【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.更多还原

【Abstract】 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 CRKⅠ,CRKⅡ and CRKL,were cloned and found to have strong interaction with PTPN4 respectively.We also verified that the interaction is rely on the N-terminal 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-localization assays.Phosphotyrosine antibody was used to prove the CRKⅠ dephosphorylation when co-transfected with PTPN4 into 293T cell line,which provide an important evidence that CRKⅠ is a physiological target of this phosphatase in cells.PTPN4 can mediate the function of CRKⅠ by dephosphorylating the protein tyrosine kinase.This result provided a clue to explore the molecular mechanism of PTPN4 and CRKⅠ.更多还原