爪蟾p21活化激酶2参与卵母细胞胞质分裂过程不依赖于Cdc42
摘要
前言
爪蟾卵母细胞减数分裂过程中，极体形成是一种不对称的细胞分裂，有助于母体的胞浆物质和基因组的储存。目前调节极体形成的机制所知甚少，有研究表明，细胞皮质层肌动蛋白极化，中期Ⅰ纺锤体与卵母细胞皮质层接触对于后期Ⅰ的启动和极体的形成非常重要。从酵母到哺乳动物细胞，P21活化激酶(p21-activated kinase，PAK)与它的激活子，小GTP酶—Cdc42在细胞极化和不对称分裂中具有进化保守功能。例如出芽酵母的出芽过程类似不对称的细胞分裂过程，需要纺锤体一端与出芽的皮质层接触从而启动胞质分裂，PAK(Cla4和Ste20)与其激活子Cdc42在肌动蛋白极化、促进出芽和胞质分裂中发挥重要作用。PAK功能的缺失和抑制导致酵母细胞极化失败，出芽不能完成。Ma和Cheng等研究证明，在爪蟾卵母细胞中，一种小GTP酶Cdc42调控肌动蛋白极化和不对称细胞分裂，所以我们假设，PAK在爪蟾卵母细胞成熟过程中同样参与了胞质分裂和极体形成。
PAK是一类进化上保守的丝氨酸/苏氨酸蛋白激酶，为Rho家族小鸟苷三磷酸酶(Rho-GTPase)Cdc42/Rac、生长因子信号的主要靶蛋白，参与许多重要细胞活动。到目前为止共发现6个PAK家族成员，根据其结构相似性，将它们分为两类，Ⅰ类包括PAK1，PAK2和PAK3，Ⅱ类包括PAK4，PAK5和PAK6。Ⅰ类PAK在N-端含有数个有争议的Src相似序列3(SH3)-结合基序和p21蛋白结合域(p21binding domain，PBD)，C-端激酶域为高度保守的序列。Ⅰ类PAK通过结合Cdc42或Rac发挥生物学活性。Ⅱ类PAK在N-端同样包含PBD，C-端也为激酶域，同样可以结合Rac和Cdc42，但是活性不依赖与它们结合，目前认为Cdc42的结合对于Ⅱ类PAK的定位更重要而非其活性。
PAK2是Rho家族中GTP酶的重要效应物。PAK2通过对肌动蛋白细胞骨架的调节，在细胞形态学和动力学的调控上起重要作用。PAK2具有高度保守的C-端激酶域和N-端Cdc42/Rac相互作用域(Cdc42/Rac interactive bindingdomain，CRIB)，又名GTP酶结合域。该区域与PAK2 N-端的自身抑制域(auto-inhibitorydomain，AID)部分重叠，调节PAK2的激酶活性。非活性PAK2自身折叠使AID与C-端激酶域结合，活化环无法暴露，抑制其激酶活性。当Cdc42与PAK2结合时，导致PAK2构象改变，这个过程会缓解PAK2自身抑制并且激活PAK2活性。
在爪蟾卵母细胞中目前已经发现四种PAK，Ⅰ类PAK 1-3，Ⅱ类PAK5。早期的研究表明PAK1过表达抑制孕酮诱导的爪蟾卵母细胞成熟。在G2期的爪蟾卵母细胞中，PAK1保持无活性状态，在卵母细胞整个成熟过程保持无活性，对Cdc42信号无应答。PAK2在爪蟾G2期的卵母细胞中保持活化状态。在爪蟾卵母细胞和成熟卵细胞中未检测到PAK3，PAK3只在爪蟾胚胎发育的肠胚期后期被表达。PAK5只出现在爪蟾胚胎中，能结合肌动蛋白和微管网络，在肠胚期以Ca~(2+)依赖性方式调节细胞骨架的会聚延伸。Cau等研究证明在爪蟾卵母细胞成熟过程中显微注射Cdc42V12(constitutively active Cdc42，活化型Cdc42)后，PAK2活性增高约50倍，提示PAK2可能是Cdc42优先选择的效应器，参与爪蟾卵母细胞成熟过程。本研究着重探讨在爪蟾卵母细胞分裂过程中PAK2是否参与胞质分裂和极体形成及与Cdc42的关系。
实验材料
1、PAK2、Cdc42相关质粒及分子生物学试剂。
2、蛋白质免疫印迹检测试剂。
3、荧光标记的相关分子生物学试剂
实验方法
一、爪蟾卵母细胞的准备及裂解
爪蟾皮下注射孕马血清促性腺激素(serum gonadotrophin，PMSG)50IU，3日后处死爪蟾，取卵巢组织，在显微镜下将其分离为15个卵左右的碎片。用胶原酶(1mg/mL)消化卵母细胞3h，取大小相似、发育成熟Ⅵ期卵母细胞，置于1×OR2(无Ca~(2+))培养液(83 mmol/L NaCl，2.5 mmol/L KCI，1 mmol/L MgCl_2，1 mmol/LNa_2HPO_4，5 mmol/L HEPES pH 7.8)中，室温震荡培养4h。10μL冷裂解缓冲液(EB缓冲液：20mM pH 7.3 HEPES，80mmol/L磷酸甘油，20mmol/L EGTA，15mmol/LMgCl_2，1mmol/L二硫苏糖醇，101×mol/L ATP，150mmol/L NaF，10μg/mL抑(蛋白)酶醛肽，200μmol/L苯甲磺酰氟，25μg/mL苯甲脒)裂解卵母细胞，4℃13，000g离心5min，上清液与2×SDS样品缓冲液、β-巯基乙醇混合，-20℃贮存。
二、爪蟾卵母细胞成熟过程中PAK2磷酸化状态观察
抗p-PAK2(Thr402)蛋白质印迹检测：孕酮刺激后不同时间点(0h、1h、2h、3h、3.5h)以及胚泡破裂(germinal vesicle breakdown，GVBD)发生后不同时间点(0min、30min、60min、90min、120min、150min、180min、12h)的爪蟾卵母细胞裂解液在15％SDS-PAGE电泳中分离，电转移至硝酸纤维素膜，10％BSA封闭1h；与抗p-PAK2抗体孵育2h，相应的二抗孵育1h，ECL发光法检测。
三、体外合成PAK2-NT、PAK2-NTm、GFP-WGBD mRNA
将PCS2-HA-PAK2-NT(PCS2-HA-PAK2-N-terminal)、PCS2-HA-PAK2-NTm(PCS2-HA-PAK2-N-terminal mutation)、GFP-wGBD质粒用特异性限制性内切酶进行酶切线性化，1.5％琼脂糖凝胶电泳进行鉴定，以线性化DNA为模板、合成PAK2-NT、PAK2-NTm、GFP-wGBD mRNA，-70℃保存备用。
四、荧光显微镜观察爪蟾卵母细胞DNA染色
观察对照组、PAK2-NT mRNA注射组和PAK2-NTm mRNA注射组爪蟾卵母细胞GVBD发生情况；按GVBD发生时间，收集单个卵母细胞，转移到新鲜配置的1×OR2培养液中。GVBD发生后70min，将卵母细胞放置在含有Hoechst染料(1：5000)的溶液中，染色10min后，荧光显微镜延迟摄影法(time-lapse)观察三组卵母细胞染色体的变化。
五、共聚焦显微镜观察爪蟾卵母细胞胞质分裂过程
15nL Alexa-488-phalloidin(4U/ml)和10nL Rhodamine-tubulin(3mg/ml)共同显微注射PAK2-NT mRNA、PAK2-NTm mRNA注射组及无注射对照组爪蟾卵母细胞。室温孵育过夜，翌日1μmol/L孕酮溶液刺激卵母细胞，利用共聚焦显微镜观察两组卵母细胞的形态变化，time-lapse方法观察。
用20nL 0.25mg/ml GFP-wGBD mRNA和10nL Rhodamine-tubulin(3mg/ml)共同注射入对照组、PAK2-NT mRNA和PAK2-NTm mRNA注射组的爪蟾卵母细胞，室温孵育过夜，翌日1μmol/L孕酮溶液刺激卵母细胞。利用共聚焦显微镜观察三组卵母细胞的形态变化，time-lapse方法观察。
六、统计学分析
比较正常爪蟾卵母细胞、PAK2-NT mRNA注射组、PAK2-NTm mRNA注射组的爪蟾卵母细胞极体形成百分率，采用SPSS11.5统计学软件进行统计分析，P<0.05认为有统计学意义。
实验结果
一、爪蟾卵母细胞成熟过程中PAK2磷酸化状态观察
利用p-PAK2(Thr402)抗体检测爪蟾卵母细胞成熟过程中内源性PAK2磷酸化活性变化。结果表明GV期PAK2处于无活性状态，在卵母细胞GVBD发生后，PAK2处于磷酸化活性状态，并且在卵母细胞成熟过程中保持活性。
PAK2-NT mRNA注射组，GV期未见PAK2磷酸化，GVBD发生后PAK2呈现弱磷酸化状态，12h后未见PAK2磷酸化。用抗HA-tag抗体检测显微注射PAK2-NT mRNA的卵母细胞是否表达PAK2-NT融合蛋白，结果显示，PAK2-NTmRNA注射组，GV和GVBD发生后均有PAK2-NT蛋白表达，而对照组无，说明显微注射PAK2-NT mRNA的卵母细胞表达PAK2-NT。
二、荧光显微镜观察爪蟾卵母细胞DNA染色结果
爪蟾卵母细胞GVBD 70min后Hoechst DNA染色结果：对照组卵母细胞，正常胞质分裂并且形成极体；PAK2-NT mRNA和PAK2-NTm mRNA注射组爪蟾卵母细胞染色体完成复制，未见极体形成。
三、PAK2-NT、PAK2-NTm抑制爪蟾卵母细胞胞质分裂
Alexa-488-phalloidin和Rhodamine-tubulin共同注射三组爪蟾卵母细胞，time-lapse实时观察发现：对照组卵母细胞中，F-肌动蛋白聚集并环绕于纺锤体上方和周边，形成收缩环，收缩环向内切断纺锤体，形成极体；PAK2-NT mRNA注射组和PAK2-NTm mRNA注射组未见F-肌动蛋白聚集，收缩环和极体形成，可见纺锤体逐渐拉长，最终未完成胞质分裂。
四、共聚焦显微镜观察爪蟾卵母细胞胞质分裂过程中Cdc42活性变化
GFP-wGBD探针可观察爪蟾卵母细胞内Cdc42活性变化。对照组卵母细胞，GV和GVBD发生后，未观察到Cdc42活性变化。在极体释放数分钟前，纺锤体上方可见微弱的Cdc42活性，活性逐渐增强，随后收缩环形成，胞质分裂发生。Cdc42活性向下扩展，包绕整个极体；PAK2-NT mRNA注射组未见明显的Cdc42活性变化，但可见纺锤体发生与对照组相似的变化，最终未形成极体；PAK2-NTmmRNA注射组可见Cdc42的活性出现在纺锤体上方，持续数分钟，但是没有形成收缩环，未见极体形成。
五、统计学分析
采用SPSS11.5统计学软件进行统计分析，正常对照组爪蟾卵母细胞、PAK2-NT mRNA注射组、PAK2-NTm mRNA注射组的爪蟾卵母细胞极体形成百分率的差异有统计学意义(P<0.05)。与正常对照组卵母细胞相比，PAK2-NT mRNA注射组、PAK2-NTm mRNA注射组的卵母细胞极体形成百分率明显降低。
实验结论
1、爪蟾卵母细胞GV期PAK2处于无活性状态，在卵母细胞GVBD发生后，PAK2处于磷酸化活性状态，并且在卵母细胞成熟过程中保持活性。
2、注射了PAK2-NT mRNA和PAK2-NTm mRNA的爪蟾卵母细胞GV和GVBD未见明显形态学变化，孕酮诱导的GVBD也未见明显异常。
3、PAK2-NT mRNA和PAK2-NTm mRNA注射组的爪蟾卵母细胞胞质分裂和极体形成障碍。
4、在爪蟾卵母细胞中，PAK2参与胞质分裂和极体形成可能是不依赖于Cdc42活性。
INTRODUCTION
Polar body formation during oocyte meiotic maturation is an extreme case of asymmetric cell division, ensuring the reservation of maternal cytoplasmic stores in oocyte while halving the genome. The mechanism regulating the formation of polar body is poorly understood. As discussed above, the polarization of cortical actin patches and the attachment of metaphase I spindle to the animal pole cortex are necessary for anaphase I initiation and the first polar body formation. The p21-activated kinases (PAKs) and their activator, small GTPase Cdc42, have an evolutionary conserved function in the establishment of actin polarity and asymmetric cell division from yeast to mammals. For example, in the bud formation process of budding yeast, another type of asymmetric cell division, it is also necessary for one pole of the mitotic spindle (the daughter pole) to attach the bud cortex to initiate cytokinesis. The PAK kinases (Cla4 and Ste20) also play an essential role in establishing the actin polarity and promoting budding and cytokinesis together with their activator, Cdc42. Loss or inhibition of PAKs function leads to a complete loss of polarization and prevents bud formation. Ma et al. have already demonstrated that in frog oocyte, inhibition of Cdc42 completely blocked the first polar body formation. So here I tested the hypothesis that Xenopus PAK kinases (X-PAKs) might also be involved in the first polar body formation during frog oocyte maturation.
