Cdk11 Family Kinase Phosphorylation Of Pak1 Regulation And Function Of The Cell Cycle

The classic CDK(cyclin dependent kinase) family kinases take part in the regulation of of cell cycle.CDK11 family belongs to p34cdc2 superfamily and has some specificic functions in cells compared with classic CDKs.There have three yet indentified CDK11 members:CDK11p110, CDK11p58 and CDK11p46.CDK11p110 is ubquitiously expressed in various tissues and cell lines examined whose function has been linked with transcription regulation and pre-mRNA splicing.During apoptosis, caspase cleavage of the CDK11p110 isoforms produces the CDK11p46 catalytic domain isoform of CDK11,which lacks the first 52 amino acids found in CDK11p58 isoform.CDK11p46 is considered to participate in apoptotic signal transduction.In our lab's early report,CDK11p46 can be induced in NIH3T3 cells during the process of anoikis.What's more, CDK11p46 can bind with PAK1(p21 activated kinase 1) and repress the kinase activity of PAK1 in a kinase dependent manner.Further analysis showed that CDK11p46 repression of PAK1 downregulates the phosphorylation level of BAD at 2 sites,Ser112 and Ser116,which makes the translocation of BAD from cytoplasm to mitochondrial,subsequently followed by cytochorome c realease and cell death.Kinase dead form of CDK11p46 is unable to cause apoptosis.The first identified CDK11 isoform is CDK11p58.The cDNA was isolated from an expression library screen using a galactosyltransferase polyclonal antiserum.CDK11p58 could bind withβ-1,4 galactosyltransferase 1 and stimulates its activity.In cells CDK11p58 is specifically expressed in G2/M phase.Overexpresison of CDK11p58 isoform could delay the growth kinetic of the cells,cause the abnormality in cell cycle,mitosis and finally the G2/M arrest and then apoptosis.Mouse knockout models of cdc2L gene(encoding CDK11 proteins) exhibited early embryonic lethality at the blastocyst stage of embryonic development,due in part,to cellular proliferative failure.CDK11p58 also seems to be essential for the maintenance of sister chromatid cohesion,as well as the centrosome maturation and biopolar spindle formation.Althrough CDK11p58 seems to play important roles in mitotic events,little is known about the exact underlying molecular mechanisms.P21 activated kinase 1(PAK1) is a PAK/STE family Ser/Thr protein kinase. Upon binding to the activated form(GTP binding) of Rho GTPase family members Rac or cdc42 with its amino-terminal PBD domain,PAK1 undergoes a conformational change,which leads to autophosphorylation at several sites,including Thr423,and activation of kinase activity.PAK1 is reported to participate in a wide range of signal transducing pathways and physiological processes including cell survival,apoptosis, proliferation,cytoskeleton dynamics,cell cycle control, transcriptional regulation and tumorgenesis.PAK1 is phosphorylated on T212 by cdc2 in cells undergoing mitosis.Moreover,activated PAK1 can accumulate at chromosomes at prophase and move to contract ring during cytokinesis.Further analysis made the finding that PAK1 can phosphorylate histone H3 in breast cancer cells during mitosis,thus it is conceivable that PAK1 may play a role in mitotic events.But the link between PAK1 and CDK11 family proteins has not been reported.Here we identified p21 activated kinase 1(PAK1) as a new CDK11p58 substrate and we mapped a new phosphorylation site of Ser174 on PAK1.By mutagenesis we created PAK1174A and PAK1174E,which mimic the dephosphorylated and phosphorylated form of PAK1,respectively.And we found PAK1174E localized the same positions like wild type PAK1 as previously reported,while PAK1174A exhibited an impaired localization pattern.Further analysis showed PAK1174E could be recruited to myosin V motor complex through binding to DLC2.PAK1174E could accelerate the mitosis progression in a nocodazole blocked cell model,while PAK1174A exhibited an opposite role.Our results indicated CDK11p58 phosphorylation created a new docking site and caused the translocation of PAK1,thus facilitating its regulation of mitosis progression.In order to further analysis the link between CDK11 family isoforms and PAK1,large amunts of proteins with physiological activity and modification is required.We used baculovirus expression system to express recombinant CDK11p46 and PAK1 in forms of His-tagged fusion proteins.First we cloned CDK11p46 and PAK1 cDNA into linarized AcNPV DNA, generating recombinant cyclic AcNPV DNA.Then we got P1 recombinant virus by transfecting sf9 cells with cyclic AcNPV DNA.After futher infection and amplification high titer P3 virus was obtained.By western blot analysis of infected sf9 cells,both CDK11p46 and PAK1 recombinant proteins'expression in sf9 cells was confirmed.We further the optimized the expression condition of the two proteins by infecting sf9 cells with different amount of virus at different time points.Finally we purified recombinant His-CDK11p46 proteins by Ni-NTA sepharose.Our research helps to further investigate the functional links between CDK11 proteins and PAK1.