CRK是PTPN4新的相互作用蛋白  

Identification of a Novel Interactional Protein of PTPN4: CRK

作　　者：周娟[1] 万兵兵[1] 单敬轩[1] 吴冬华[1] 施慧莉[1] 霍克克[1] 

机构地区：[1]复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室,上海200433

出　　处：《复旦学报（自然科学版）》2008年第5期576-584,F0002,共10页Journal of Fudan University：Natural Science

基　　金：国家高技术研究发展计划"八六三"资助项目(2006AA02A310);上海市科技创新团队计划资助项目(03DZ14024)

摘　　要：以PTPN4为“诱饵”，利用酵母双杂交系统筛选人肝cDNA文库，获得了一个与PTPN4相互作用的蛋白：CRKⅠ．从cDNA文库中通过PCR得到CRK家族的3个成员的基因，把这3个基因与PTPN4共转酵母，发现CRKⅠ、CRKⅡ和CRKL均能与PTPN4发生很强的相互作用．通过截断体证实它们之间的相互作用是通过CRKs的SH3结构域与PTPN4的462-468位的脯氨酸富集区介导的．免疫共沉淀以及免疫荧光共定位实验进一步证实了CRKI与PTPN4的相互作用．通过酪氨酸磷酸化特异性抗体检测发现PTPN4可以明显降低CRKI的磷酸化水平．By using PTPN4 as a ＂bait＂ to screen a Gal4 BD fused human liver cDNA library, CRK was identified to be able to interact with PTPN4. Three members of CRK family, inculding CRK I , CRK II and CRKL, were cloned and found to have strong interaction with PTPN4 respectively. We also verified that the interaction is rely on the N-teminal SH3 domain of CRK and the proline-rich domain between amino acids 462 and 468 in PTPN4. The interactions were further confirmed by co-immunoprecipitation and co-lcrMization assays. Phosphotyrosine antibody was used to prove the CRK I dephosphorylation when co-transfected with PTPN4 into 293T cell line, which provide an important evidence that CRK I is a physiological target of this phosphatase in cells, PTPN4 can mediate the function of CRK I by dephosphorylating the protein tyrmine kinase. This result provided a clue to explore the molecular mechanism of PTPN4 and CRK I .

关 键 词：PTPN4 CRK Ⅰ CRKⅡ CRKL 酵母双杂交 免疫荧光定位 酪氨酸磷酸化 

分 类 号：Q71[生物学—分子生物学]