The p21-activated kinases (PAKs) are a highly conserved family of protein kinases whose activities are stimulated by binding of active Rac and Cdc42 GTPases. They have some common functions in activation of MAPK cascades and regulation of cell polarity and motility by acting on the actin and tubulin cytoskeletons.PAKs have a similar primary structure, which contains an N-terminal p21 GTPase (Rac or Cdc42) binding domain (PBD or CRIB) and a C-terminal protein kinase domain, except that some PAKs (Cla4, Skm1 and Pak2) from yeast contain an N-terminal pleckstrin homology (PH) domain. Based on the structural organization and activity regulation, the PAK family is divided into two subfamilies in higher eukaryotes: group A (PAKs 1-3) and group B (PAKs 4-6). PAKs of Group A are serine/threonine protein kinases with a significantly conserved sequence homology in their catalytic domains. They contain several putative Src homology 3 (SH3)-binding motifs in the N terminus, a PBD and a C-terminal kinase domain. They bind both Cdc42 and Rac, and are strongly activated by the bindings. Group B PAKs contain a PBD at the extreme N terminus and a C-terminal kinase domain. They bind Cdc42 and Rac, but are not appreciably activated upon binding. It is thought that binding by Cdc42 is more important for their localization rather than activation of group B PAKs.
PAKs of group A exist as a homodimer. Cdc42 or Rac binding disrupts dimerization, releases autoinhibition and obtains kinase activity by phosphorylation at several sites. For example, PAK1 exists as a homodimer in a trans-inhibited conformation where the N-terminal autoinhibitory domain of one PAK1 molecule binds and inhibits the C-terminal kinase domain of the other. Binding of active Cdc42 or Rac leads to a conformation change, dissociates the dimer and unlocks both kinase domains, as the PBD overlaps the autoinhibitory domain. Phosphorylation of several sites both in C and N termini fully activates the kinase and prevents reversal of these steps. Phosphorylation of Thr423 in the kinase domain is very important for full catalytic function toward exogenous substrates. PAK2 also has similar activation mechanism.
In Xenopus, four PAKs have been discovered. X-PAKs 1-3 belong to group A, whereas X-PAK5 belongs to group B. Earlier studies indicated that over-expression of X-PAK1-Cter could prevent progesterone-induced oocyte maturation. However, X-PAK1 was inactive in G2 oocytes and remained inactive through oocyte maturation and appeared not responsive to Cdc42 in Xenopus oocytes. In contrast, X-PAK2 was active in G2 oocytes but became inactive at GVBD due to its phosphorylation. And it was suggested to be involved in the control of G2/M transition as the downstream effector of Cdc42. X-PAK3 was undetectable in oocyte or eggs and began to be expressed at late gastrula stages of embryonic development in the neuroectoderm. X-PAK5 was found in the embryos to bind to actin and microtubule networks and regulate convergent extension movements during gastrula in a calcium-dependent way. Therefore, it seems that only X-PAK2 is involved in oocyte maturation and can response to its activator, Cdc42, in oocytes. So I focused mainly on the functions of X-PAK2 in the first polar body formation during frog oocyte maturation.
MATERIALS
1、PAK2 ,Cdc42 plasmids and reagents for molecular biology
2、Western blot reagents
3、Related fluorescently-labeled biology reagents.
METHODS
1、Animal and oocyte manipulation
Frogs were primed by gonadotropin (PMSG, 50IU per frog) 3 days before operations. Ovarian fragments were removed surgicallyand teared 15 fragments under microscope. Ovary sections were treated in collagenase solution for 3h.Choose the same size and mature stage VI oocytes ,put them in OR2 medium(free-Ca~(2+)) and shake them for 4h. Oocytes were lysed in ice-cold extraction (EB) buffer(10μl lysis buffer per oocyte),. Following centrifugation (13000g for 5 min, 4℃), the clarified extract was removed, mixed with 2X SDS sample buffer plus beta-mercaptoethanol (β-Me), and stored in -20℃.
2、PAK2 phosphorylation assay during oocyte development
PAK2 phosphorylation status: Time course of the endogenous PAK2 (recognized by p-PAK2 antibody) phosphorylation during oocyte maturation. After the addition of progesterone, three oocytes were picked randomly at different time and lysed, single oocytes were picked from that group at different time after GVBD and individually lysed. The extracts were subjected to immunoblotting with p-PAK2 antibody.
3、PAK2-NT ,PAK2-NTm and GFP-wGBD mRNA Synthesis
Messenger RNAs were in vitro transcripted by using mMESSAGE mMACHINE(?) T7/SP6 Kit (Ambion). The synthesized mRNA was dissolved in nuclease-free water, aliquoted, and stored in -80°C until injection.
4、Xenopus oocyte DNA staining under fluorescence microscope
Observe GVBD process of normal oocytes,oocytes injected with PAK2-NT mRNA and oocytes injected with PAK2-NTm.According the time of GVBD,collect single oocyte,and transfer to fresh calcium-free OR2 medium.After 70min of GVBD,oocytes were put in Hoechst dye (l:5000),and stained for 10min. Observe the chromosome changes of oocytes with time-lapse under fluorescence microscope.
