EVOLUTIONARY HOMOLOGS part 2/3 || part 3/3 || part 1/3

Targets of PAKs

Although the ability of Cdc42 and Rac GTPases to activate Pak is well established, relatively little else is known about Pak regulation or the identity of Pak cellular targets. Two closely related Pak3-binding proteins, possibly arising from alternative splicing, have been isolated and designated p50(Cool-1) and p85(Cool-1), where Cool-1 stands for 'cloned out of library'. Both isoforms of Cool contain a Src homology 3 domain that directly mediates interaction with Pak3 and tandem Dbl homology and pleckstrin homology domains. Despite the presence of the Dbl homology-pleckstrin homology motif (a characteristic of Rho family activators), activation of Cdc42 or Rac by Cool is not detectable. Instead, the binding of p50(Cool-1), but not p85(Cool-1), to Pak3 represses Pak3's activation by upstream activators such as the Dbl oncoprotein, indicating a novel mechanism for regulation of Pak signaling (Bagrodia, 1998).

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. A new class of Rho-p21 guanine nucleotide exchange factor has been purified and cloned that binds 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. These results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex (Manser, 1998).

The serine/threonine kinase p21-activated kinase (PAK) has been implicated as a downstream effector of the small GTPases Rac and Cdc42. While these GTPases evidently induce a variety of morphological changes, the role(s) of PAK remains elusive. Overexpression of betaPAK in PC12 cells induces a Rac phenotype, including cell spreading/membrane ruffling, and increased lamellipodia formation at growth cones and shafts of nerve growth factor-induced neurites. These effects are still observed in cells expressing kinase-negative or Rac/Cdc42 binding-deficient PAK mutants, indicating that kinase- and p21-binding domains are not involved. Furthermore, lamellipodia formation in all cell lines, including those expressing Rac binding-deficient PAK, is inhibited significantly by dominant-negative RacN17. Equal inhibition is achieved by blocking PAK interaction with the guanine nucleotide exchange factor PIX using a specific N-terminal PAK fragment. It is concluded that PAK, via its N-terminal non-catalytic domain, acts upstream of Rac mediating lamellipodia formation through interaction with PIX (Obermeier, 1998).

Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. p21-activated kinase (Pak1) phosphorylates LIM-kinase (see Drosophila LIM-kinase1) 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 (Edwards, 1999).

Inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and Raf is the critical target of PI 3-kinase regulation. To investigate how PI 3-kinase regulates Raf, sites on Raf1 required for regulation by PI 3-kinase were examined and the mechanisms involved in this regulation were explored. Serine 338 (Ser338), which 1s critical for fibronectin stimulation of Raf1, is phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) is independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, is also dependent on PI 3-kinase activity, and expression of a kinase-inactive Pak-1 mutant blocks phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins (Chaudhary, 2000).

The formation of new branched actin filament networks at the cell cortex of migrating cells is choreographed by the actin-related protein (Arp) 2/3 complex. Despite the fundamental role of the Arp2/3 complex in actin nucleation and branching, upstream signals that control the functions of p41-Arc, a putative regulatory component of the mammalian Arp2/3 complex, remain unidentified. p41-Arc is shown to interacts with p21-activated kinase 1 (Pak1) both in vitro and in vivo. Pak1 phosphorylation of p41-Arc regulates its localization with the Arp2/3 complex in the cortical nucleation regions of cells. Pak1 phosphorylates p41-Arc on threonine 21 in the first WD repeat, and its mutation has functional implications in vivo. Threonine 21 phosphorylation by Pak1 is required for both constitutive and growth-factor-induced cell motility. Pak1 regulation of p41-Arc activation status represents a novel mechanism by which signalling pathways may influence the functions of the Arp2/3 complex, leading to motility in mammalian cells (Vadlamudi, 2004).

Rho GTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. A member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G(12/13) family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ RhoGEF and abolishes its ability to mediate the accumulation of Rho-GTP by Galpha13. Moreover, when overexpressed, active PAK4 dramatically decreases Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases (Barac, 2004).

