Residues in Cdc42 that specify binding to individual CRIB effector proteins

Cdc42 is a member of the Rho family of small G proteins. Signal transduction events emanating from Cdc42 lead to cytoskeletal rearrangements, cell proliferation, and cell differentiation. Many effector proteins have been identified for Cdc42; however, it is not clear how certain effectors specifically recognize and bind to Cdc42, as opposed to Rac or Rho, or in many cases, which effector controls what cellular events. Mutations were introduced into Cdc42 at residues: Met1, Val8, Phe28, Tyr32, Val33, Thr35, Val36, Phe37, Asp38, Tyr40, Val42, Met45, Ile46, Glu127, Ala130, Asn132, Gln134, Lys135, and Leu174. Measurements were made of their equilibrium binding constants to the Cdc42 binding domains of the CRIB effectors ACK, PAK, and WASP and to the GTPase-activating protein Rho GAP. Generally, mutations in the effector loop have an equally deleterious effect on binding to all CRIB proteins tested, though the F37A mutation resulted in significant selectivity. Residues outside the effector loop were found to be important for binding of Cdc42 to CRIB containing proteins and also to contribute to selectivity. Mutations such as V42A and L174A resulted in large, selective changes in binding to specific CRIB effectors. Neither mutation resulted in alteration in PAK binding, whereas both severely disrupt binding to ACK and only L174A disrupted binding to WASP. These mutations are interpreted using the structures of the Cdc42/ACK and Cdc42/WASP complexes to give insight into how effectors can specifically recognize Cdc42. Those mutations in Cdc42 that inhibit certain interactions, while retaining others, should aid investigations of the role of specific effectors in Cdc42 signaling in vivo.     

Localization of the PAK1-, WASP-, and IQGAP1-specifying regions of Cdc42.

The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.     

Structure of Cdc42 bound to the GTPase binding domain of PAK

The Rho family GTPases, Cdc42, Rac and Rho, regulate signal transduction pathways via interactions with downstream effector proteins. We report here the solution structure of Cdc42 bound to the GTPase binding domain of alphaPAK, an effector of both Cdc42 and Rac. The structure is compared with those of Cdc42 bound to similar fragments of ACK and WASP, two effector proteins that bind only to Cdc42. The N-termini of all three effector fragments bind in an extended conformation to strand beta2 of Cdc42, and contact helices alpha1 and alpha5. The remaining residues bind to switches I and II of Cdc42, but in a significantly different manner. The structure, together with mutagenesis data, suggests reasons for the specificity of these interactions and provides insight into the mechanism of PAK activation.     

The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: interfaces differ between the complexes

IQGAP1 contains a domain related to the catalytic portion of the GTPase-activating proteins (GAPs) for the Ras small G proteins, yet it has no RasGAP activity and binds to the Rho family small G proteins Cdc42 and Rac1. It is thought that IQGAP1 is an effector of Rac1 and Cdc42, regulating cell-cell adhesion through the E-cadherin-catenin complex, which controls formation and maintenance of adherens junctions. This study investigates the binding interfaces of the Rac1-IQGAP1 and Cdc42-IQGAP1 complexes. We mutated Rac1 and Cdc42 and measured the effects of mutations on their affinity for IQGAP1. We have identified similarities and differences in the relative importance of residues used by Rac1 and Cdc42 to bind IQGAP1. Furthermore, the residues involved in the complexes formed with IQGAP1 differ from those formed with other effector proteins and GAPs. Relatively few mutations in switch I of Cdc42 or Rac1 affect IQGAP1 binding; only mutations in residues 32 and 36 significantly decrease affinity for IQGAP1. Switch II mutations also affect binding to IQGAP1 although the effects differ between Rac1 and Cdc42; mutation of either Asp-63, Arg-68, or Leu-70 abrogate Rac1 binding, whereas no switch II mutations affect Cdc42 binding to IQGAP1. The Rho family "insert loop" does not contribute to the binding affinity of Rac1/Cdc42 for IQGAP1. We also present thermodynamic data pertaining to the Rac1/Cdc42-RhoGAP complexes. Switch II contributes a large portion of the total binding energy to these complexes, whereas switch I mutations also affect binding. In addition we identify "cold spots" in the Rac1/Cdc42-RhoGAP/IQGAP1 interfaces. Competition data reveal that the binding sites for IQGAP1 and RhoGAP on the small G proteins overlap only partially. Overall, the data presented here suggest that, despite their 71% identity, Cdc42 and Rac1 appear to have only partially overlapping binding sites on IQGAP1, and each uses different determinants to achieve high affinity binding.     