5、Xenopus Oocyte cytokinesis processs under confocal microscope
15nL Alexa-488-phalloidin (4U/ml) and 10nL Rhodamine-tubulin (3mg/ml) were microinjected into oocytes injecte with PAK2-NT mRNA and oocytes injected with PAK2-NTm.All oocytes were incubated at rome temperature.Under this condition,most oocytes processed GVBD,but did not form M I spindles.Observe the all oocytes morphologic changes with time-lapse under confocal microscopy.
10nL 0.25mg/ml GFP-wGBD mRNA and 10nL Rhodamine-tubulin (3mg/ml) were microinjecte into oocytes injecte with PAK2-NT mRNA and oocytes injected with PAK2-NTm.All oocytes were incubated at rome temperature.The next day,oocytes were stimulated in progesterone solution.3h later, observe the all oocytes morphologic changes with time-lapse under confocal microscopy.
6、Statistic analysis
Compare cytokinesis and polar body formation percentage of normal oocytes,oocytes injected with PAK2-NT mRNA,oocytes injected with PAK2-NTm mRNA.SPSS 11.5 software was emplyed, and P<0.05 was considered different significantly.
RESULTS
1、PAK2 phosphorylation assay during Xenopus oocyte development
We employed a phospho-specific antibody (p-PAK (Thr402)) to examine the phosphorylation and activity status of endogenous X-PAK2 during oocyte maturation. By using this phospho-specific antibody, we discovered that a possible endogenous X-PAK was phosphorylated at GVBD and remained phosphorylated until metaphase II arrest. This suggested that the endogenous PAK2 was inactive in GV oocytes, but fully activated upon GVBD and remained active throughout the rest of oocyte maturation process. In oocytes injected with PAK2-NT,at GV,PAK2 was unphosphorylated,at GVBD PAK2 remained weak phosphorylated, 12h later,PAK2 phosphorylation disappeared.
Employ HA-tag antibody to detect PAK2-NT fusion protein exoression in normal oocyte and oocytes injected with PAK2-NT mRNA.Results showed that PAK2-NT were expressed at GV and GVBD in oocytes injected with PAK2-NT mRNA,but normal oocytes none.It indicated that infusion proteins were expressed in oocytes injected with PAK2-NT mRNA.
2、Observation of DNA changes under fluorescent microscopy
Normal oocytes showed cytokinesis and polar body formation,but PAK2-NT mRNA and PAK2-NTm mRNA injected oocytes completed chromosome replication ,but no polar body formation.
3、PAK2-NT、PAK2-NTm inhibits oocyte cytokinesis
Alexa-488-phalloidin and Rhodamine-tubulin were microinjected into two group oocytes,time-lapse results indicated:in normal oocyte,F-actin accumulated around and over spindle and then narrowed as it contracted inward beneath the forming polar body. In PAK2-NT and PAK2-NTm mRNA injected oocytes,no F-actin accumulation,and no contract ring and polar body formation. Spindle elonged gradually,but failed to completed cytokinesis.
4、Cdc42 activity changes in controls and injection groups under confocal microscopy
To determine whether Cdc42 activation occurred at the time of polar body formation,we utilized the GFP-wGBD probe previously developed for visualization of Cdc42 activity in Xenopus oocytes.In control oocytes, Cdc42 was undetectable in GV oocytes or GVBD oocytes until several minutes before emission of the polar body,at which point a faint patch of increased Cdc42 activity appeared overlaying the spindle microtubules.This patch increased in intensity and,within a few min after the appearance of the patch,cytokinesis ensued,as the cap spread downward in concert with formation of the polar body and eventually surrounded the entire polar body as it was pinched off.
PAK2-NT mRNA injected oocytes showed no Cdc42 activity changes,but spindle morphology the same as the control oocyte,but failed to form polar body.In PAK2-NTm mRNA injected oocytes,Cdc42 activty appeaed above and around the spindle,last a few minutes,but did not form contract ring and polar body.showed
5、Statistic analysis
SPSS 11.5 software was emplyed, and P<0.05 was considered different significantly.Compared with control oocytes,cytokinesis and polar body formation percentages of PAK2-NT mRNA injected oocytes and PAK2-NTm mRNA injected oocytes decreased significantly.
CONCLUSIONS
1、In Xenopus oocytes, the endogenous PAK2 was inactive in GV oocytes, but fully activated upon GVBD and remained active throughout the rest of oocyte maturationprocess.
2、Compared with control oocytes, PAK2-NT mRNA and PAK2-NTm mRNAinjection and its expression does not affect the GV and progesterone induced GVBD.
3、PAK2-NT mRNA and PAK2-NTm mRNA injection inhibit oocyte cytokinesis andpolar body formation
4、PAK2 involved in cytokinesis independent of Cdc42 during Xenopus oocytematuration.
爪蟾卵母细胞减数分裂过程中，极体形成是一种不对称的细胞分裂，有助于母体的胞浆物质和基因组的储存。目前调节极体形成的机制所知甚少，有研究表明，细胞皮质层肌动蛋白极化，中期Ⅰ纺锤体与卵母细胞皮质层接触对于后期Ⅰ的启动和极体的形成非常重要。从酵母到哺乳动物细胞，P21活化激酶(p21-activated kinase，PAK)与它的激活子，小GTP酶—Cdc42在细胞极化和不对称分裂中具有进化保守功能。例如出芽酵母的出芽过程类似不对称的细胞分裂过程，需要纺锤体一端与出芽的皮质层接触从而启动胞质分裂，PAK(Cla4和Ste20)与其激活子Cdc42在肌动蛋白极化、促进出芽和胞质分裂中发挥重要作用。PAK功能的缺失和抑制导致酵母细胞极化失败，出芽不能完成。Ma和Cheng等研究证明，在爪蟾卵母细胞中，一种小GTP酶Cdc42调控肌动蛋白极化和不对称细胞分裂，所以我们假设，PAK在爪蟾卵母细胞成熟过程中同样参与了胞质分裂和极体形成。
PAK是一类进化上保守的丝氨酸/苏氨酸蛋白激酶，为Rho家族小鸟苷三磷酸酶(Rho-GTPase)Cdc42/Rac、生长因子信号的主要靶蛋白，参与许多重要细胞活动。到目前为止共发现6个PAK家族成员，根据其结构相似性，将它们分为两类，Ⅰ类包括PAK1，PAK2和PAK3，Ⅱ类包括PAK4，PAK5和PAK6。Ⅰ类PAK在N-端含有数个有争议的Src相似序列3(SH3)-结合基序和p21蛋白结合域(p21binding domain，PBD)，C-端激酶域为高度保守的序列。Ⅰ类PAK通过结合Cdc42或Rac发挥生物学活性。Ⅱ类PAK在N-端同样包含PBD，C-端也为激酶域，同样可以结合Rac和Cdc42，但是活性不依赖与它们结合，目前认为Cdc42的结合对于Ⅱ类PAK的定位更重要而非其活性。
PAK2是Rho家族中GTP酶的重要效应物。PAK2通过对肌动蛋白细胞骨架的调节，在细胞形态学和动力学的调控上起重要作用。PAK2具有高度保守的C-端激酶域和N-端Cdc42/Rac相互作用域(Cdc42/Rac interactive bindingdomain，CRIB)，又名GTP酶结合域。该区域与PAK2 N-端的自身抑制域(auto-inhibitorydomain，AID)部分重叠，调节PAK2的激酶活性。非活性PAK2自身折叠使AID与C-端激酶域结合，活化环无法暴露，抑制其激酶活性。当Cdc42与PAK2结合时，导致PAK2构象改变，这个过程会缓解PAK2自身抑制并且激活PAK2活性。
在爪蟾卵母细胞中目前已经发现四种PAK，Ⅰ类PAK 1-3，Ⅱ类PAK5。早期的研究表明PAK1过表达抑制孕酮诱导的爪蟾卵母细胞成熟。在G2期的爪蟾卵母细胞中，PAK1保持无活性状态，在卵母细胞整个成熟过程保持无活性，对Cdc42信号无应答。PAK2在爪蟾G2期的卵母细胞中保持活化状态。在爪蟾卵母细胞和成熟卵细胞中未检测到PAK3，PAK3只在爪蟾胚胎发育的肠胚期后期被表达。PAK5只出现在爪蟾胚胎中，能结合肌动蛋白和微管网络，在肠胚期以Ca~(2+)依赖性方式调节细胞骨架的会聚延伸。Cau等研究证明在爪蟾卵母细胞成熟过程中显微注射Cdc42V12(constitutively active Cdc42，活化型Cdc42)后，PAK2活性增高约50倍，提示PAK2可能是Cdc42优先选择的效应器，参与爪蟾卵母细胞成熟过程。本研究着重探讨在爪蟾卵母细胞分裂过程中PAK2是否参与胞质分裂和极体形成及与Cdc42的关系。
