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.     

Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141

Pak2, a member of the p21-activated protein kinase (Pak) family, is activated in response to a variety of stresses and is directly involved in the induction of cytostasis. At the molecular level Pak2 binds Cdc42(GTP), translocating Pak2 to the endoplasmic reticulum where it is autophosphorylated and activated. Pak2 is autophosphorylated at eight sites; Ser-141 and Ser-165 in the regulatory domain and Thr-402 in the activation loop are identified as key sites in activation of the protein kinase. The function of phosphorylation of Ser-141 and Ser-165 on the activation was analyzed with wild-type (WT) and mutants of Pak2. With S141A, the level of autophosphorylation was reduced to 65% as compared with that of WT and S141D with a concomitant 45% reduction in substrate phosphorylation, indicating that phosphorylation at Ser-141 is required for optimal activity. Autophosphorylation inhibited the interaction between WT Pak2 and Cdc42(GTP). In 293T cells, WT Pak2, S141A, and S141D formed a stable complex with the constitutively active mutant Cdc42 L61, but not with the dominant negative Cdc42 N17. As shown in glutathione S-transferase pull-down assays, S141A bound to Cdc42(GTP) at a 6-fold higher level than that of S141D. In contrast, the S165A and S165D mutants had no effect on autophosphorylation, binding to Cdc42, or activation of Pak2. In summary, autophosphorylation of Ser-141 was required for activation of Pak2 and down-regulated the interaction of Pak2 with Cdc42. A model is proposed suggesting that binding of Cdc42 localizes Pak2 to the endoplasmic reticulum, where autophosphorylation alters association of the two proteins.     

Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics

Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. Here we show that p21-activated kinase (Pak1) phosphorylates LIM-kinase at threonine residue 508 within LIM-kinase's activation loop, and increases LIM-kinase-mediated phosphorylation of the actin-regulatory protein cofilin tenfold in vitro. In vivo, activated Rac or Cdc42 increases association of Pak1 with LIM-kinase; this association requires structural determinants in both the amino-terminal regulatory and the carboxy-terminal catalytic domains of Pak1. A catalytically inactive LIM-kinase interferes with Rac-, Cdc42- and Pak1-dependent cytoskeletal changes. A Pak1-specific inhibitor, corresponding to the Pak1 autoinhibitory domain, blocks LIM-kinase-induced cytoskeletal changes. Activated GTPases can thus regulate actin depolymerization through Pak1 and LIM-kinase.     

Pak1 kinase homodimers are autoinhibited in trans and dissociated upon activation by Cdc42 and Rac1.

Pak1, a serine/threonine kinase that regulates the actin cytoskeleton, is an effector of the Rho family GTPases Cdc42 and Rac1. The crystal structure of Pak1 revealed an autoinhibited dimer that must dissociate upon GTPase binding. We show that Pak1 forms homodimers in vivo and that its dimerization is regulated by the intracellular level of GTP-Cdc42 or GTP-Rac1. The dimerized Pak1 adopts a trans-inhibited conformation: the N-terminal inhibitory portion of one Pak1 molecule in the dimer binds and inhibits the catalytic domain of the other. One GTPase interaction can result in activation of both partners. Another ligand, betaPIX, can stably associate with dimerized Pak1. Dimerization does not facilitate Pak1 trans-phosphorylation. We conclude that the functional significance of dimerization is to allow trans-inhibition.     

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.     

The Cdc42 binding and scaffolding activities of the fission yeast adaptor protein Scd2

The small GTP-binding protein Cdc42, the guanine nucleotide exchange factor Scd1, the p21-activated kinase Shk1, and the adaptor protein Scd2 are involved in the Cdc42-dependent signaling cascade in fission yeast. In the present study, we analyzed the Cdc42 binding and scaffolding activities of Scd2 by co-precipitation assays. We found that two SH3-containing regions, amino acid residues 1-87 (CB1 (Cdc42-binding region 1)) and 110-266 (CB2), of Scd2 can bind to the GTP-bound form of Cdc42. CB2 is cryptic because of the intramolecular binding between the SH3 domain in CB2 (SH3(C)) and the PX domain and binds to Cdc42 only when the Scd2 PB1 domain binds to the PC motif-containing region (residues 760-872) of Scd1. This CB2.Cdc42 association, which would stabilize the open configuration of Scd2, enables the SH3(C) domain to bind to the polyproline motif of Shk1. We also found that the GTP-bound form of Cdc42 binds to the CRIB motif of Shk1 more strongly than to Scd2. Thus, Scd2 functions as a scaffold to form a protein complex, and the GTP-bound Cdc42 might be transferred effectively from the upstream activator Scd1 to the downstream effector Shk1 via Scd2.     

