Crystal structure of the SH3 domain of betaPIX in complex with a high affinity peptide from PAK2

The p21-activated kinases (PAKs) are important effector proteins of the small GTPases Cdc42 and Rac and control cytoskeletal rearrangements and cell proliferation. The direct interaction of PAKs with guanine nucleotide exchange factors from the PIX/Cool family, which is responsible for the localization of PAK kinases to focal complexes in the cell, is mediated by a 24-residue peptide segment in PAKs and an N-terminal src homology 3 (SH3) domain in PIX/Cool. The SH3-binding segment of PAK contains the atypical consensus-binding motif PxxxPR, which is required for unusually high affinity binding. In order to understand the structural basis for the high affinity and specificity of the PIX-PAK interaction, we solved crystal structures for the N-terminal SH3 domain of betaPIX and for the complex of the atypical binding segment of PAK2 with the N-terminal SH3 domain of betaPIX at 0.92 A and 1.3A resolution, respectively. The asymmetric unit of the crystal contains two SH3 domains and two peptide ligands. The bound peptide adopts a conformation that allows for intimate contacts with three grooves on the surface of the SH3 domain that lie between the n-Src and RT-loops. Most notably, the arginine residue of the PxxxPR motif forms a salt-bridge and is tightly coordinated by a number of residues in the SH3 domain. This arginine-specific interaction appears to be the key determinant for the high affinity binding of PAK peptides. Furthermore, C-terminal residues of the peptide engage in additional interactions with the surface of the RT-loop, which significantly increases binding specificity. Compared to a recent NMR structure of a similar complex, our crystal structure reveals an alternate binding mode. Finally, we compare our crystal structure with the recently published betaPIX/Cbl-b complex structure, and suggest the existence of a molecular switch.     

PAK5在凋亡级联反应中的调节作用

PAK5为PAKs家族中新近发现的成员. PAKs是一类通过与Rac和Cdc42结合而激活的高度保守的蛋白酶.PAKs在细胞骨架、神经生长、激素信号传导、基因转录等生理活动中起着重要的调控作用. PAK5在大脑组织中高度表达,在细胞中定位于线粒体. PAK5 与神经生长、增强微管的稳定性以及阻止细胞凋亡等活动密切相关.本文就PAK5的结构、表达部位和功能以及其在调控凋亡级联反应中的作用等方面做了简要综述.     

p21-Activated kinase 3 (PAK3) protein regulates synaptic transmission through its interaction with the Nck2/Grb4 protein adaptor

Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.     

The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both alpha- and betaPAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation, a phosphorylation of alphaPAK serine 144 or betaPAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.     

Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness

The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility.     

Interaction between PAK and nck: a template for Nck targets and role of PAK autophosphorylation

The kinase PAK binds tightly to the SH3 domain of its partner PIX via a central proline-rich sequence. A different N-terminal sequence allows alphaPAK to bind an SH3 domain of the adaptor Nck. The Nck SH3[2] domain interacts equally with an 18-mer PAK-derived peptide and full-length alphaPAK. Detailed analysis of this binding by saturation substitution allows related Nck targets to be accurately identified from sequence characteristics alone. All Nck SH3[2] binding proteins, including PAK, NIK, synaptojanin, PRK2, and WIP, possess the motif PXXPXRXXS; in the case of PAK, serine phosphorylation at this site negatively regulates binding. We show that kinase autophosphorylation blocks binding by both Nck and PIX to alphaPAK, thus providing a mechanism to regulate PAK interactions with its SH3-containing partners. One cellular consequence of the regulatable binding of PAK is facilitation of its cycling between cytosolic and focal complex sites.     

Signaling,Regulation, and Specificity of the Type II p21-activated Kinases

The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases.     

P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.     

Identification of an intramolecular interaction important for the regulation of GIT1 functions

G-protein coupled receptor kinase-interacting protein (GIT) proteins include an N-terminal Arf GTPase-activating protein domain, and a C terminus that binds proteins regulating adhesion and motility. Given their ability to form large molecular assemblies, the GIT1 protein must be tightly regulated. However, the mechanisms regulating GIT1 functions are poorly characterized. We found that carboxy-terminal-truncated fragments of GIT1 bind their partners with higher efficiency compared with the full-length GIT1. We have explored the hypothesis that GIT1 is regulated by an intramolecular mechanism, and we identified two distinct intramolecular interactions between the N and C terminus of GIT1. The release of these interactions increases binding of GIT1 to paxillin and liprin-alpha, and it correlates with effects on cell spreading. Analysis of cells plated on fibronectin has shown that different deletion mutants of GIT1 either enhance or inhibit spreading, depending on their subcellular localization. Moreover, although the association between betaPIX and GIT1 is insufficient to activate GIT1 binding to paxillin, binding of a PAK1 fragment including the betaPIX-binding domain enhances paxillin binding to betaPIX/GIT1, indicating that p21-activated kinase can activate the binding of paxillin to GIT1 by a kinase-independent mechanism. The release of the identified intramolecular interaction seems to be an important mechanism for the regulation of GIT1 functions.     

