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

乳腺癌组织中表达的CDK变异体的分子克隆和功能分析

基本信息

批准号：
09671263
负责人：
FUKUDA Mamoru
金额：
$ 1.79万
依托单位：
St.Marianna University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671263/
关键词：
breast cancer cell cycle CDC2ΔT CDK2ΔT variant T-loop CDK2ΔT cdc2 cdk2 p21 Δcdc2 Δcdk2 RT-PCR

项目摘要

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.

我们从乳腺癌组织中克隆了一个T环缺失突变体CDC 2（CDC 2 ΔT：在本基金申请表中简称为Δ CDC 2），并对其功能进行了研究，同时探讨了其他CDK家族中存在类似突变体的可能性。CDC 2 ΔT-HA与cyclin B1和/或21 CDK抑制剂在Hela细胞中瞬时共表达。用免疫沉淀法（IP）和免疫蛋白质印迹法（IP-western）检测蛋白质间的相互作用，用组织蛋白激酶（Histo-H1 kinase）法检测免疫复合物的激酶活性。虽然CDC 2 ΔT具有保守的细胞周期蛋白结合位点和ATP结合位点，但它不能与细胞周期蛋白B1或p21复合，并且缺乏希斯顿H1激酶活性。结果表明，T环不仅在保持游离CDK处于失活状态中起关键作用，而且通过促进细胞周期蛋白结合而促进CDK活化。通过进一步研究CDK家族中的其他变体，我们克隆了CDK 2变体，其T环C端部分缺少34个氨基酸。(CDK2ΔT，#AB012305）这些变异在细胞周期或致癌作用中的生物学意义目前尚不清楚。进一步研究CDC 2和CDK 2各结构域的详细功能可能有助于阐明这些变异体的作用。