实验材料
1、PAK2、Cdc42相关质粒及分子生物学试剂。
2、蛋白质免疫印迹检测试剂。
3、荧光标记的相关分子生物学试剂
实验方法
一、爪蟾卵母细胞的准备及裂解
爪蟾皮下注射孕马血清促性腺激素(serum gonadotrophin，PMSG)50IU，3日后处死爪蟾，取卵巢组织，在显微镜下将其分离为15个卵左右的碎片。用胶原酶(1mg/mL)消化卵母细胞3h，取大小相似、发育成熟Ⅵ期卵母细胞，置于1×OR2(无Ca~(2+))培养液(83 mmol/L NaCl，2.5 mmol/L KCI，1 mmol/L MgCl_2，1 mmol/LNa_2HPO_4，5 mmol/L HEPES pH 7.8)中，室温震荡培养4h。10μL冷裂解缓冲液(EB缓冲液：20mM pH 7.3 HEPES，80mmol/L磷酸甘油，20mmol/L EGTA，15mmol/LMgCl_2，1mmol/L二硫苏糖醇，101×mol/L ATP，150mmol/L NaF，10μg/mL抑(蛋白)酶醛肽，200μmol/L苯甲磺酰氟，25μg/mL苯甲脒)裂解卵母细胞，4℃13，000g离心5min，上清液与2×SDS样品缓冲液、β-巯基乙醇混合，-20℃贮存。
二、爪蟾卵母细胞成熟过程中PAK2磷酸化状态观察
抗p-PAK2(Thr402)蛋白质印迹检测：孕酮刺激后不同时间点(0h、1h、2h、3h、3.5h)以及胚泡破裂(germinal vesicle breakdown，GVBD)发生后不同时间点(0min、30min、60min、90min、120min、150min、180min、12h)的爪蟾卵母细胞裂解液在15％SDS-PAGE电泳中分离，电转移至硝酸纤维素膜，10％BSA封闭1h；与抗p-PAK2抗体孵育2h，相应的二抗孵育1h，ECL发光法检测。
三、体外合成PAK2-NT、PAK2-NTm、GFP-WGBD mRNA
将PCS2-HA-PAK2-NT(PCS2-HA-PAK2-N-terminal)、PCS2-HA-PAK2-NTm(PCS2-HA-PAK2-N-terminal mutation)、GFP-wGBD质粒用特异性限制性内切酶进行酶切线性化，1.5％琼脂糖凝胶电泳进行鉴定，以线性化DNA为模板、合成PAK2-NT、PAK2-NTm、GFP-wGBD mRNA，-70℃保存备用。
四、荧光显微镜观察爪蟾卵母细胞DNA染色
观察对照组、PAK2-NT mRNA注射组和PAK2-NTm mRNA注射组爪蟾卵母细胞GVBD发生情况；按GVBD发生时间，收集单个卵母细胞，转移到新鲜配置的1×OR2培养液中。GVBD发生后70min，将卵母细胞放置在含有Hoechst染料(1：5000)的溶液中，染色10min后，荧光显微镜延迟摄影法(time-lapse)观察三组卵母细胞染色体的变化。
五、共聚焦显微镜观察爪蟾卵母细胞胞质分裂过程
15nL Alexa-488-phalloidin(4U/ml)和10nL Rhodamine-tubulin(3mg/ml)共同显微注射PAK2-NT mRNA、PAK2-NTm mRNA注射组及无注射对照组爪蟾卵母细胞。室温孵育过夜，翌日1μmol/L孕酮溶液刺激卵母细胞，利用共聚焦显微镜观察两组卵母细胞的形态变化，time-lapse方法观察。
用20nL 0.25mg/ml GFP-wGBD mRNA和10nL Rhodamine-tubulin(3mg/ml)共同注射入对照组、PAK2-NT mRNA和PAK2-NTm mRNA注射组的爪蟾卵母细胞，室温孵育过夜，翌日1μmol/L孕酮溶液刺激卵母细胞。利用共聚焦显微镜观察三组卵母细胞的形态变化，time-lapse方法观察。
六、统计学分析
比较正常爪蟾卵母细胞、PAK2-NT mRNA注射组、PAK2-NTm mRNA注射组的爪蟾卵母细胞极体形成百分率，采用SPSS11.5统计学软件进行统计分析，P<0.05认为有统计学意义。
实验结果
一、爪蟾卵母细胞成熟过程中PAK2磷酸化状态观察
利用p-PAK2(Thr402)抗体检测爪蟾卵母细胞成熟过程中内源性PAK2磷酸化活性变化。结果表明GV期PAK2处于无活性状态，在卵母细胞GVBD发生后，PAK2处于磷酸化活性状态，并且在卵母细胞成熟过程中保持活性。
PAK2-NT mRNA注射组，GV期未见PAK2磷酸化，GVBD发生后PAK2呈现弱磷酸化状态，12h后未见PAK2磷酸化。用抗HA-tag抗体检测显微注射PAK2-NT mRNA的卵母细胞是否表达PAK2-NT融合蛋白，结果显示，PAK2-NTmRNA注射组，GV和GVBD发生后均有PAK2-NT蛋白表达，而对照组无，说明显微注射PAK2-NT mRNA的卵母细胞表达PAK2-NT。
二、荧光显微镜观察爪蟾卵母细胞DNA染色结果
爪蟾卵母细胞GVBD 70min后Hoechst DNA染色结果：对照组卵母细胞，正常胞质分裂并且形成极体；PAK2-NT mRNA和PAK2-NTm mRNA注射组爪蟾卵母细胞染色体完成复制，未见极体形成。
三、PAK2-NT、PAK2-NTm抑制爪蟾卵母细胞胞质分裂
Alexa-488-phalloidin和Rhodamine-tubulin共同注射三组爪蟾卵母细胞，time-lapse实时观察发现：对照组卵母细胞中，F-肌动蛋白聚集并环绕于纺锤体上方和周边，形成收缩环，收缩环向内切断纺锤体，形成极体；PAK2-NT mRNA注射组和PAK2-NTm mRNA注射组未见F-肌动蛋白聚集，收缩环和极体形成，可见纺锤体逐渐拉长，最终未完成胞质分裂。
四、共聚焦显微镜观察爪蟾卵母细胞胞质分裂过程中Cdc42活性变化
GFP-wGBD探针可观察爪蟾卵母细胞内Cdc42活性变化。对照组卵母细胞，GV和GVBD发生后，未观察到Cdc42活性变化。在极体释放数分钟前，纺锤体上方可见微弱的Cdc42活性，活性逐渐增强，随后收缩环形成，胞质分裂发生。Cdc42活性向下扩展，包绕整个极体；PAK2-NT mRNA注射组未见明显的Cdc42活性变化，但可见纺锤体发生与对照组相似的变化，最终未形成极体；PAK2-NTmmRNA注射组可见Cdc42的活性出现在纺锤体上方，持续数分钟，但是没有形成收缩环，未见极体形成。
五、统计学分析
采用SPSS11.5统计学软件进行统计分析，正常对照组爪蟾卵母细胞、PAK2-NT mRNA注射组、PAK2-NTm mRNA注射组的爪蟾卵母细胞极体形成百分率的差异有统计学意义(P<0.05)。与正常对照组卵母细胞相比，PAK2-NT mRNA注射组、PAK2-NTm mRNA注射组的卵母细胞极体形成百分率明显降低。
实验结论
1、爪蟾卵母细胞GV期PAK2处于无活性状态，在卵母细胞GVBD发生后，PAK2处于磷酸化活性状态，并且在卵母细胞成熟过程中保持活性。
2、注射了PAK2-NT mRNA和PAK2-NTm mRNA的爪蟾卵母细胞GV和GVBD未见明显形态学变化，孕酮诱导的GVBD也未见明显异常。
3、PAK2-NT mRNA和PAK2-NTm mRNA注射组的爪蟾卵母细胞胞质分裂和极体形成障碍。
4、在爪蟾卵母细胞中，PAK2参与胞质分裂和极体形成可能是不依赖于Cdc42活性。
INTRODUCTION
Polar body formation during oocyte meiotic maturation is an extreme case of asymmetric cell division, ensuring the reservation of maternal cytoplasmic stores in oocyte while halving the genome. The mechanism regulating the formation of polar body is poorly understood. As discussed above, the polarization of cortical actin patches and the attachment of metaphase I spindle to the animal pole cortex are necessary for anaphase I initiation and the first polar body formation. The p21-activated kinases (PAKs) and their activator, small GTPase Cdc42, have an evolutionary conserved function in the establishment of actin polarity and asymmetric cell division from yeast to mammals. For example, in the bud formation process of budding yeast, another type of asymmetric cell division, it is also necessary for one pole of the mitotic spindle (the daughter pole) to attach the bud cortex to initiate cytokinesis. The PAK kinases (Cla4 and Ste20) also play an essential role in establishing the actin polarity and promoting budding and cytokinesis together with their activator, Cdc42. Loss or inhibition of PAKs function leads to a complete loss of polarization and prevents bud formation. Ma et al. have already demonstrated that in frog oocyte, inhibition of Cdc42 completely blocked the first polar body formation. So here I tested the hypothesis that Xenopus PAK kinases (X-PAKs) might also be involved in the first polar body formation during frog oocyte maturation.
The p21-activated kinases (PAKs) are a highly conserved family of protein kinases whose activities are stimulated by binding of active Rac and Cdc42 GTPases. They have some common functions in activation of MAPK cascades and regulation of cell polarity and motility by acting on the actin and tubulin cytoskeletons.PAKs have a similar primary structure, which contains an N-terminal p21 GTPase (Rac or Cdc42) binding domain (PBD or CRIB) and a C-terminal protein kinase domain, except that some PAKs (Cla4, Skm1 and Pak2) from yeast contain an N-terminal pleckstrin homology (PH) domain. Based on the structural organization and activity regulation, the PAK family is divided into two subfamilies in higher eukaryotes: group A (PAKs 1-3) and group B (PAKs 4-6). PAKs of Group A are serine/threonine protein kinases with a significantly conserved sequence homology in their catalytic domains. They contain several putative Src homology 3 (SH3)-binding motifs in the N terminus, a PBD and a C-terminal kinase domain. They bind both Cdc42 and Rac, and are strongly activated by the bindings. Group B PAKs contain a PBD at the extreme N terminus and a C-terminal kinase domain. They bind Cdc42 and Rac, but are not appreciably activated upon binding. It is thought that binding by Cdc42 is more important for their localization rather than activation of group B PAKs.