The small GTPase Rac and the second messenger cGMP (guanosine 3',5'-cyclic monophosphate) are critical regulators of diverse cell functions. When activated by extracellular signals via membrane signaling receptors, Rac executes its functions through engaging downstream effectors such as p21-activated kinase (PAK), a serine/threonine protein kinase. However, the molecular mechanism by which membrane signaling receptors regulate cGMP levels is not known. A signaling pathway linking Rac to the increase of cellular cGMP has been uncovered. Rac uses PAK to directly activate transmembrane guanylyl cyclases (GCs), leading to increased cellular cGMP levels. This Rac/PAK/GC/cGMP pathway is involved in platelet-derived growth factor-induced fibroblast cell migration and lamellipodium formation. These findings connect two important regulators of cellular physiological functions and provide a general mechanism for diverse receptors to modulate physiological responses through elevating cellular cGMP levels (Guo, 2007).

Signaling downstream of PAKs

The family of p21-activated protein kinases (PAKs) appears to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. A Drosophila PAK has been shown to be enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure, where it colocalizes with structures resembling focal complexes. In epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions, which indicates that focal adhesions are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras have also been obtained. Plasmids encoding these different constitutively active alpha-PAKs cause loss of stress fibers when introduced into both HeLa cells and fibroblasts, which is similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions are also observed. These combined effects result in retraction of the cell periphery after plasmid microinjection. These data support suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes (Manser, 1997).

The Rho-related GTP-binding proteins Cdc42 and Rac1 have been shown to regulate signaling pathways involved in cytoskeletal reorganization and stress-responsive JNK (Jun N-terminal kinase) activation. However, to date, the GTPase targets that mediate these effects have not been identified. PAK defines a growing family of mammalian kinases that are related to yeast Ste20 and are activated in vitro through binding to Cdc42 and Rac1. Clues to PAK function have come from studies of Ste20, which controls the activity of the yeast mating mitogen-activated protein (MAP) kinase cascade, in response to a heterotrimeric G protein and Cdc42. To initiate studies of mammalian Ste20-related kinases, a novel human PAK isoform, hPAK1, has been identified. When expressed in yeast, hPAK1 is able to replace Ste20 in the pheromone response pathway. Chemical mutagenesis of a plasmid encoding hPAK1, followed by transformation into yeast, has led to the identification of a potent constitutively active hPAK1 with a substitution of a highly conserved amino-acid residue (L107F) in the Cdc42-binding domain. Expression of the hPAK1(L107F) allele in mammalian cells leads to specific activation of the Jun N-terminal kinase MAP kinase pathway, but not the mechanistically related extracellular signal-regulated MAP kinase pathway. These results demonstrate that hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway in mammalian cells, as Ste20 does in yeast. Thus, PAK and Ste20 kinases play key parts in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation (Brown, 1996).

Activation of the canonical mitogen-activated protein kinase (MAPK) cascade by soluble mitogens is blocked in non-adherent cells. It is also blocked in cells in which the cAMP-dependent protein kinase (PKA) is activated. Inhibition of PKA allows anchorage-independent stimulation of the MAPK cascade by growth factors. This effect is transient, and its duration correlates with sustained tyrosine phosphorylation of paxillin and focal-adhesion kinase (FAK) in non-adherent cells. The effect is sensitive to cytochalasin D, implicating the actin cytoskeleton as an important factor in mediating this anchorage-independent signaling. Interestingly, constitutively active p21-activated kinase (PAK) also allows anchorage-independent MAPK signaling. Furthermore, PKA negatively regulates PAK in vivo, and whereas the induction of anchorage-independent signaling resulting from PKA suppression is blocked by dominant negative PAK, it is markedly prolonged by constitutively active PAK. These observations indicate that PKA and PAK are important regulators of anchorage-dependent signal transduction (Howe, 2000).

The T-cell antigen receptor (TCR) triggers a signaling cascade initiated by the tyrosine kinase Lck and requiring the proto-oncogene p95(vav). Vav is activated by Lck and can function as a guanine nucleotide exchange factor for the Rho-family GTPases, Rac1 and Cdc42. To investigate the involvement of these GTPases in TCR signaling, a focus was placed on their well characterized effector, Pak1. This serine/threonine kinase is activated by GTP-bound Rac1 or Cdc42. However, its role in mediating downstream signaling events is controversial. There is a rapid, TCR-dependent activation of Pak1 and TCR-inducible association of Pak1 with Nck, which is tyrosine phosphorylated following stimulation. Pak1 activation occurs independent of Ras activation or calcium flux, but is dependent on the Lck tyrosine kinase, and is downstream of Vav and Cdc42. Dominant negative Pak1 or Nck specifically inhibits TCR-mediated activation of the nuclear factor of activated T cells (NFAT) transcription factor. TCR-mediated activation of Erk2 is also inhibited by dominant negative Pak. However, Pak1 activation is neither necessary nor sufficient for TCR-dependent c-Jun N-terminal kinase (JNK) activation. Therefore, Pak1 acts downstream of Vav and is required for activation of Erk2 and NFAT by a JNK-independent pathway. This is the first demonstration of a requirement for Pak to mediate the regulation of gene expression by an extracellular ligand (Yablonski, 1998).

