miR-133 is a key negative regulator of CDC42–PAK pathway in gastric cancer

Cell division cycle 42 (CDC42), an important member of the Ras homolog (Rho) family, plays a key role in regulating multiple cellular processes such as cell cycle progression, migration, cell cytoskeleton organization, cell fate determination and differentiation. Among the downstream effectors of CDC42, P21-activated kinases (PAKs) obtain the most attention. Although a large body of evidence indicates that CDC42/PAKs pathway plays important role in tumor growth, invasion and metastasis, the mechanism of their negative regulation remains unclear. Here, we identified CDC42, a PAKs activating factor, was a target of miR-133. Ectopic overexpression of miRNAs not only downregulated CDC42 expression and PAKs activation, but also inhibited cancer cell proliferation and migration. We also found that miR-133 was down-regulated in 180 pairs gastric cancer tissues. miR-133 expression was negatively associated with tumor size, invasion depth and peripheral organ metastasis. Besides, dysfunction of miR-133 was an independent prognosis factor for overall survival. Our findings could provide new insights into the molecular mechanisms of gastric carcinogenesis, and may help facilitating development of CDC42/PAK-based therapies for human cancer.     

p21活化激酶的生物学活性及其与肿瘤的关系

p21活化激酶(p21-activatedkinase,PAK),为一类进化上保守的丝氨酸/苏氨酸蛋白激酶。PAK在许多组织中广泛表达,作为小G蛋白Rho家族Cdc42和Rac1的下游靶蛋白,可以被生长因子及其他胞外信号通过GTP酶依赖的信号通路或非GTP酶依赖的信号通路活化,发挥多种生物学效应。PAK作为一种重要的生物学调节因子,在哺乳动物一系列细胞功能中具有重要作用,如:细胞运动、细胞生存、细胞周期、血管生成、基因转录调节及癌细胞的侵袭转移。通过对PAK家族成员信号转导机制的研究,为癌症治疗提供分子靶标。     

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.     

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 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.     

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.     

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.     

p21-activated protein kinase: a crucial component of morphological signaling?

The mechanisms by which Rho family GTPases (Rho, Rac and Cdc42) regulate coordinated changes to the actin cytoskeleton are being elucidated. This review will focus on the current evidence that the p21-activated kinases (PAKs) are involved in regulating some of the diverse cytoskeletal changes induced by Rac and Cdc42. PAKs have been shown to be required for processes including neurite formation and axonal guidance, development of cell polarity and motile responses. Signaling molecules interacting with PAKs that might contribute to the regulation of such processes have recently been identified.     

Ste20-related kinases: effectors of signaling and morphogenesis in fungi

The family of Ste20-related kinases is conserved from yeast to mammals and includes the p21 activated kinases (PAKs) and germinal centre kinases (GCKs). These kinases have been shown to be involved in signaling through mitogen activated protein kinase (MAPK) pathways and in morphogenesis through the regulation of cytokinesis and actin-dependent polarized growth. This review concentrates on the role of Ste20-related kinases in fungi where recent research has revealed roles for both PAKs and GCKs in the regulation of cytokinesis and in previously unidentified roles in promoting hyphal growth and differentiation of asexual development structures. In particular, the importance of PAKs during pathogenesis will be examined.     

Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. Here, we have identified a novel function for the group I PAKs in cell–cell fusion. We show that the two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. We propose a model whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion.     

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.     

NMR binding and crystal structure reveal that intrinsically-unstructured regulatory domain auto-inhibits PAK4 by a mechanism different from that of PAK1

Six human PAK members are classified into groups I (PAKs 1–3) and II (PAK4–6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs.     

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.     

Emerging from the Pak: the p21-activated protein kinase family

The p21-activated protein kinases (PAKs) are members of a growing family of regulatory enzymes that may play roles in diverse phenomena such as cellular morphogenesis, the stress response and the pathogenesis of AIDS. PAKs were initially discovered as binding partners for small (21 kDa) GTPases that regulate actin polymerization, and recent evidence has shown that some members of the PAK family may be effectors for related GTPases that are involved in intracellular vesicle trafficking. Because the downstream signalling pathways for all such GTPases are poorly understood, intense studies are under way to discern the role of PAK and its cousins. In this review, the authors highlight some of the established properties of the extended PAK family and discuss current controversies regarding their possible roles as GTPase effectors.     

Systemic analysis of the expression and prognostic significance of PAKs in breast cancer

PAKs (p21-activated kinases) are reported to play crucial roles in a variety of cellular processes and participate in the progression of human cancers. However, the expression and prognostic values of PAKs remain poorly explored in breast cancers. In our study, we examined the mRNA and protein expression levels of PAKs and the prognostic value. We also analyzed the interaction network, genetic alteration, and functional enrichment of PAKs. The results showed that the mRNA levels of PAK1, PAK2, PAK4 and PAK6 were significantly up-regulated in breast cancer compared with normal tissues, while the reverse trend for PAK3 and PAK5 was found, furthermore, the proteins expression of PAK1, PAK2 and PAK4 in breast cancer tissues were higher than that in normal breast tissues. Survival analysis revealed breast cancer patients with low mRNA expression of PAK3 and PAK5 showed worse RFS, conversely, elevated PAK4 levels predicted worse RFS. In addition, the breast cancer patients with PAKs genetic alterations correlated with worse OS. These results indicated that PAKs might be promising potential biomarkers for breast cancer.     

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.