PAKs of group A exist as a homodimer. Cdc42 or Rac binding disrupts dimerization, releases autoinhibition and obtains kinase activity by phosphorylation at several sites. For example, PAK1 exists as a homodimer in a trans-inhibited conformation where the N-terminal autoinhibitory domain of one PAK1 molecule binds and inhibits the C-terminal kinase domain of the other. Binding of active Cdc42 or Rac leads to a conformation change, dissociates the dimer and unlocks both kinase domains, as the PBD overlaps the autoinhibitory domain. Phosphorylation of several sites both in C and N termini fully activates the kinase and prevents reversal of these steps. Phosphorylation of Thr423 in the kinase domain is very important for full catalytic function toward exogenous substrates. PAK2 also has similar activation mechanism.
In Xenopus, four PAKs have been discovered. X-PAKs 1-3 belong to group A, whereas X-PAK5 belongs to group B. Earlier studies indicated that over-expression of X-PAK1-Cter could prevent progesterone-induced oocyte maturation. However, X-PAK1 was inactive in G2 oocytes and remained inactive through oocyte maturation and appeared not responsive to Cdc42 in Xenopus oocytes. In contrast, X-PAK2 was active in G2 oocytes but became inactive at GVBD due to its phosphorylation. And it was suggested to be involved in the control of G2/M transition as the downstream effector of Cdc42. X-PAK3 was undetectable in oocyte or eggs and began to be expressed at late gastrula stages of embryonic development in the neuroectoderm. X-PAK5 was found in the embryos to bind to actin and microtubule networks and regulate convergent extension movements during gastrula in a calcium-dependent way. Therefore, it seems that only X-PAK2 is involved in oocyte maturation and can response to its activator, Cdc42, in oocytes. So I focused mainly on the functions of X-PAK2 in the first polar body formation during frog oocyte maturation.
MATERIALS
1、PAK2 ,Cdc42 plasmids and reagents for molecular biology
2、Western blot reagents
3、Related fluorescently-labeled biology reagents.
METHODS
1、Animal and oocyte manipulation
Frogs were primed by gonadotropin (PMSG, 50IU per frog) 3 days before operations. Ovarian fragments were removed surgicallyand teared 15 fragments under microscope. Ovary sections were treated in collagenase solution for 3h.Choose the same size and mature stage VI oocytes ,put them in OR2 medium(free-Ca~(2+)) and shake them for 4h. Oocytes were lysed in ice-cold extraction (EB) buffer(10μl lysis buffer per oocyte),. Following centrifugation (13000g for 5 min, 4℃), the clarified extract was removed, mixed with 2X SDS sample buffer plus beta-mercaptoethanol (β-Me), and stored in -20℃.
2、PAK2 phosphorylation assay during oocyte development
PAK2 phosphorylation status: Time course of the endogenous PAK2 (recognized by p-PAK2 antibody) phosphorylation during oocyte maturation. After the addition of progesterone, three oocytes were picked randomly at different time and lysed, single oocytes were picked from that group at different time after GVBD and individually lysed. The extracts were subjected to immunoblotting with p-PAK2 antibody.
3、PAK2-NT ,PAK2-NTm and GFP-wGBD mRNA Synthesis
Messenger RNAs were in vitro transcripted by using mMESSAGE mMACHINE(?) T7/SP6 Kit (Ambion). The synthesized mRNA was dissolved in nuclease-free water, aliquoted, and stored in -80°C until injection.
4、Xenopus oocyte DNA staining under fluorescence microscope
Observe GVBD process of normal oocytes,oocytes injected with PAK2-NT mRNA and oocytes injected with PAK2-NTm.According the time of GVBD,collect single oocyte,and transfer to fresh calcium-free OR2 medium.After 70min of GVBD,oocytes were put in Hoechst dye (l:5000),and stained for 10min. Observe the chromosome changes of oocytes with time-lapse under fluorescence microscope.
5、Xenopus Oocyte cytokinesis processs under confocal microscope
15nL Alexa-488-phalloidin (4U/ml) and 10nL Rhodamine-tubulin (3mg/ml) were microinjected into oocytes injecte with PAK2-NT mRNA and oocytes injected with PAK2-NTm.All oocytes were incubated at rome temperature.Under this condition,most oocytes processed GVBD,but did not form M I spindles.Observe the all oocytes morphologic changes with time-lapse under confocal microscopy.
10nL 0.25mg/ml GFP-wGBD mRNA and 10nL Rhodamine-tubulin (3mg/ml) were microinjecte into oocytes injecte with PAK2-NT mRNA and oocytes injected with PAK2-NTm.All oocytes were incubated at rome temperature.The next day,oocytes were stimulated in progesterone solution.3h later, observe the all oocytes morphologic changes with time-lapse under confocal microscopy.
6、Statistic analysis
Compare cytokinesis and polar body formation percentage of normal oocytes,oocytes injected with PAK2-NT mRNA,oocytes injected with PAK2-NTm mRNA.SPSS 11.5 software was emplyed, and P<0.05 was considered different significantly.
RESULTS
1、PAK2 phosphorylation assay during Xenopus oocyte development
We employed a phospho-specific antibody (p-PAK (Thr402)) to examine the phosphorylation and activity status of endogenous X-PAK2 during oocyte maturation. By using this phospho-specific antibody, we discovered that a possible endogenous X-PAK was phosphorylated at GVBD and remained phosphorylated until metaphase II arrest. This suggested that the endogenous PAK2 was inactive in GV oocytes, but fully activated upon GVBD and remained active throughout the rest of oocyte maturation process. In oocytes injected with PAK2-NT,at GV,PAK2 was unphosphorylated,at GVBD PAK2 remained weak phosphorylated, 12h later,PAK2 phosphorylation disappeared.
Employ HA-tag antibody to detect PAK2-NT fusion protein exoression in normal oocyte and oocytes injected with PAK2-NT mRNA.Results showed that PAK2-NT were expressed at GV and GVBD in oocytes injected with PAK2-NT mRNA,but normal oocytes none.It indicated that infusion proteins were expressed in oocytes injected with PAK2-NT mRNA.
2、Observation of DNA changes under fluorescent microscopy
Normal oocytes showed cytokinesis and polar body formation,but PAK2-NT mRNA and PAK2-NTm mRNA injected oocytes completed chromosome replication ,but no polar body formation.
3、PAK2-NT、PAK2-NTm inhibits oocyte cytokinesis
Alexa-488-phalloidin and Rhodamine-tubulin were microinjected into two group oocytes,time-lapse results indicated:in normal oocyte,F-actin accumulated around and over spindle and then narrowed as it contracted inward beneath the forming polar body. In PAK2-NT and PAK2-NTm mRNA injected oocytes,no F-actin accumulation,and no contract ring and polar body formation. Spindle elonged gradually,but failed to completed cytokinesis.
4、Cdc42 activity changes in controls and injection groups under confocal microscopy
To determine whether Cdc42 activation occurred at the time of polar body formation,we utilized the GFP-wGBD probe previously developed for visualization of Cdc42 activity in Xenopus oocytes.In control oocytes, Cdc42 was undetectable in GV oocytes or GVBD oocytes until several minutes before emission of the polar body,at which point a faint patch of increased Cdc42 activity appeared overlaying the spindle microtubules.This patch increased in intensity and,within a few min after the appearance of the patch,cytokinesis ensued,as the cap spread downward in concert with formation of the polar body and eventually surrounded the entire polar body as it was pinched off.
PAK2-NT mRNA injected oocytes showed no Cdc42 activity changes,but spindle morphology the same as the control oocyte,but failed to form polar body.In PAK2-NTm mRNA injected oocytes,Cdc42 activty appeaed above and around the spindle,last a few minutes,but did not form contract ring and polar body.showed
5、Statistic analysis
SPSS 11.5 software was emplyed, and P<0.05 was considered different significantly.Compared with control oocytes,cytokinesis and polar body formation percentages of PAK2-NT mRNA injected oocytes and PAK2-NTm mRNA injected oocytes decreased significantly.
CONCLUSIONS
1、In Xenopus oocytes, the endogenous PAK2 was inactive in GV oocytes, but fully activated upon GVBD and remained active throughout the rest of oocyte maturationprocess.
2、Compared with control oocytes, PAK2-NT mRNA and PAK2-NTm mRNAinjection and its expression does not affect the GV and progesterone induced GVBD.
3、PAK2-NT mRNA and PAK2-NTm mRNA injection inhibit oocyte cytokinesis andpolar body formation
4、PAK2 involved in cytokinesis independent of Cdc42 during Xenopus oocytematuration.
