase-kinase interaction, but only in the case of PAK do we have a good molecular understanding of kinase regulation. The ability of Rho GTPases to co-ordinate spatial and temporal phosphorylation events explains in part their prominent role in eukaryotic cell biology.

The kinase PAK binds tightly to the SH3 domain of its partner PIX via a central proline-rich sequence. A different N-terminal sequence allows ␣PAK to bind an SH3 domain of the adaptor Nck. The Nck SH3[2] domain interacts equally with an 18-mer PAK-derived peptide and full-length ␣PAK. Detailed analysis of this binding by saturation substitution allows related Nck targets to be accurately identified from sequence characteristics alone. All Nck SH3[2] binding proteins, including PAK, NIK, synaptojanin, PRK2, and WIP, possess the motif PXXPXRXXS; in the case of PAK, serine phosphorylation at this site negatively regulates binding. We show that kinase autophosphorylation blocks binding by both Nck and PIX to ␣PAK, thus providing a mechanism to regulate PAK interactions with its SH3-containing partners. One cellular consequence of the regulatable binding of PAK is facilitation of its cycling between cytosolic and focal complex sites.Signal transduction pathways often utilize protein-protein interaction modules whose domain structures are conserved at the primary or secondary structural level. Two domains frequently found on signaling molecules are Src homology 2 and 3 domains (41). In Src, these domains not only regulate association with other proteins but also intramolecular functions, including protein tyrosine kinase activity (35). The adaptor signaling molecules contain no catalytic domain (41). The best studied of these is Grb2, whose SH3 domains complex to the Ras guanine nucleotide exchange factor (GEF) SOS. Upon stimulation, tyrosine kinase receptors that engage Grb2 through binding to its SH2 domains recruit the Grb2-SOS complex, thus causing Ras activation (7).We have recently described a new class of Rac1 GEF whose SH3 domain binds selectively to a nonconventional prolinerich binding sequence present in all mammalian PAKs (32). Because these PAK-interacting exchange (PIX) proteins are complexed to PAK, the kinase has roles both upstream and downstream of Rac and/or Cdc42 (39). Thus, recruitment of the complex via PAK leads to Rac activation, while PIX itself is known to play a role in localizing PAK to focal complexes (FCs) and activating the kinase (32). Although Cdc42 or Rac directly activate PAKs, the ubiquitous adaptor protein Nck, which binds to an N-terminally located proline-rich sequence (2,5,14), can also activate PAK by recruitment to the plasma membrane (27). PAK activation by Nck is mimicked when membrane-localizing signals are directly attached to PAK (30). Nck contains three tandem SH3 domains and a C-terminally located SH2 domain (8,9,25). The Drosophila Nck homologue, Dock, plays a role in axonal guidance: both DPak and Dock are highly expressed in the nervous system (15, 18). Membrane-tethered DPak acts as a dominant gain-of-function protein in dock mutants, restoring the normal pattern of R-cell connectivity; thus, DPak is a key downstream partner of Dock (19).The structures of many SH3 domains have been determined by crystallographic and nuclear magnetic resonance protocols.These analyse...