Bellis S L, Perrotta J A, Curtis M S, Turner C E
Department of Anatomy and Cell Biology, State University of New York Health Science Center, 750 E. Adams Street, Syracuse, NY 13210, USA.
Biochem J. 1997 Jul 15;325 ( Pt 2)(Pt 2):375-81. doi: 10.1042/bj3250375.
Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation.
粘着斑激酶（FAK）介导的桩蛋白酪氨酸磷酸化被认为是一种与细胞粘附和细胞骨架重组相关的信号转导机制。桩蛋白丝氨酸磷酸化在这些事件中的潜在作用尚未得到充分表征。在本研究中，我们检测了桩蛋白在体外和体内的磷酸化情况。通过在体外沉淀激酶试验中使用谷胱甘肽S-转移酶-桩蛋白融合蛋白，我们观察到一个跨越桩蛋白氨基酸残基54 - 313且包含FAK结合位点的融合蛋白，从平滑肌裂解物中沉淀出大量丝氨酸激酶活性以及FAK活性。这些激酶共同使桩蛋白的酪氨酸残基118（该位点先前已被确定为FAK磷酸化的靶点）以及丝氨酸残基188和/或190发生磷酸化。丝氨酸激酶的结合位点（其身份目前未知）进一步定位到桩蛋白的残基168 - 191。为了评估体外磷酸化的这些位点的生理相关性，我们对在纤连蛋白上接种成纤维细胞刺激下体内桩蛋白的磷酸化情况进行了表征。正如预期的那样，将细胞接种在纤连蛋白上可增强桩蛋白的酪氨酸磷酸化。然而，桩蛋白96%的磷酸化发生在丝氨酸残基上。通过二维磷酸肽分析比较表明，体外试验中检测到的酪氨酸和丝氨酸磷酸化的主要位点与体内因将细胞接种在纤连蛋白上而磷酸化的桩蛋白衍生的磷酸肽共同迁移。这些发现支持酪氨酸激酶和丝氨酸激酶在将整合素激活与桩蛋白磷酸化联系起来的信号转导途径中均发挥作用。