引文
1． Bokoch GM.Biology of the p21-activated kinases[J].Annu Rev Biochem.2003,72:743-781．
2． Chu PC,Wu J,Liao XC,et al.A novel role for p21-activated protein kinase 2 in T cell activation[J].J Immunol.2004,172(12) ,7324-7334．
3． Kumar R,Vadlamudi RK.Emerging functions of p21-activated kinases in human cancer cells[J].J Cell Physiol.2002,193(2) :133-144．
4． Manser E,Leung T,Salihuddin H,et al.A Brain serine/threonine protein kinase activated by Cdc42 and Racl[J].Nature.1994,367(6458) :40-46．
5． Bagrodia S,Cerione RA.PAK to the future[J].Trends Cell Biol.1999,9(9) :350-355．
6． Kanus UG,Bokoch GM.The p21Rac/Cdc42-activated kinases(PAKs)[J].Int J Biol Cell Biol.1998,30(8) :857-862．
7． Sells MA,Chernoff J.Emerging from the Pak:the p21-activated protein kinase family[J].Trends Cell Biol.1997,193(2) :162-167．
8． Abo A,Qu J,Cammarano MS,et al.PAK4,a novel effector for Cdc42Hs,is implicated in the reorganization of the actin cytoskeleton and in the formation of fílopodia[J].EMBO J.1998,17(22) :6527-6540．
9． Yang F,Li X,Sharma M,et al.Androgen receptor specifically interacts with a novel p21-activated kinase,PAK6[J].J Biol Chem.2001,276(18) :15345-15353．
10． Lee SR,Ramos SM,Ko A,et al.AR and ER interaction with a p21-activated kinase(PAK6) [J].Mol Endocrinol.2002,16(1) :85-99．
11． Pandey A,Dan I,Kristiansen TZ,et al.Cloning and characterization of PAK5,a novel member of mammalian p21-activated kinase-Ⅱ subfamily that is predominantly expressed in brain[J].Oncogene.2002,21(24) :3939-3948．
12． Taylor SS,Knighton DR,Zheng J,et al.Structural framework for the protein kinase family[J].Annu Rev Cell Biol.1992,8:429-462．
13． Jaffer ZM,Chernoff J.p21-activated kinases:three more join the Pak[J].Int J Biochem Cell Biol.2002,34(7) :713-717
14． Weisz Hubsman M,Volinsky N,Manser E,et al.Autophosphorylation-dependent degradation of Pakl,triggered by the Rho-family GTPase,Chp[J].Biochem J.2007 Mar 13;[Epub ahead of print].
15． Chan WH,Yu JS,Yang SD.PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism:evidence for the involvement of oxidative stress[J].J Cell Physiol.1999,178(3) :397-408．
16． Vadlamudi RK.Adam L,Wang RA,et al.Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells[J].J Biol Chem.2000,275(46) :36238-36244．
17． Banerjee M,Worth D,Prowse DW,et al.Pakl phosphorylation on t212 affects microtubules in cells undergoing mitosis[J].Curr Biol.2002,12(14) :1233-1239．
18． Li F,Adam L,Vadlamudi RK,et al.p21-activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells[J].EMBO Rep.2002,3(8) :767-773．
19． Zhao ZS,Manser E,Loo TH,et al.Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly[J].Mol Cell Biol.2000,20(17) :6354-6363．
20． Balasenthil S,Sahin AA,Barnes CJ,et al.p21-activated kinase-1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells[J].J Biol Chem.2004,279(2) :1422-1428．
21． Ma C,Ha Benink,Cheng DY,et al.Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation[J].Curr Biol,2006,16(2) :214-220
22． Cvrckova F,De Virgilio C.Manser E.Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast[J].Genes dev,1995,9(15) :1817-1830．
23． Brown DD.A tribute to the Xenopus laevis oocyte and egg[J].J Biol Chem.2004,279(44) :45291-45299．
24． Leveleki L,Mahlert M,Sandrock B,et al.The PAK family kinase Cla4 is required for budding and morphogenesis in Ustilago maydis[J].Mol Microbiol.2004,54(2) :396-406．
25． Sagata N.What does Mos do in oocytes and somatic cells[J]?Bioessays.1997,19(1) :13-21．
26． Faure S,Vigneron S,Doree M,et al.A member of the Ste20/PAK family of protein kinases is involved in both arrest of Xenopus oocytes at G2/prophase of the first meiotic cell cycle and in prevention of apoptosis[J].EMBO J.1997,16(18) :5550-5561．
27． Faure S,Vigneron S,Galas S,et al.Control of G2/M transition in Xenopus by a member of the p21-activated kinase(PAK)family:a link between protein kinase A and PAK signaling pathways[J]?J Biol Chem.1999,274(6) :3573-3579．
28． Leberer E,Dignard D,Harcus D,et al.The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components[J].EMBO J.1992,11(13) :4815-4824．
29． Wightman R,Bates S,Amornrrattanapan P,et al.In Candida albicans,the Nim1 kinases Gin4 and Hsl1 negatively regulate pseudohypha formation and Gin4 also controls septin organization[J].2004,164(4) :581-591．
30． Cau J,Faure S,Vigneron S,et al.Regulation of Xenopus p21-activated kinase(X-PAK2) by Cdc42 and maturation-promoting factor controls Xenopus oocyte maturation[J].J Biol Chem,2000,275(4) :2367-2375
31． Thiel D,Reeder M,Pfaff A,et al.Cell cycle-regulated phosphorylation of p21-activated kinase 1[J].Curr Biol.2002,12:1227-1232．
32． Stofega MR,Sanders LC,Gardiner EM,et al.Constitutive p21-activated kinase(PAK)activation in breast cancer cells as a result of mislocalization of PAK to focal adhesions[J].Mol Biol Cell.2004,15(6) :2965-2977．
33． Joberty G,Perlungher RR,Sheffield PJ.Borg proteins control septin organization and are negatively regulated by Cdc42[J].Nat Cell Biol,2001,3(10) :861-866．
34． Drechsel DN,Hyman AA,Hall A,et al.A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos[J].Curr Biol.1997,7(1) :12-23．
35． Cau J,Faure S,Comps M,et al.A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization[J].J Cell Biol.2001,155(6) :1029-1042．
36． Souopgui J,SolterM,Pieler T.XPak3 promotes cell cycle withdrawal during primary neurogenesis in Xenopus laevis[J].Embo J.2002,21(23) :6429-6439．
37． Faure S,Cau J,de Santa Barbara P.Xenopus p21-activated kinase 5 regulates blastomeres adhesive properties during convergent extension movements[J].Dev Biol.2005,277(2) :472-492．
38． Zenke FT,King CC,Bohl BP,et al.Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity[J].J Biol Chem.1999,274(46) :32565-32573．
39． Lei M,Lu W,Meng W,et al.Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch[J].Cell.2000,102(3) :387-397．
40． Jung JH,Traugh JA.Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141[J].J Biol Chem.2005,280(48) :40025-40031．
41． Versele M,Thorner T.Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK,Cla4[J].J Cell Biol,2004,164(5) :701-715．
42． Crane RF,Rudeman JV.Using Xenopus oocyte extracts to study signal transduction[J].Methods Mol Biol.2006;322:435-443．
43． Shin EY,Shin KS,Lee CS,et al.Phosphorylation of p85 beta PIX,a Rac/Cdc42-specific guanine nucleotide exchange factor,via the Ras/ERK/PAK2 pathway is required for basic fibroblast growth factor-induced neurite outgrowth[J].J Biol Chem.2002,277(46) :44417-44430．
44． Nebreda AR,Porras A,Santos E.p21ras-induced meiotic maturation of Xenopus oocytes in the absence of protein synthesis:MPF activation is preceded by activation of MAP and S6 kinases[J].Oncogene.1993,8(2) :467-77．
45． Na J,Zernicka-Goetz M.Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function[J].Curr Biol.2006,16(12) :1249-1254．
46． Osmani N,Vitale N,Borg JP,et al.Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration[J].Curr Biol.2006,16(24) :2395-2405．
47． Darenfed H,Mandato CA.Wound-induced contractile ring:a model for cytokinesis[J].Biochem Cell Biol.2005,83(6) :711-720．
48． Zhao ZS,Manser E,Chen XQ,et al.A conserved negative regulating region in alphaPAK:inhibition of PAK kinases reveals their morphological roles downstream of Cdc42 and Rac1[J].Mol Cell Biol.1998,18(4) :2153～2163．