Using degenerate polymerase chain reaction, a human cDNA encoding a protein kinase homologous to STE20 has been isolated. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK is a serine/threonine protein kinase that specifically activates the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it does not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increases AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation is inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibits HGK-induced JNK activation. Dominant-negative HGK mutants inhibit TNF-alpha-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK --> TAK1 --> MKK4, MKK7 --> JNK kinase cascade may mediate the TNF-alpha signaling pathway (Yao, 1999).

A betaPIX-PAK2 complex confers protection against Scrib-dependent and cadherin-mediated apoptosis

During epithelial morphogenesis, a complex comprising the βPIX (PAK-interacting exchange factor β) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian ortholog of the Drosophila polarity determinant and tumor suppressor Scribble, binds βPIX directly. Scrib is also targeted to adherens junctions by E-cadherin, where Scrib strengthens cadherin-mediated cell-cell adhesion. Although a role for the Scrib-βPIX-PAK signaling complex in promoting membrane protrusion at wound edges has been elucidated, a function for this complex at adherens junctions remains unknown. This study establish, in cultured mammalian cells, that Scrib targets βPIX and PAK2 to adherens junctions where a βPIX-PAK2 complex counterbalances apoptotic stimuli transduced by Scrib and elicited by cadherin-mediated cell-cell adhesion. Moreover, it was shown that this signaling pathway regulates cell survival in response to osmotic stress. Finally, it was determined that in suspension cultures, the Scrib-βPIX-PAK2 complex functions to regulate anoikis elicited by cadherin engagement, with Scrib promoting and the βPIX-PAK2 complex suppressing anoikis, respectively. These findings demonstrate that the Scrib-βPIX-PAK2 signaling complex functions as an essential modulator of cell survival when localized to adherens junctions of polarized epithelia. The activity of this complex at adherens junctions is thereby essential for normal epithelial morphogenesis and tolerance of physiological stress. Furthermore, when localized to adherens junctions, the Scrib-βPIX-PAK2 signaling complex serves as a key determinant of anoikis sensitivity, a pivotal mechanism in tumor suppression. Thus, this work also reveals the need to expand the definition of anoikis to include a central role for adherens junctions (Frank, 2012).

This study has established that differentiated epithelial cells rely on a survival signaling network associated with adherens junctions that is distinct from that used at cell-matrix adhesions. Specifically, it was demonstrated that epithelial cells are critically dependent on Scrib-mediated localization of a βPIX-PAK2 complex to adherens junctions to counterbalance the apoptosis-promoting effects of E-cadherin engagement. As a result, disruption of βPIX-PAK2 signaling results in a near complete loss of epithelial viability at confluent density. Consistent with an established role in phosphorylating cellular targets involved in apoptosis, a functional kinase domain is required for PAK2-dependent survival signaling. In subconfluent cultures, where the βPIX-PAK2 complex localizes to focal adhesions, the complex is dispensable for cell survival. Thus, as epithelial cells form cell-cell junctions and polarize, they become critically dependent on a prosurvival signal provided by the βPIX-PAK2 complex at adherens junctions (Frank, 2012).

To date, the only direct evidence for the function of PAKs at adherens junctions is in the regulation of cadherin adhesiveness and actin-dependent cell contractility. While invoked by prior findings, the current results demonstrate for the first time that Scrib via its association with βPIX promotes targeting of PAK2 to adherens junctions. In cultured human keratinocytes, PAK1 has been reported to augment cadherin adhesiveness in response to activated Rac. PAK1 and PAK2 have also been reported to enhance and suppress, respectively, loss of cell-cell contacts in response to hepatocyte growth factor. The current results suggest that neither PAK1 nor PAK2 play a major role in steady state junctional integrity in MDCK cells. However, because MDCK cells express both PAK1 and PAK2, the possibility cannot be excluded that they may function redundantly in regulation of E-cadherin adhesiveness and/or junctional remodeling (Frank, 2012).