Crystal Structures of the p21-activated kinases PAK4, PAK5, and PAK6 reveal catalytic domain plasticity of active group II PAKs

p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4-6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix alphaC, a key regulatory element of kinase function, resulted in an additional helical turn at the alphaC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, alphaC, and the activation segment and firmly anchor alphaC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5.     

Evaluation of the catalytic mechanism of the p21-activated protein kinase PAK2

The p21-activated kinases (PAKs) play important roles in diverse cellular processes. In the present study, we provide an in-depth kinetic analysis of one of the PAK family members, PAK2, in phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate derived from LIM kinase, LIMKtide. Steady-state kinetic analysis of the initial reaction velocity of PAK2 phosphorylation of MBP is consistent with both randomly and compulsorily ordered mechanisms. Further kinetic studies carried out in various concentrations of sucrose revealed that solvent viscosities had no effect on k(cat)/K(m) for either ATP or MBP while k(cat) was highly sensitive to solvent viscosity, indicating that the enzymatic phosphorylation by PAK2 can be best interpreted by a rapid-equilibrium random bi-bi reaction model, and k(cat) is partially limited by both phosphoryl group transfer (31 s(-)(1)) and the product release (19 s(-)(1)). In the phosphorylation of LIMKtide, both k(cat) and k(cat)/K(m) were insensitive to solvent viscosity, and the product release (86 s(-)(1)) was much faster than the phosphoryl group transfer step (19 s(-)(1)). These studies suggest that the release of phospho-MBP product is likely partially rate determining for the PAK2-catalyzed reaction since the dissociation rate of products from the PAK2 active site for LIMKtide phosphorylation differs from that of MBP significantly. Such a mechanism is in contrast to the previously established kinetics for the phosphorylation of peptide substrates by cAMP-dependent kinase, in which this process is limited by the release of ADP but not the phospho-peptide product. These results complement previous structure-function studies of PAKs and provide important insight for mechanistic interpretation of the kinase functions.     

P21活化激酶在肿瘤的作用

p21活化激酶（p21-activated kinase,PAKs）是小G蛋白Rac和细胞分裂调控蛋白42（Cdc42）的一类效应蛋白质. PAKs是一类进化保守的丝氨酸/苏氨酸蛋白激酶,在细胞骨架重排、细胞增生、细胞存活及增殖等方面发挥重要作用. 在哺乳动物中根据结构特征可将PAKs分为2个亚家族I类（A组）和Ⅱ类（B组）：I类包括PAK1、PAK2和PAK3,Ⅱ类包括PAK4,PAK5和PAK6. 近年来,对PAKs在肿瘤发生发展中作用的研究成为焦点.本文对PAKs中各成员的结构功能,及其在肿瘤发生发展过程中的作用等方面进行简要综述.     

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.     

Human immunodeficiency virus type 1 Nef selectively associates with a catalytically active subpopulation of p21-activated kinase 2 (PAK2) independently of PAK2 binding to Nck or beta-PIX

We have recently identified the Nef-associated serine-threonine kinase (NAK) as the p21-activated kinase 2 (PAK2). Here we have taken advantage of the possibility to manipulate the functional properties of NAK by transfecting PAK2 cDNA or its mutant derivatives in order to further characterize the Nef-NAK complex. To exclude the possibility that some Nef variants might interact with PAK1 instead of PAK2, we also examined the identity of NAK complexed with divergent human immunodeficiency virus type 1 HIV-1 Nef proteins. All tested Nef proteins, including SF2, NL4-3, BH10, and HAN-2, associated with PAK2 but not with PAK1. By exchanging different regions between these two PAK proteins, the selective ability of PAK2 to associate with Nef could be mapped to the carboxy-terminal part of its regulatory domain. Binding of PAK2 with the adapter protein Nck or beta-PIX was found to be dispensable for the assembly of the Nef-PAK2 complex, whereas an intact Cdc42-Rac1 interactive binding motif was required. Most importantly, we found that NAK represented a distinct subpopulation of the total cellular PAK2 characterized by a high specific kinase activity. Thus, although only a small fraction of cellular PAK2 could be found in complex with Nef, NAK represented a major part of cellular PAK2 activity.     

Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly

The p21-activated kinase PAK is targeted to focal complexes (FCs) through interactions with the SH3 domains of the PAK-interacting exchange factor PIX and Nck. PIX is a Rac GTP exchange factor that also binds the G-protein-coupled receptor kinase-interacting protein known as GIT1. Overexpression of GIT1 in fibroblasts or epithelial cells causes a loss of paxillin from FCs and stimulates cell motility. This is due to the direct interaction of a C-terminal 125-residue domain of GIT1 with paxillin, under the regulation of PIX. In its activated state, GIT1 can promote FC disassembly independent of actin-myosin contractile events. Additionally, GIT directly couples to a key component of FCs, focal adhesion kinase (FAK), via a conserved Spa2 homology domain. We propose that GIT1 and FAK cooperate to promote motility both by directly regulating focal complex dynamics and by the activation of Rac.     

The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity

p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The pak3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.