Double mutant cycle thermodynamic analysis of the hydrophobic Cdc42-ACK protein-protein interaction

Protein-protein interactions such as those between small G proteins and their effector proteins control most cell signaling pathways and thereby govern many cellular processes in both normal and disease states. Each small G protein interacts with several effectors, some shared between similar G proteins and others unique to a single GTPase. Although there is knowledge of the structural basis of these interactions, there is limited understanding of their thermodynamic basis. This is particularly significant because of the intrinsic conformational flexibility of the interacting partners. Here we have conducted a double mutant thermodynamic cycle for two key hydrophobic interactions in the Cdc42-ACK interface: Val42Cdc42-Ile463ACK and Leu174Cdc42-Leu449ACK. Val42 and Leu174 are known to be energetically important in this complex from previous thermodynamic studies, and their respective partners were predicted from the structure of the complex. Such a study has not been hitherto performed on any hydrophobic protein-protein interaction. The results confirm that a significant proportion of the overall interaction is dependent upon these residues, but in neither case is the direct interaction between the side chains the predominant energetic force. Indeed, the interaction of the side chains of Val42 and Ile463 appears to exert an energetic penalty. Rather, the stabilization of the complex, which requires the presence of these two pairs of residues, appears to be due to conformational changes, or interactions, that are not easily visualized in the structure of the complexes. In this respect, it is noteworthy that isolated Cdc42 shows regions of disorder and isolated ACK has no stable tertiary structure, whereas the Cdc42-ACK complex has a well-defined quaternary structure. Such changes may well be critical for the known selectivity of Cdc42 and related proteins such as Rho and Rac, for their wide range of effectors.     

Identification of the region in Cdc42 that confers the binding specificity to activated Cdc42-associated kinase

The Rho family small G-protein Cdc42 has been implicated in a diversity of biological functions. Multiple downstream effectors have been identified. How Cdc42 discriminates the interaction with its multiple downstream effectors is not known. Activated Cdc42-associated tyrosine kinase (ACK) is a very specific effector of Cdc42. To delineate the Cdc42 signaling pathway mediated by ACK, we set about to identify the specific ACK-binding region in Cdc42. We utilized TC10, another member of the Rho family of G-proteins that is 66.7% identical to Cdc42, to construct TC10/Cdc42 chimeras for screening the specific ACK-binding region in Cdc42. A region between switch I and switch II has been identified as the specific ACK-binding (AB) region. The replacement of the AB region with the corresponding region in TC10 resulted in the complete loss of ACK-binding ability but did not affect the binding to WASP, suggesting that the AB region confers the binding specificity to ACK. On the other hand, replacement of the corresponding region of TC10 with the AB region enabled TC10 to acquire ACK-binding ability. Eight residues are different between the AB region and the corresponding region of TC10. The mutational analysis indicated that all eight residues contribute to the binding to ACK2. The assays for the Cdc42-mediated activation of ACK2 indicated that the AB region is essential for Cdc42 to activate ACK2 in cells. Thus, our studies have defined a specific ACK-binding region in Cdc42 and have provided a molecular basis for generating ACK binding-defective mutants of Cdc42 to delineate ACK-mediated signaling pathway.     

The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway

The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.     

Conformational Switch and Role of Phosphorylation in PAK Activation

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.     

Phosphorylation of WASP by the Cdc42-associated kinase ACK1: dual hydroxyamino acid specificity in a tyrosine kinase

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.     