1． Bokoch GM.Biology of the p21-activated kinases[J].Annu Rev Biochem.2003,72:743-781
2． Jaffer ZM,Chernoff J.p21-activated kinases:three more join the Pak[J].Int J Biochem Cell Biol.2002,34(7) :713-717
3． kumar R,Vadlamudi RK.Emerging functions of p21-activated kinases in human cancer cells[J].J Cell Physiol.2002,193(2) :133-144
4． Zenke FT,King CC,Bohl BP,et al.Identification of a central phosphor-ylation site in p21-activated regulating autoinhibition and kinase activity[J].J Biol Chem.1999,274(46) :32565-32573
5． Tapon N,Hall C.Rho,Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton[J].Curr Opin Cell Biol.1997,9(1) :86-92
6． Reeder MK,Serebriiskii IG,Golemis EA,et al.Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42[J].J Biol Chem.2001,276(44) :40606-40613
7． Knaus UG,Wang Y,Reilly AM,et al.Structure requirements for PAK activation by Rac GTPase[J].J Biol Chem.1998,273:21512-21518
8． King CC,Gardiner EMM,Zenke FT,et al.P2l-activated kinase(PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1(PDK1) [J].J Biol Chem.2000,275:41201-41209
9． Tang Y,Zhou H,Chen A,et al.The Akt proto-oncogene links Ras to PAK and cell survival signals[J].J Biol Chem.2000,275(13) :9106-9109
10． Koh CG,Tan EJ,Manser E,et al.The p21-activated kinase(PAK)is regatively regulated by POPX1 and POPX2,a pair of serine/threonine phosphatase of the PP2C family[J].Curr Biol.2002,12(4) :317-321
11． Edwards DC,Sanders LC,Bokoch GM,et al.Activation of LIM-kinase by PAK1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics[J].Nat Cell Biol.1999,1(5) :253-259
12． Vadlamudi RK,Li F,Adam L,et al.Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1[J].Nat Cell Biol.2002,4:681-690
13． Li F,Adam L,Vadlamudi RK,et al.p21-activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells[J].EMBO 2002,3(8) :767-773
14． Tang Y,Chen Z,Ambrose D,et al.Kinase-defìcient PAK1 mutants inhibit Ras transformation of Rat-1 fíbroblasts[J].Mol Cell Biol.1997,17(8) :4454-4464
15． Lu W,Katz S,Gupta R,et al.Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck[J].Curr Biol.1997,7(2) :85-94
16． Tang Y,Yu J,Field J.Signals from the Ras,Rac,and Rho GTPases converge on the Pak protein kinase in Rat-1 fíbroblasts[J].Mol Cell Biol.1999,19(3) :1881-1891
17． Gallagher ED,Xu S,Moomaw C,et al.Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation[J].J Biol Chem.2002,277(48) :45785-45792
18． Mira JP,Bernard V,Groffen J,et al.Endogenous,hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway[J].Proc Natl Acad Sci USA.2000,97(1) :185-189
19． Gnesutta N,Qu J,Minden A.The serine/threonine kinase PAK4 prevents caspase activation and protects cells from apoptosis[J].J Biol Chem.2001,276(17) :14414-14419
20． Schneeberger D,Raabe T.Mbt,a Drosophila PAK protein,combines with Cdc42 to regulate photoreceptor cell morphogenesis[J].Development.2003,130(3) :427-437
21． Wu H,Zheng Y,Wang ZX.Evaluation of the catalytic mechanism of the p21-activated protein kinase PAK2[J].Biochemistry.2003,42(4) :1129-1139
22． Chan WH,Yu JS,Yang SD.PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism:evidence for the involvement of oxidative stress[J].J Cell Physiol.1999,178(3) :397-408
23． Rudel T,Bokoch GM.Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2[J].Science.1997,276(5318) :1571-1574
24． Jin S,Zhuo Y,Guo W,et al.p21-activated Kinase 1(Pak1) -dependent phosphory lation of Raf-1 regulates its mitochondrial localization,phosphorylation of BAD,and Bcl-2 association[J].J Biol Chem.2005,280(26) :24698-24705
25． Zha J,Harada H,Yang E,et al.Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L)[J].Cell.1996,87(4) :619-628
26． Chiang CW,Harris G,Ellig C,et al.Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin-3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation[J].Blood.2001,97(5) :1289-1291
27． Wolf D,Witte V,Laffert B,et al.HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals[J].Nat Med.2001,7(11) :1217-1224
28． Cotteret S,Jaffer ZM,Beeser A,et al.p21-Activated kinase 5(Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD[J].Mol Cell Biol.2003,23(16) :5526-5539
29． Lee SR,Ramos SM,Ko A,et al.AR and ER interaction with a p21-activated kinase(PAK6) [J].Mol Endocrinol.2002,16(1) :85-99
30． Yang F,Li X,Shama M,et al.Androgen receptor specifically interacts with a novel p21-activated kinase,PAK6[J].J Biol Chem.2001,276(18) :15345-15353
31． Bagheri-Yamand R,Mandal M,Jalud AH,et al.Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells[J].J Biol Chem.2001,276(31) :29403-29409
32． Mazumdan A,Kumar R.Estrogen regulation of Pakl and FKHR pathways in breast cancer cells[J].FEBS Lett.2003,535(1-3) :6-10
33． Bames CJ,Bagheri-Yamand R,Mandal M,et al.Suppression of epidermal growth factor receptor,mitogen-activated protein kinase,and Pak1 pathways and invasiveness of human cutaneous squamous cancer cells by the tyrosine kinase inhibitor ZD1839(Iressa)[J].Mol Cancer Ther.2003,2(4) :345-351
34． Kawazoe N,Watabe M,Masuda Y,et al.Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells[J].Oncogene.1999,18(15) :2413-2421
35． Stoletov KV,Ratcliffe KE,Spring SC,et al.NCK and PAK participate in the signaling pathway by which vascular endothelial growth factor stimulates the assembly of focal adhesions[J].J Biol Chem.2001,276(25) :22748-22755
2． Chu PC,Wu J,Liao XC,et al.A novel role for p21-activated protein kinase 2 in T cell activation[J].J Immunol.2004,172(12) ,7324-7334．
3． Kumar R,Vadlamudi RK.Emerging functions of p21-activated kinases in human cancer cells[J].J Cell Physiol.2002,193(2) :133-144．
4． Manser E,Leung T,Salihuddin H,et al.A Brain serine/threonine protein kinase activated by Cdc42 and Racl[J].Nature.1994,367(6458) :40-46．
5． Bagrodia S,Cerione RA.PAK to the future[J].Trends Cell Biol.1999,9(9) :350-355．
6． Kanus UG,Bokoch GM.The p21Rac/Cdc42-activated kinases(PAKs)[J].Int J Biol Cell Biol.1998,30(8) :857-862．
7． Sells MA,Chernoff J.Emerging from the Pak:the p21-activated protein kinase family[J].Trends Cell Biol.1997,193(2) :162-167．
8． Abo A,Qu J,Cammarano MS,et al.PAK4,a novel effector for Cdc42Hs,is implicated in the reorganization of the actin cytoskeleton and in the formation of fílopodia[J].EMBO J.1998,17(22) :6527-6540．
9． Yang F,Li X,Sharma M,et al.Androgen receptor specifically interacts with a novel p21-activated kinase,PAK6[J].J Biol Chem.2001,276(18) :15345-15353．
10． Lee SR,Ramos SM,Ko A,et al.AR and ER interaction with a p21-activated kinase(PAK6) [J].Mol Endocrinol.2002,16(1) :85-99．
11． Pandey A,Dan I,Kristiansen TZ,et al.Cloning and characterization of PAK5,a novel member of mammalian p21-activated kinase-Ⅱ subfamily that is predominantly expressed in brain[J].Oncogene.2002,21(24) :3939-3948．
12． Taylor SS,Knighton DR,Zheng J,et al.Structural framework for the protein kinase family[J].Annu Rev Cell Biol.1992,8:429-462．
13． Jaffer ZM,Chernoff J.p21-activated kinases:three more join the Pak[J].Int J Biochem Cell Biol.2002,34(7) :713-717
14． Weisz Hubsman M,Volinsky N,Manser E,et al.Autophosphorylation-dependent degradation of Pakl,triggered by the Rho-family GTPase,Chp[J].Biochem J.2007 Mar 13;[Epub ahead of print].
