Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness

Mol Biol Cell. 2005 Sep;16(9):4316-28. doi: 10.1091/mbc.e05-02-0131. Epub 2005 Jul 6.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Cell Cycle Proteins / metabolism*
  • Cell Line
  • Cell Movement / physiology*
  • Focal Adhesion Protein-Tyrosine Kinases / physiology*
  • Focal Adhesions / enzymology*
  • Focal Adhesions / metabolism
  • GTPase-Activating Proteins / metabolism*
  • Genes, Reporter
  • Intercellular Signaling Peptides and Proteins
  • Mice
  • Oncogene Proteins / metabolism
  • Paxillin / metabolism
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship
  • Tyrosine / metabolism
  • src-Family Kinases / genetics
  • src-Family Kinases / physiology*

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • GTPase-Activating Proteins
  • Git2 protein, mouse
  • Intercellular Signaling Peptides and Proteins
  • Nck protein
  • Oncogene Proteins
  • Paxillin
  • Phosphoproteins
  • Pxn protein, mouse
  • Recombinant Fusion Proteins
  • Tyrosine
  • Focal Adhesion Protein-Tyrosine Kinases
  • src-Family Kinases