CRK是PTPN4新的相互作用蛋白

Identification of a Novel Interactional Protein of PTPN4: CRK

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 .