Bellis S L, Miller J T, Turner C E
Department of Anatomy and Cell Biology, State University of New York Health Science Center, Syracuse 13210, USA.
J Biol Chem. 1995 Jul 21;270(29):17437-41. doi: 10.1074/jbc.270.29.17437.
The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion.
粘着斑蛋白桩蛋白（paxillin）和酪氨酸激酶粘着斑激酶（FAK）在包括整合素介导的细胞粘附在内的多种刺激下会伴随酪氨酸磷酸化，这表明桩蛋白磷酸化与FAK活性密切相关。在本研究中，我们在桩蛋白中鉴定出一个特定的酪氨酸残基，即酪氨酸118（Tyr-118），它是体外FAK磷酸化的主要位点。通过免疫沉淀FAK并进行体外激酶测定来确定该位点为FAK磷酸化的靶点，使用桩蛋白序列中含有截短的谷胱甘肽S-转移酶（GST）-桩蛋白融合蛋白或酪氨酸残基被苯丙氨酸取代的融合蛋白作为底物。在Tyr-118处含有苯丙氨酸取代（Y118F）的GST-桩蛋白不会被FAK免疫沉淀物磷酸化；然而，该突变体与野生型融合蛋白一样能很好地结合FAK。作为评估Tyr-118处桩蛋白磷酸化功能的第一步，将Y118F桩蛋白cDNA构建体瞬时转染到NIH 3T3细胞中。与野生型桩蛋白类似，突变的桩蛋白定位于粘着斑，这表明Tyr-118处桩蛋白的磷酸化对于桩蛋白募集到细胞粘附位点并非必需。