βPIX and PAKs have been most extensively characterized for their role in focal adhesion dynamics, where they play a coordinated role in regulating turnover of these integrin attachment sites and promoting directional motility. PAK1 and PAK2 appear to have nonredundant functions in cell invasion and motility. Moreover, there is evidence to suggest that when localized to focal adhesions, the βPIX-PAK complex promotes mitogenic signaling and that redistribution of the complex from focal adhesions to adherens junctions contributes to the cessation of epithelial proliferation and establishment of contact inhibition. Scrib also shuttles between adherens junctions and protrusive membrane structures where it promotes βPIX-PAK complex-dependent cell motility. It will be interesting to determine whether Scrib will play a role in redistribution of the βPIX-PAK2 complex from focal adhesions to adherens junctions, which occurs as cells undergo contact inhibition. In brief, taken together with the present work, these studies suggest that PAKs have distinct roles when localized to focal adhesions and adherens junctions. When localized to focal adhesions, PAKs participate in signaling pathways that control proliferation and motility, whereas at adherens junctions PAKs regulate adhesiveness and survival. As such, the translocation of the βPIX-PAK complex from to lateral membranes upon formation of stable adherens junctions likely plays a fundamental role in the transition from motile and mitogenic states to a nonmotile and quiescent state (Frank, 2012).

E-cadherin is a potent inhibitor of multiple signaling pathways and plays a fundamental role in suppression of motility and proliferation upon establishment of cell-cell contact. The ability of Scrib to promote E-cadherin adhesiveness may underlie some of its tumor suppressive potential in mammalian cells. However, increasing evidence suggests that Scrib also plays E-cadherin-independent roles in the regulation of signaling pathways, such as inhibition of ERK and AKT, as well as activation of Hippo signaling. These Scrib-dependent effects would be predicted to sensitize cells to apoptosis; a prediction supported by the present results, which furthermore establish that Scrib-mediated apoptosis is counterbalanced by its recruitment of active PAK2. Taken together, these findings demonstrate that Scrib transduces both pro- and anti-apoptotic stimuli (Frank, 2012).

Metastasis requires that cells tolerate the loss of matrix adhesion, i.e., that they are protected against anoikis. However, in spite of seminal work suggesting a role for cell-cell adhesion in modulating anoikis, the role for epithelial architecture in anoikis remains unappreciated and poorly characterized. Nevertheless, there is growing evidence indicating that loss of E-cadherin function suffices to abrogate anoikis. The current results support a key role of adherens junctions in modulating anoikis. In simple polarized epithelia, which likely rely on the summation of survival signals emanating from both focal adhesions and adherens junctions, apoptotic stimuli emanating from E-cadherin will sensitize cells to anoikis. In contrast, suprabasal cells in stratified epithelia depend exclusively on survival signals from adherens junctions. PAK2 is highly expressed in suprabasal keratinocytes, which may suggest that upregulation of PAK2-dependent survival signals emanating from adherens junctions are required to offset loss of integrin-mediated survival signaling. In summary, the current results indicate that E-cadherin does not function simply as a prosurvival or proapoptotic factor but rather as a regulatory node to coordinate death and survival signaling. The balance between pro- and antiapoptotic signaling emanating from adherens junctions plays a key role in epithelial cell viability and is likely essential in regulating diverse processes, including epithelial morphogenesis, wound healing, physiological stress, and metastasis (Frank, 2012).

PAKs and cytoskeletal rearrangement

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 (Ottilie, 1995).

The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors. In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers. The serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity towards exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors. Microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles. The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures. This phenotype resembles that seen in motile fibroblasts. The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains. Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton. These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility (Sells, 1997).

Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established. Mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton (Eby, 1998).

The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, a novel two-hybrid system, the Ras recruitment system (RRS), was used that aims to detect protein-protein interactions at the inner surface of the plasma membrane. The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homolog of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains, and the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicate Chp in the induction of lamellipodia and show that Chp activates the JNK MAP kinase cascade (Aronheim, 1998).

PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, the mechanism of mutational activation of PAK1 have been examined, and the effects of different active PAK1 proteins on cytoskeletal structure in vivo have been characterized. PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain. Two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, activation of PAK1 by these mutations is shown to result from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When the morphological effects of these different forms of PAK1 were examined in vivo, PAK1 kinase activity was found to be associated with disassembly of focal adhesions and actin stress fibers and it was found that activity may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, is independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain. Thus the differential effects of PAK1-activating mutations reveals both activity-dependent and activity-independent effects on cytoskeletal regulation (Frost, 1998).