The active conformation of the PAK1 kinase domain

The p21-activated kinases (PAKs) participate in cytoskeletal control networks, downstream of Rho-family GTPases. A structure of PAK1 in an autoregulated, "off" state showed that a regulatory region, N-terminal to the kinase domain, forces the latter into an inactive conformation, prevents phosphorylation of Thr423 in the activation loop, and promotes dimerization. We have now determined structures at 1.8 A resolution for the free PAK1 kinase domain, with a mutation in the active site that blocks enzymatic activity, and for the same domain with a "phosphomimetic" mutation in the activation loop. The two very similar structures show that even in the absence of a phosphorylated Thr423, the kinase has an essentially active conformation. When Cdc42 binds the regulatory region and dissociates the dimer, PAK1 will be in an "intermediate-active" state, with a capacity to phosphorylate itself or other substrates even prior to modification of its activation loop.     

Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.     

Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity.

p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in vivo by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphingosine, which stimulate autophosphorylation and phosphorylation of exogenous substrates. The mechanism of Pak activation by these agents remains unclear. We investigated Pak kinase activation in more detail to gain insight into the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation. We present biochemical evidence that an autoinhibitory domain (ID) contained within amino acid residues 67-150 of Pak1 interacts with the carboxyl-terminal kinase domain and that this interaction is regulated in a GTPase-dependent fashion. Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited in trans by recombinant ID peptide, indicating similarities in their mode of activation. However, Pak1, which was autophosphorylated in response to either GTPase or sphingosine, is highly active and is insensitive to inhibition by the ID peptide. We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and enzymatic activity. Phosphorylation studies suggested that the stimulatory effect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation.     

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.     

Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase Acts as a Cdc42 Effector in Promoting Cytoskeletal Reorganization

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ~190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCKα and Cdc42^V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCKα resulted in actin and myosin reorganization. Expression of kinase-dead MRCKα blocked Cdc42^V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCKα mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCKα plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCKα mutants. These results suggest that MRCKα may act as a downstream effector of Cdc42 in cytoskeletal reorganization.     

Fission yeast pak1+ encodes a protein kinase that interacts with Cdc42p and is involved in the control of cell polarity and mating.

A STE20/p65pak homolog was isolated from fission yeast by PCR. The pak1+ gene encodes a 72 kDa protein containing a putative p21-binding domain near its amino-terminus and a serine/threonine kinase domain near its carboxyl-terminus. The Pak1 protein autophosphorylates on serine residues and preferentially binds to activated Cdc42p both in vitro and in vivo. This binding is mediated through the p21 binding domain on Pak1p and the effector domain on Cdc42p. Overexpression of an inactive mutant form of pak1 gives rise to cells with markedly abnormal shape with mislocalized actin staining. Pak1 overexpression does not, however, suppress lethality associated with cdc42-null cells or the morphologic defeat caused by overexpression of mutant cdc42 alleles. Gene disruption of pak1+ establishes that, like cdc42+, pak1+ function is required for cell viability. In budding yeast, pak1+ expression restores mating function to STE20-null cells and, in fission yeast, overexpression of an inactive form of Pak inhibits mating. These results indicate that the Pak1 protein is likely to be an effector for Cdc42p or a related GTPase, and suggest that Pak1p is involved in the maintenance of cell polarity and in mating.     

alphaPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.     

Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20

The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G beta gamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.     

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.     

Oncogenic Dbl, Cdc42, and p21-activated kinase form a ternary signaling intermediate through the minimum interactive domains

Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.     

Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase

Selective activation of Rac GTPase signaling pathways requires the specific release of Rac from RhoGDI complexes. We identified a RhoGDI kinase from bovine brain as p21-activated kinase (Pak). Pak1 binds and phosphorylates RhoGDI both in vitro and in vivo at Ser101 and Ser174. This resulted in dissociation of Rac1-RhoGDI, but not RhoA-RhoGDI, complexes, as determined by in vitro assays of complexation and in vivo by coimmunoprecipitation analysis. We observed that Cdc42-induced Rac1 activation is inhibited by expression of Pak1 autoinhibitory domain. The dissociation of Rac1 from RhoGDI and its subsequent activation stimulated by PDGF or EGF is also attenuated by Pak1 autoinhibitory domain, and this is dependent on the ability of RhoGDI to be phosphorylated at Ser101/174. These results support a role for Pak1-mediated RhoGDI phosphorylation as a mechanism for Cdc42-mediated Rac activation, and suggest the possibility of Rac-induced positive feed-forward regulation of Rac activity.     

Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20

The Saccharomyces cerevisiae PAK (p21-activated kinase) family kinase Ste20 functions in several signal transduction pathways, including pheromone response, filamentous growth, and hyperosmotic resistance. The GTPase Cdc42 localizes and activates Ste20 by binding to an autoinhibitory motif within Ste20 called the CRIB domain. Another factor that functions with Ste20 and Cdc42 is the protein Bem1. Bem1 has two SH3 domains, but target ligands for these domains have not been described. Here we identify an evolutionarily conserved binding site for Bem1 between the CRIB and kinase domains of Ste20. Mutation of tandem proline-rich (PxxP) motifs in this region disrupts Bem1 binding, suggesting that it serves as a ligand for a Bem1 SH3 domain. These PxxP motif mutations affect signaling additively with CRIB domain mutations, indicating that Bem1 and Cdc42 make separable contributions to Ste20 function, which cooperate to promote optimal signaling. This PxxP region also binds another SH3 domain protein, Nbp2, but analysis of bem1Delta versus nbp2Delta strains shows that the signaling defects of PxxP mutants result from impaired binding to Bem1 rather than from impaired binding to Nbp2. Finally, the PxxP mutations also reduce signaling by constitutively active Ste20, suggesting that postactivation functions of PAKs can be promoted by SH3 domain proteins, possibly by colocalizing PAKs with their substrates. The overall results also illustrate how the final signaling function of a protein can be governed by combinatorial addition of multiple, independent protein-protein interaction modules.     

Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp

The Paks (p21-activated kinases) Pak1, Pak2 and Pak3 are among the most studied effectors of the Rho-family GTPases, Rac, Cdc42 (cell division cycle 42) and Chp (Cdc42 homologous protein). Pak kinases influence a variety of cellular functions, but the process of Pak down-regulation, following activation, is poorly understood. In the present study, we describe for the first time a negative-inhibitory loop generated by the small Rho-GTPases Cdc42 and Chp, resulting in Pak1 inhibition. Upon overexpression of Chp, we unexpectedly observed a T-cell migration phenotype consistent with Paks inhibition. In line with this observation, overexpression of either Chp or Cdc42 caused a marked reduction in the level of Pak1 protein in a number of different cell lines. Chp-induced degradation was accompanied by ubiquitination of Pak1, and was dependent on the proteasome. The susceptibility of Pak1 to Chp-induced degradation depended on its p21-binding domain, kinase activity and a number of Pak1 autophosphorylation sites, whereas the PIX- (Pak-interacting exchange factor) and Nck-binding sites were not required. Together, these results implicate Chp-induced kinase autophosphorylation in the degradation of Pak1. The N-terminal domain of Chp was found to be required for Chp-induced degradation, although not for Pak1 activation, suggesting that Chp provides a second function, distinct from kinase activation, to trigger Pak degradation. Collectively, our results demonstrate a novel mechanism of signal termination mediated by the Rho-family GTPases Chp and Cdc42, which results in ubiquitin-mediated degradation of one of their direct effectors, Pak1.