Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness.
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.
Brown
Michael C
MC
Department of Cell and Developmental Biology, College of Medicine, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
Cary
Leslie A
LA
Jamieson
Jennifer S
JS
Cooper
Jonathan A
JA
Turner
Christopher E
CE
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000502:physiology*; D022001:Focal Adhesions / Q000201:enzymology* / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D015513:Oncogene Proteins / Q000378:metabolism; D051419:Paxillin / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; C120767:Git2 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases