FRAX597, a Small Molecule Inhibitor of the p21-activated Kinases,Inhibits Tumorigenesis of Neurofibromatosis Type 2 (NF2)-associated Schwannomas

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.     

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.     

Group I and II mammalian PAKs have different modes of activation by Cdc42

p21‐activated kinases (PAKs) are Cdc42 effectors found in metazoans, fungi and protozoa. They are subdivided into PAK1‐like (group I) or PAK4‐like (group II) kinases. Human PAK4 is widely expressed and its regulatory mechanism is unknown. We show that PAK4 is strongly inhibited by a newly identified auto‐inhibitory domain (AID) formed by amino acids 20 to 68, which is evolutionarily related to that of other PAKs. In contrast to group I kinases, PAK4 is constitutively phosphorylated on Ser 474 in the activation loop, but held in an inactive state until Cdc42 binding. Thus, group II PAKs are regulated through conformational changes in the AID rather than A‐loop phosphorylation.     

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.     

The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.     

The role of p21-activated kinases in hepatocellular carcinoma metastasis

The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.     

The Caenorhabditis elegans P21-activated kinases are differentially required for UNC-6/netrin-mediated commissural motor axon guidance

P21 activated kinases (PAKs) are major downstream effectors of rac-related small GTPases that regulate various cellular processes. We have identified the new PAK gene max-2 in a screen for mutants disrupted in UNC-6/netrin-mediated commissural axon guidance. There are three Caenorhabditis elegans PAKs. We find that each C. elegans PAK represents a distinct group previously identified in other species. Here we examine their roles in the postembryonic migration of the P cell neuroblasts and the axon guidance of the ventral cord commissural motoneurons (VCCMNs). We find that the two PAKs, max-2 and pak-1, are redundantly required for P cell migration and function with UNC-73/Trio and the rac GTPases (CED-10 and MIG-2). During axon guidance of the VCCMNs, PAK-1 also acts with the rac GTPases, CED-10 and MIG-2, and is completely redundant with MAX-2. Interestingly, we find that unlike MAX-2 activity during P cell migration, for motoneuron axon guidance max-2 is also required in parallel to this PAK-1 pathway, independent of rac GTPase signaling. Finally, we provide evidence that MAX-2 functions downstream of the UNC-6/netrin receptor UNC-5 during axon repulsion and is an integral part of its signaling.     

p21-Activated kinases are required for transformation in a cell-based model of neurofibromatosis type 2

Background

NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.

Methodology/Principal Findings

Using a specific peptide inhibitor of group I Paks, we show that loss of Pak activity restores normal cell movement in cells lacking Merlin function. In addition, xenografts of such cells form fewer and smaller tumors than do cells without Pak inhibition. However, in tumors, loss of Pak activity does not reduce Erk or Akt activity, two signaling proteins that are thought to mediate Pak function in growth factor pathways.

Conclusions/Significance

These results suggest that Pak functions in novel signaling pathways in NF2, and may serve as a useful therapeutic target in this disease.     

Substrate and Inhibitor Specificity of the Type II p21-Activated Kinase,PAK6

The p21-activated kinases (PAKs) are important effectors of Rho-family small GTPases. The PAK family consists of two groups, type I and type II, which have different modes of regulation and signaling. PAK6, a type II PAK, influences behavior and locomotor function in mice and has an ascribed role in androgen receptor signaling. Here we show that PAK6 has a peptide substrate specificity very similar to the other type II PAKs, PAK4 and PAK5 (PAK7). We find that PAK6 catalytic activity is inhibited by a peptide corresponding to its N-terminal pseudosubstrate. Introduction of a melanoma-associated mutation, P52L, into this peptide reduces pseudosubstrate autoinhibition of PAK6, and increases phosphorylation of its substrate PACSIN1 (Syndapin I) in cells. Finally we determine two co-crystal structures of PAK6 catalytic domain in complex with ATP-competitive inhibitors. We determined the 1.4 Å co-crystal structure of PAK6 with the type II PAK inhibitor PF-3758309, and the 1.95 Å co-crystal structure of PAK6 with sunitinib. These findings provide new insights into the structure-function relationships of PAK6 and may facilitate development of PAK6 targeted therapies.     

P21-activated kinase in inflammatory and cardiovascular disease

P-21 activated kinases, or PAKs, are serine–threonine kinases that serve a role in diverse biological functions and organ system diseases. Although PAK signaling has been the focus of many investigations, still our understanding of the role of PAK in inflammation is incomplete. This review consolidates what is known about PAK1 across several cell types, highlighting the role of PAK1 and PAK2 in inflammation in relation to NADPH oxidase activation. This review explores the physiological functions of PAK during inflammation, the role of PAK in several organ diseases with an emphasis on cardiovascular disease, and the PAK signaling pathway, including activators and targets of PAK. Also, we discuss PAK1 as a pharmacological anti-inflammatory target, explore the potentials and the limitations of the current pharmacological tools to regulate PAK1 activity during inflammation, and provide indications for future research. We conclude that a vast amount of evidence supports the idea that PAK is a central molecule in inflammatory signaling, thus making PAK1 itself a promising prospective pharmacological target.     

PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.     

p21 activated kinase 5 activates Raf-1 and targets it to mitochondria

Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at mitochondria.     

Interaction of paxillin with p21-activated Kinase (PAK). Association of paxillin alpha with the kinase-inactive and the Cdc42-activated forms of PAK3

p21-activated kinases (PAKs) are implicated in integrin signalings, and have been proposed to associate with paxillin indirectly. We show here that paxillin can bind directly to PAK3. We examined several representative focal adhesion proteins, and found that paxillin is the sole protein that associates with PAK3. PAK3 associated with the alpha and beta isoforms of paxillin, but not with gamma. We also show that paxillin alpha associated with both the kinase-inactive and the Cdc42-activated forms of PAK3 in vivo, without affecting the activation states of the kinase. A number of different functions have been ascribed to PAKs; and PAKs can bind directly to growth factor signaling-adaptor molecule, Nck, and a guanine nucleotide exchanger, betaPIX. Our results revealed that paxillin alpha can compete with Nck and betaPIX in the binding of PAK3. Moreover, paxillin alpha can be phosphorylated by PAK3 at serine. Therefore, paxillin alpha, but not gamma, appears to be capable of linking both the kinase-inactive and activated forms of PAK3 to integrins independent of Nck and betaPIX, as Nck links PAK1 to growth factor receptors. Our results also revealed that paxillin is involved in highly complexed protein-protein interactions in integrin signaling.     

p21-Activated kinase 5: A pleiotropic kinase

The PAKs (p21-activated kinases) are highly conserved serine/threonine protein kinases which comprise six mammalian PAKs. PAK5 (p21-activated kinase 5) is the least understood member of PAKs that regulate many intracellular processes when they are stimulated by activated forms of the small GTPases Cdc42 and Rac. PAK5 takes an important part in multiple signal pathways in mammalian cells and controls a variety of cellular functions including cytoskeleton organization, cell motility and apoptosis. The main goal of this review is to describe the structure, mechanisms underlying its activity regulation, its role in apoptosis and the likely directions of further research.     

Phosphorylation of Merlin Regulates its Stability and Tumor Suppressive Activity

The neurofibromatosis-2 (NF2) tumor suppressor protein, merlin or schwannomin, inhibits cell proliferation by modulating the growth activities of its binding partners, including the cell surface glycoprotein CD44, membrane-cytoskeleton linker protein ezrin and PIKE (PI 3-kinase Enhancer) GTPase etc. Merlin exerts its growth suppressive activity through a folded conformation that is tightly controlled through phosphorylation by numerous protein kinases including PAK, PKA and Akt. Merlin inhibits PI 3-kinase activity through binding to PIKE-L. Now, we show that merlin is a physiological substrate of Akt, which phosphorylates merlin on both T230 and S315 residues. This phosphorylation abolishes the folded conformation of merlin and inhibits its association with PIKE-L, provoking merlin polyubiquitination and proteasome-mediated degradation. This finding demonstrates a negative feed-back loop from merlin/PIKE-L/PI 3-kinase to Akt in tumors. The proliferation repressive activity of merlin is also partially regulated by S518 phosphorylation. Thus, Akt-mediated merlin T230/S315 phosphorylation, combined with S518 phosphorylation by PAK and PKA, provides new insight into abrogating merlin function in the absence of merlin mutational inactivation.     

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.     

Activation loop phosphorylation of ERK3/ERK4 by group I p21-activated kinases (PAKs) defines a novel PAK-ERK3/4-MAPK-activated protein kinase 5 signaling pathway

Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinase-activated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway.     

Molecular and functional characterization of EhPAK3, a p21 activated kinase from Entamoeba histolytica

p21-activated kinases (PAKs) are a family of serine/threonine kinases whose activity is regulated by the binding of the small Rho family GTPases as well as by RhoGTPase independent mechanisms. PAKs have wide-ranging functions which include cytoskeletal organisation, cell motility, cell proliferation and survival. We have identified a PAK from Entamoeba histolytica - EhPAK3 that is distributed in the cytoplasm of unstimulated cells and localizes to the caps after induction of capping with Concanavalin A. EhPAK3 contains a GTPase interacting (CRIB) domain, an N-terminal pleckstrin homology (PH) domain and a C-terminal kinase domain. Among the PAKs of E. histolytica studied so far, EhPAK3 bears the maximum similarity to Dictyostelium discoideum PAKC (DdPAKC). Phylogenetic analysis showed that EhPAK3 was closely related to DdPAKC and forms a group with DdPAKA, Dd Myosin I heavy chain kinase (DdMIHCK), and a PAK reported earlier from E. histolytica EhPAK2. Recombinant full-length EhPAK3 undergoes auotophosphorylation and phosphorylates histone H1 in vitro in the absence of any small GTPase. This is the first comprehensive characterization of a PAK protein from E. histolytica, which has constitutive activity and has demonstrated a strong involvement in receptor capping.