P21-activated Kinase 2 Involved In Cytokinesis Independent Of Cdc42 During Xenopus Oocyte Maturation

INTRODUCTIONPolar 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.MATERIALS1,PAK2 ,Cdc42 plasmids and reagents for molecular biology2,Western blot reagents3,Related fluorescently-labeled biology reagents.METHODS1,Animal and oocyte manipulationFrogs 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-Ca²⁺) 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 SynthesisMessenger 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 microscopeObserve 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 microscope15nL 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.RESULTS1,PAK2 phosphorylation assay during Xenopus oocyte developmentWe 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 microscopyNormal 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 cytokinesisAlexa-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 microscopyTo 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.showed5,Statistic analysisSPSS 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.CONCLUSIONS1,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 formation4,PAK2 involved in cytokinesis independent of Cdc42 during Xenopus oocytematuration.