15． Chan WH,Yu JS,Yang SD.PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism:evidence for the involvement of oxidative stress[J].J Cell Physiol.1999,178(3) :397-408．
16． Vadlamudi RK.Adam L,Wang RA,et al.Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells[J].J Biol Chem.2000,275(46) :36238-36244．
17． Banerjee M,Worth D,Prowse DW,et al.Pakl phosphorylation on t212 affects microtubules in cells undergoing mitosis[J].Curr Biol.2002,12(14) :1233-1239．
18． Li F,Adam L,Vadlamudi RK,et al.p21-activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells[J].EMBO Rep.2002,3(8) :767-773．
19． Zhao ZS,Manser E,Loo TH,et al.Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly[J].Mol Cell Biol.2000,20(17) :6354-6363．
20． Balasenthil S,Sahin AA,Barnes CJ,et al.p21-activated kinase-1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells[J].J Biol Chem.2004,279(2) :1422-1428．
21． Ma C,Ha Benink,Cheng DY,et al.Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation[J].Curr Biol,2006,16(2) :214-220
22． Cvrckova F,De Virgilio C.Manser E.Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast[J].Genes dev,1995,9(15) :1817-1830．
23． Brown DD.A tribute to the Xenopus laevis oocyte and egg[J].J Biol Chem.2004,279(44) :45291-45299．
24． Leveleki L,Mahlert M,Sandrock B,et al.The PAK family kinase Cla4 is required for budding and morphogenesis in Ustilago maydis[J].Mol Microbiol.2004,54(2) :396-406．
25． Sagata N.What does Mos do in oocytes and somatic cells[J]?Bioessays.1997,19(1) :13-21．
26． Faure S,Vigneron S,Doree M,et al.A member of the Ste20/PAK family of protein kinases is involved in both arrest of Xenopus oocytes at G2/prophase of the first meiotic cell cycle and in prevention of apoptosis[J].EMBO J.1997,16(18) :5550-5561．
27． Faure S,Vigneron S,Galas S,et al.Control of G2/M transition in Xenopus by a member of the p21-activated kinase(PAK)family:a link between protein kinase A and PAK signaling pathways[J]?J Biol Chem.1999,274(6) :3573-3579．
28． Leberer E,Dignard D,Harcus D,et al.The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components[J].EMBO J.1992,11(13) :4815-4824．
29． Wightman R,Bates S,Amornrrattanapan P,et al.In Candida albicans,the Nim1 kinases Gin4 and Hsl1 negatively regulate pseudohypha formation and Gin4 also controls septin organization[J].2004,164(4) :581-591．
30． Cau J,Faure S,Vigneron S,et al.Regulation of Xenopus p21-activated kinase(X-PAK2) by Cdc42 and maturation-promoting factor controls Xenopus oocyte maturation[J].J Biol Chem,2000,275(4) :2367-2375
31． Thiel D,Reeder M,Pfaff A,et al.Cell cycle-regulated phosphorylation of p21-activated kinase 1[J].Curr Biol.2002,12:1227-1232．
32． Stofega MR,Sanders LC,Gardiner EM,et al.Constitutive p21-activated kinase(PAK)activation in breast cancer cells as a result of mislocalization of PAK to focal adhesions[J].Mol Biol Cell.2004,15(6) :2965-2977．
33． Joberty G,Perlungher RR,Sheffield PJ.Borg proteins control septin organization and are negatively regulated by Cdc42[J].Nat Cell Biol,2001,3(10) :861-866．
34． Drechsel DN,Hyman AA,Hall A,et al.A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos[J].Curr Biol.1997,7(1) :12-23．
35． Cau J,Faure S,Comps M,et al.A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization[J].J Cell Biol.2001,155(6) :1029-1042．
36． Souopgui J,SolterM,Pieler T.XPak3 promotes cell cycle withdrawal during primary neurogenesis in Xenopus laevis[J].Embo J.2002,21(23) :6429-6439．
37． Faure S,Cau J,de Santa Barbara P.Xenopus p21-activated kinase 5 regulates blastomeres adhesive properties during convergent extension movements[J].Dev Biol.2005,277(2) :472-492．
38． Zenke FT,King CC,Bohl BP,et al.Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity[J].J Biol Chem.1999,274(46) :32565-32573．
39． Lei M,Lu W,Meng W,et al.Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch[J].Cell.2000,102(3) :387-397．
40． Jung JH,Traugh JA.Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141[J].J Biol Chem.2005,280(48) :40025-40031．
41． Versele M,Thorner T.Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK,Cla4[J].J Cell Biol,2004,164(5) :701-715．
42． Crane RF,Rudeman JV.Using Xenopus oocyte extracts to study signal transduction[J].Methods Mol Biol.2006;322:435-443．
43． Shin EY,Shin KS,Lee CS,et al.Phosphorylation of p85 beta PIX,a Rac/Cdc42-specific guanine nucleotide exchange factor,via the Ras/ERK/PAK2 pathway is required for basic fibroblast growth factor-induced neurite outgrowth[J].J Biol Chem.2002,277(46) :44417-44430．
44． Nebreda AR,Porras A,Santos E.p21ras-induced meiotic maturation of Xenopus oocytes in the absence of protein synthesis:MPF activation is preceded by activation of MAP and S6 kinases[J].Oncogene.1993,8(2) :467-77．
45． Na J,Zernicka-Goetz M.Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function[J].Curr Biol.2006,16(12) :1249-1254．
46． Osmani N,Vitale N,Borg JP,et al.Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration[J].Curr Biol.2006,16(24) :2395-2405．
47． Darenfed H,Mandato CA.Wound-induced contractile ring:a model for cytokinesis[J].Biochem Cell Biol.2005,83(6) :711-720．
48． Zhao ZS,Manser E,Chen XQ,et al.A conserved negative regulating region in alphaPAK:inhibition of PAK kinases reveals their morphological roles downstream of Cdc42 and Rac1[J].Mol Cell Biol.1998,18(4) :2153～2163．
1． Bokoch GM.Biology of the p21-activated kinases[J].Annu Rev Biochem.2003,72:743-781
2． Jaffer ZM,Chernoff J.p21-activated kinases:three more join the Pak[J].Int J Biochem Cell Biol.2002,34(7) :713-717
3． kumar R,Vadlamudi RK.Emerging functions of p21-activated kinases in human cancer cells[J].J Cell Physiol.2002,193(2) :133-144
4． Zenke FT,King CC,Bohl BP,et al.Identification of a central phosphor-ylation site in p21-activated regulating autoinhibition and kinase activity[J].J Biol Chem.1999,274(46) :32565-32573
5． Tapon N,Hall C.Rho,Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton[J].Curr Opin Cell Biol.1997,9(1) :86-92
6． Reeder MK,Serebriiskii IG,Golemis EA,et al.Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42[J].J Biol Chem.2001,276(44) :40606-40613
7． Knaus UG,Wang Y,Reilly AM,et al.Structure requirements for PAK activation by Rac GTPase[J].J Biol Chem.1998,273:21512-21518
8． King CC,Gardiner EMM,Zenke FT,et al.P2l-activated kinase(PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1(PDK1) [J].J Biol Chem.2000,275:41201-41209
9． Tang Y,Zhou H,Chen A,et al.The Akt proto-oncogene links Ras to PAK and cell survival signals[J].J Biol Chem.2000,275(13) :9106-9109
10． Koh CG,Tan EJ,Manser E,et al.The p21-activated kinase(PAK)is regatively regulated by POPX1 and POPX2,a pair of serine/threonine phosphatase of the PP2C family[J].Curr Biol.2002,12(4) :317-321
11． Edwards DC,Sanders LC,Bokoch GM,et al.Activation of LIM-kinase by PAK1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics[J].Nat Cell Biol.1999,1(5) :253-259
12． Vadlamudi RK,Li F,Adam L,et al.Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1[J].Nat Cell Biol.2002,4:681-690
13． Li F,Adam L,Vadlamudi RK,et al.p21-activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells[J].EMBO 2002,3(8) :767-773
14． Tang Y,Chen Z,Ambrose D,et al.Kinase-defìcient PAK1 mutants inhibit Ras transformation of Rat-1 fíbroblasts[J].Mol Cell Biol.1997,17(8) :4454-4464
15． Lu W,Katz S,Gupta R,et al.Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck[J].Curr Biol.1997,7(2) :85-94
16． Tang Y,Yu J,Field J.Signals from the Ras,Rac,and Rho GTPases converge on the Pak protein kinase in Rat-1 fíbroblasts[J].Mol Cell Biol.1999,19(3) :1881-1891
17． Gallagher ED,Xu S,Moomaw C,et al.Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation[J].J Biol Chem.2002,277(48) :45785-45792
18． Mira JP,Bernard V,Groffen J,et al.Endogenous,hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway[J].Proc Natl Acad Sci USA.2000,97(1) :185-189
19． Gnesutta N,Qu J,Minden A.The serine/threonine kinase PAK4 prevents caspase activation and protects cells from apoptosis[J].J Biol Chem.2001,276(17) :14414-14419
20． Schneeberger D,Raabe T.Mbt,a Drosophila PAK protein,combines with Cdc42 to regulate photoreceptor cell morphogenesis[J].Development.2003,130(3) :427-437
21． Wu H,Zheng Y,Wang ZX.Evaluation of the catalytic mechanism of the p21-activated protein kinase PAK2[J].Biochemistry.2003,42(4) :1129-1139
22． Chan WH,Yu JS,Yang SD.PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism:evidence for the involvement of oxidative stress[J].J Cell Physiol.1999,178(3) :397-408
23． Rudel T,Bokoch GM.Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2[J].Science.1997,276(5318) :1571-1574
24． Jin S,Zhuo Y,Guo W,et al.p21-activated Kinase 1(Pak1) -dependent phosphory lation of Raf-1 regulates its mitochondrial localization,phosphorylation of BAD,and Bcl-2 association[J].J Biol Chem.2005,280(26) :24698-24705
25． Zha J,Harada H,Yang E,et al.Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L)[J].Cell.1996,87(4) :619-628
26． Chiang CW,Harris G,Ellig C,et al.Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin-3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation[J].Blood.2001,97(5) :1289-1291
27． Wolf D,Witte V,Laffert B,et al.HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals[J].Nat Med.2001,7(11) :1217-1224
28． Cotteret S,Jaffer ZM,Beeser A,et al.p21-Activated kinase 5(Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD[J].Mol Cell Biol.2003,23(16) :5526-5539
29． Lee SR,Ramos SM,Ko A,et al.AR and ER interaction with a p21-activated kinase(PAK6) [J].Mol Endocrinol.2002,16(1) :85-99
30． Yang F,Li X,Shama M,et al.Androgen receptor specifically interacts with a novel p21-activated kinase,PAK6[J].J Biol Chem.2001,276(18) :15345-15353
31． Bagheri-Yamand R,Mandal M,Jalud AH,et al.Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells[J].J Biol Chem.2001,276(31) :29403-29409
32． Mazumdan A,Kumar R.Estrogen regulation of Pakl and FKHR pathways in breast cancer cells[J].FEBS Lett.2003,535(1-3) :6-10
33． Bames CJ,Bagheri-Yamand R,Mandal M,et al.Suppression of epidermal growth factor receptor,mitogen-activated protein kinase,and Pak1 pathways and invasiveness of human cutaneous squamous cancer cells by the tyrosine kinase inhibitor ZD1839(Iressa)[J].Mol Cancer Ther.2003,2(4) :345-351
34． Kawazoe N,Watabe M,Masuda Y,et al.Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells[J].Oncogene.1999,18(15) :2413-2421
35． Stoletov KV,Ratcliffe KE,Spring SC,et al.NCK and PAK participate in the signaling pathway by which vascular endothelial growth factor stimulates the assembly of focal adhesions[J].J Biol Chem.2001,276(25) :22748-22755