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Vol. 16, Issue 9, 4316-4328, September 2005
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* Department of Cell and Developmental Biology, College of Medicine, State University of New York Upstate Medical University, Syracuse, NY 13210;
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
Submitted February 16, 2005;
Revised June 7, 2005;
Accepted June 29, 2005
Monitoring Editor: Clare Waterman-Storer
The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility.
Abbreviations used: FAK, focal adhesion kinase; GAP, GTPase-activating protein; GFP, green fluorescent protein; GIT, G protein-coupled receptor kinase-interacting protein; GST, glutathione S-transferase; MEF, mouse embryo fibroblast; PAK, p21-activated kinase; PBS, paxillin binding subdomain; PKL, paxillin kinase linker; SHD, SpaII homology domain; SYF, Src/Yes/Fyn; WT, wild-type.
Address correspondence to: Christopher E. Turner (turnerce{at}upstate.edu).
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