Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin

被引:102
作者
Bellis, SL [1 ]
Perrotta, JA [1 ]
Curtis, MS [1 ]
Turner, CE [1 ]
机构
[1] SUNY HLTH SCI CTR, DEPT ANAT & CELL BIOL, SYRACUSE, NY 13210 USA
关键词
D O I
10.1042/bj3250375
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 ii? 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 comigrate 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.
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页码:375 / 381
页数:7
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