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. The individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL reveals a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localizes to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibits lamellipodia formation in response to insulin-like growth factor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreases cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL (Turner, 1999).

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. An ephrinB-EphB receptor trans-synaptic signaling pathway has been identified that regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin (Drosophila ortholog: Trio) to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin or dominant-negative Rac1, or inhibition of PAK each eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity (Penzes, 2003).

The role of the Rac1 effector p21-activated kinase PAK was examined. Several PAK proteins are expressed in the brain, and previous studies have shown that some of the effects of Rac1 on the cytoskeleton are mediated by PAK. In addition, genetic analysis in Drosophila has shown that PAK1 is genetically associated with Trio, the fly ortholog of kalirin, in the pathway through which Trio affects axon growth and guidance. Binding of activated Rac1 to PAK induces PAK autophosphorylation, which strongly correlates with its activation. To test whether ephrinB treatment induces activation of PAKs, an antibody detecting autophosphorylated PAK (P-PAK) was used. In addition, this experiment can be regarded as a way to visualize endogenous Rac1 activation. Treatment of hippocampal neurons with clustered ephrinB1 induce a dramatic increase in the number and size of clusters stained with the P-PAK antibody. This effect was confirmed by Western analysis with the P-PAK antibody of extracts of 4-week-old high-density cortical neurons treated with ephrinB1. Moreover, in hippocampal neurons, ephrinB1 treatment induces activation of PAK at synapses, as shown by P-PAK immunostaining coincident with synaptophysin (Penzes, 2003).

To test whether kalirin-7 was required for ephrinB1-induced PAK phosphorylation, the effect was examined of overexpressing the GEF inactive kal7-mut in hippocampal neurons on the ability of clustered ephrinB1 to induce phosphorylation of PAK. Therefore, DIV7 hippocampal neurons were transfected with myc-kal7-mut, and 2 days later the neurons were treated with clustered ephrinB1 for 2 hr, followed by fixation and immunostaining for myc and P-PAK. While ephrinB1 treatment induces an increased phosphorylation of PAK in nontransfected neurons, in neurons expressing kal7-mut, the level of P-PAK is visibly reduced compared to adjacent nontransfected neurons. Quantification of the ratios of P-PAK fluorescence intensities to total cell areas of nontransfected control neurons relative to the same ratios for neurons expressing myc-kal7-mut confirmed this observation (Penzes, 2003).

PAKs phosphorylate proteins involved in regulating the actin cytoskeleton and gene expression. To test whether PAK is an essential downstream component of ephrinB signaling in spine morphogenesis, GFP-transfected hippocampal neurons were treated with a fusion protein of the PAK1 inhibitory domain (PID) fused with the cell-penetrating peptide (TAT-PID) along with ephrinB1. These neurons exhibit a reduction in the number and size of spines, compared to the ephrinB1-treated neurons, while also showing a reduced phosphorylation level of PAK, confirming its inhibition by PID. Together, these data demonstrate that Rac1 and PAK are key downstream components of ephrinB regulation of spine morphogenesis (Penzes, 2003).

During development, it is necessary to coordinate accurately the formation and location of presynaptic active zones with those of the postsynaptic structures. This could be achieved by signaling from presynaptic ephrinB, clustered at active zones on axons, to activate postsynaptic EphB2, resulting in synaptogenesis on the apposing dendrites. Even in mature neurons, dendritic spines are very dynamic structures, and recent studies have demonstrated that LTP induces morphological changes in spines, which may contribute to plasticity in adult neurons. The rapid and dramatic effect of ephrinB on spine maturation suggests that ephrinB-EphB2 signaling may be a key component in the regulation of spine morphogenesis during plasticity. Other extracellular signals have been shown to regulate spine morphogenesis, such as K+ depolarization, glutamate action on NMDA receptors, and BDNF. It is possible that kalirin mediates the intracellular effects of these signals as well (Penzes, 2003).

A FOXO-Pak1 transcriptional pathway controls neuronal polarity

Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. This study reports that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. The gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, was identified as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development (de la Torre-Ubieta, 2010).

PAKs and the cell cycle in yeast

see PAK-kinase Evolutionary homologs part 3/3 | back to part 1/3 |

PAK-kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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