ABSTRACT
The tyrosine phosphatase PTP-PEST has been implicated in the regulation of cell spreading and migration through dephosphorylation of focal adhesion proteins and inhibition of Rac GTPase activity. The focal adhesion adaptor protein paxillin is also necessary for normal cell migration and binds directly to PTP-PEST. In this study, we have utilized PTP-PEST(-/-) and paxillin(-/-) fibroblasts to demonstrate that paxillin is essential for PTP-PEST inhibition of cell spreading and membrane protrusion as well as inhibition of adhesion-induced Rac activation. Furthermore, we show that paxillin-binding is necessary for PTP-PEST stimulation of cell migration. Mutation analysis indicates that PTP-PEST function involves binding to the paxillin C-terminal LIM domains, and signaling through the tyrosine 31 and 118 phosphorylation sites, as well as the LD4 motif of the paxillin N-terminus. Using 'substrate trapping' approaches and immunoprecipitation, we show that the ARF GAP paxillin kinase linker PKL/GIT2, a paxillin LD4 binding partner, is a substrate for PTP-PEST. Additionally, the PKL-paxillin interaction was necessary for PTP-PEST inhibition of cell spreading. These data provide mechanistic insight into how the paxillin-PTP-PEST interaction contributes to integrin signaling events associated with the spatiotemporal regulation of key modulators of the cytoskeleton and cell motility machinery.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Movement/physiology , Cytoskeleton/metabolism , Fibroblasts/metabolism , Phosphoproteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Signal Transduction/physiology , Amino Acid Motifs/genetics , Animals , Cell Cycle Proteins/genetics , Cells, Cultured , Cytoskeleton/genetics , Fibroblasts/cytology , GTPase-Activating Proteins , Integrins/genetics , Integrins/metabolism , Intercellular Signaling Peptides and Proteins , Mice , Paxillin/genetics , Paxillin/metabolism , Phosphoproteins/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 12 , Protein Tyrosine Phosphatases/deficiencyABSTRACT
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.