A method to measure the interaction of Rac/Cdc42 with their binding partners using fluorescence resonance energy transfer between mutants of green fluorescent protein

Enhanced blue fluorescent protein (EBFP) and enhanced green fluorescent protein (EGFP) mutants of GFP in close proximity to one another can act as a fluorescence resonance energy transfer (FRET) pair. Unstructured amino acid linkers of varying length were inserted between EBFP and EGFP, revealing that linkers even as long as 50 amino acids can be accommodated and still allow FRET to occur. This led to the development of a novel biosensor for Rac/Cdc42 binding to their effector proteins based on the insertion of amino acids 75-118 of p21-activated kinase (PAK) between the GFP mutants. We demonstrate that this protein construct allows significant FRET between EBFP and EGFP and retains the ability to bind to Rac in its GTP-bound form with a binding affinity similar to the uncomplexed PAK fragment, and furthermore, on binding to Rac or Cdc42 a marked change in FRET takes place. This forms the basis for a simple, sensitive, and rapid method to measure binding of Rac/Cdc42 to their effector proteins. Since the signal is dependent upon the interaction with active GTP-bound forms it acts as a biosensor for the activation of Rac/Cdc42. It has the potential for use in live cells and for identifying localization of Rac/Cdc42 within subcellular compartments.     

Epidermal growth factor-dependent regulation of Cdc42 is mediated by the Src tyrosine kinase

Treatment of cells with epidermal growth factor (EGF) promotes the activation of the small GTP-binding protein Cdc42, as well as its phosphorylation in cells. The EGF-dependent phosphorylation of Cdc42 occurs at tyrosine 64 in the Switch II domain and appears to be mediated through the Src tyrosine kinase, because both the expression of a dominant-negative Src mutant (mouse Src(K297R)) and treatment of cells with the Src kinase inhibitor PP2 blocks the EGF-stimulated phosphorylation of Cdc42, whereas expression of an activated Src mutant (Src(Y529F)) promotes phosphorylation in the absence of EGF treatment. The EGF-stimulated phosphorylation of Cdc42 is not required for its activation, nor does it directly affect the interactions of activated Cdc42 with target/effector proteins including PAK, ACK, WASP, or IQGAP. However, the EGF-stimulated phosphorylation of Cdc42 is accompanied by an enhancement in the interaction of Cdc42 with the Rho-GDP dissociation inhibitor (RhoGDI). The EGF-stimulated activation of Cdc42 does require activated Src, as well as the Vav2 protein, a member of the Dbl family of guanine nucleotide exchange factors. Src catalyzes the tyrosine phosphorylation of Vav2, and overexpression of Vav2 together with activated Src (Src(Y529F)) can completely bypass the need for EGF to promote the activation of Cdc42. Thus, EGF signaling through Src appears to have dual regulatory effects on Cdc42: 1). it leads to the activation of Cdc42 as mediated by the Vav2 guanine nucleotide exchange factor, and 2). it results in the phosphorylation of Cdc42, which stimulates the binding of RhoGDI, perhaps to direct the movement of Cdc42 to a specific cellular site to trigger a signaling response, because Cdc42-RhoGDI interactions are essential for Cdc42-induced cellular transformation.     

Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo. Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation

Src homology 3 domain (SH3)-containing proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the c-Jun NH(2)-terminal kinase pathway. The mechanisms by which SPRK activity is regulated are not well understood. The small Rho family GTPases, Rac and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including SPRK, which contain Cdc42/Rac interactive binding motifs. Coexpression of SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac interactive binding-like motif contains six of the eight consensus residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is required for SPRKs association with and activation by Cdc42. However, experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which Cdc42 increases SPRKs catalytic activity involves a change in the in vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase. These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.     

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.     

Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner

The tyrosine kinase ACK1 phosphorylates and activates the guanine nucleotide exchange factor Dbl, which in turn directs the Rho family GTP-binding proteins. However, the regulatory mechanism of ACK1/Dbl signaling in response to extracellular stimuli remains poorly understood. Here we describe that epidermal growth factor stimulates the ACK1/Dbl pathway, leading to actin cytoskeletal rearrangements. The role of the two ACK1-binding proteins Cdc42 and Grb2 was assessed by overexpression of the Cdc42/Rac interactive binding domain and a dominant-negative Grb2 mutant, respectively. Specific inhibition of the interaction of ACK1 with Cdc42 or Grb2 by the use of these constructs diminished tyrosine phosphorylation of both ACK1 and Dbl in response to EGF. Therefore, the activation of ACK1 and subsequent downstream signaling require both Cdc42-dependent and Grb2-dependent processes within the cell. In addition, we show that EGF transiently induces formation of the focal complex and stress fibers when ACK1 was ectopically expressed. The induction of these structures was totally sensitive to the action of botulinum toxin C from Clostridium botulinum, suggesting a pivotal role of Rho. These results provide evidence that ACK1 acts as a mediator of EGF signals to Rho family GTP-binding proteins through phosphorylation and activation of GEFs such as Dbl.     

Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).     

Structural analysis of the SH3 domain of beta-PIX and its interaction with alpha-p21 activated kinase (PAK)

The PAK Ser/Thr kinases are important downstream effectors of the Rho family GTPases Cdc42 and Rac, partly mediating the role of these G proteins in cell proliferation and cytoskeletal rearrangements. As well as small G proteins, PAK interacts with the Cdc42/Rac exchange factor beta-PIX via the PIX SH3 domain and a nontypical Pro-rich region in PAK. This interaction is thought to affect the localization of PAK, as well as increased GTP/GDP exchange of Rac and Cdc42. We have determined the structure of the PIX-SH3/PAK peptide complex and shown that it differs from typical Src-like SH3/peptide complexes. The peptide makes contacts through the Pro-rich sequence in a similar way to standard SH3/peptide complexes, even though the Pro residue positions are not conserved. In addition, there are interactions with a Pro and Lys in the PAK, which are C-terminal to the conserved Arg found in all SH3-binding sequences. These contact a fourth binding pocket on the SH3 domain. We have measured the affinity of PIX-SH3 for the PAK peptide and found that it is of intermediate affinity. When PAK is activated, Ser-199 in the PIX-binding site is phosphorylated. This phosphorylation is sufficient to reduce the affinity for PIX 6-fold.     

Phosphorylation of RhoGDI1 by p21-activated kinase 2 mediates basic fibroblast growth factor-stimulated neurite outgrowth in PC12 cells

We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.     

Multiple Rho proteins regulate the subcellular targeting of PAK5

We investigated the regulatory mechanisms controlling the subcellular localization of p21-activated kinase 5 (PAK5) and found that the Cdc42/Rac interactive binding (CRIB) domain within PAK5 is critical for proper targeting within the cell. We also observed that PAK5 interacts with RhoD and RhoH in addition to Cdc42, and that interaction with RhoD targets PAK5 to subcellular locations that are distinct from those stimulated by Cdc42. Through deletion analysis we observed that the mitochondrial localization of PAK5 is controlled by multiple domains, providing evidence that the kinase activity of PAK5 is critical to its ability to cycle on and off mitochondria, and demonstrate that expression of kinase-inactive PAK5 elicits dramatic effects on mitochondrial morphology. These data indicate that PAK5 is directed to distinct subcellular locations by different Rho family small G proteins as well as by intrinsic targeting sequences.     

Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization

The family of p21-activated kinases (PAKs) have been implicated in the rearrangement of actin cytoskeleton by acting downstream of the small GTPases Rac and Cdc42. Here we report that even though Cdc42/Rac1 or Akt are not activated, phosphatidylinositol-3 (PI-3) kinase activation induces PAK1 kinase activity. Indeed, we demonstrate that PI-3 kinase associates with the N-terminal regulatory domain of PAK1 (amino acids 67-150) leading to PAK1 activation. The association of the PI-3 kinase with the Cdc42/Rac1 binding-deficient PAK1(H83,86L) confirms that the small GTPases are not involved in the PI-3 kinase-PAK1 interaction. Furthermore, PAK1 was activated in cells expressing the dominant-negative forms of Cdc42 or Rac1. Additionally, we show that PAK1 phosphorylates actin, resulting in the dissolution of stress fibers and redistribution of microfilaments. The phosphorylation of actin was inhibited by the kinase-dead PAK1(K299R) or the PAK1 autoinhibitory domain (PAK1(83-149)), indicating that PAK1 was responsible for actin phosphorylation. We conclude that the association of PI-3 kinase with PAK1 regulates PAK1 kinase activity through a Cdc42/Rac1-independent mechanism leading to actin phosphorylation and cytoskeletal reorganization.