Molecular Cloning and Functional Analysis of CDK Variant Expressed in Breast Cancer Tissues

We have cloned a T-loop deletion variant CDC2(CDC2ΔT：described asΔCDC2in the application form for this grant)from breast cancer tissues，and investigated its function and the possibility of similar variant in other CDK family。CDC2ΔT-HA was transiently co-expressed in Hela cells with cyclin B1 and/or21CDK inhibitor。The protein-protein interaction was studied by Ii D135 IeD1S-immunoprecipitation(IP)or IP-western，and kianse activity of the immunocomplex was studied by Histo-H1 kinase assay.Although CDC2ΔT possesses the conserved cyclin binding site and the ATP binding site，it is unable to complex with either Cyclin B1nor p21 and lacks histon H1 kinase activity。The results indicate that the T-loop not only play a key role in keeping a free CDK in its inactive state but also in facilitating CDK activation by promoting cyclin binding.Through additional study to investigate other variant in CDK family，we cloned a CDK2variant，which lacks34amino acids in the C-terminal portion of its T-loop。(CDK2ΔT，#AB012305)The biological significance of these variants in cell cycle or carcinogenesis is unclear at present。Further investigation of detail function of each domain of CDC2and CDK2may elacidate the role of these variants。

We have cloned a T-loop deletion variant CDC2(CDC2ΔT : described as ΔCDC2 in the application form for this grant) from breast cancer tissues, and investigated its function and the possibility of similar variant in other CDK family. CDC2ΔT-HA was transiently co-expressed in Hela cells with cyclin B1 and/or 21 CDK inhibitor. The protein-protein interaction was studied by ィイD135ィエD1S-immunoprecipitation(IP) or IP-western, and kianse activity of the immunocomplex was studied by Histo-H1 kinase assay. Although CDC2ΔT possesses the conserved cyclin binding site and the ATP binding site, it is unable to complex with either Cyclin B1 nor p21 and lacks histon H1 kinase activity. The results indicate that the T-loop not only play a key role in keeping a free CDK in its inactive state but also in facilitating CDK activation by promoting cyclin binding. Through additional study to investigate other variant in CDK family, we cloned a CDK2 variant, which lacks 34 amino acids in the C-terminal portion of its T-loop. (CDK2ΔT, #AB012305) The biological significance of these variants in cell cycle or carcinogenesis is unclear at present. Further investigation of detail function of each domain of CDC2 and CDK2 may elacidate the role of these variants.