SERINE AND THREONINE PHOSPHORYLATION OF THE PAXILLIN LIM DOMAINS REGULATES PAXILLIN FOCAL ADHESION LOCALIZATION AND CELL-ADHESION TO FIBRONECTIN

We have previously shown that the LIM domains of paxillin operate as t he focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to s erve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulat ed after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localiza tion of paxillin after adhesion to fibronectin was investigated. An av ian paxillin-CHO.K1 model system was used to explore the role of paxil lin phosphorylation in paxillin localization to FAs. We found that mut ations of paxillin that mimicked LIM domain phosphorylation accelerate d fibronectin-induced localization of paxillin to focal contacts. Furt her, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation signi ficantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was spe cific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in re sponse to fibronectin adhesion had no effect on the rate of FA localiz ation or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Add itionally, these results provide the first evidence that paxillin cont ributes to ''inside-out'' integrin-mediated signal transduction.