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. INTRODUCTIONCell attachment, spreading, and motility are complex processes requiring the integration of diverse signaling networks and structural assemblies (Jockusch et al., 1995;Sastry and Burridge, 2000;Juliano, 2002). One of the earliest steps in transducing extracellular cues through integrins to the cytoskeleton is the activation of the tyrosine kinases Src and FAK (Schwartz et al., 1995;Giancotti and Tarone, 2003). FAK is an integrin-binding nonreceptor tyrosine kinase that upon integrin ligation is activated to autophosphorylate Y397 and bind to the Src SH2 domain (Schaller et al., 1994;Calalb et al., 1995). Src then phosphorylates FAK on multiple residues that increase FAK kinase activity and several downstream binding partners for Src/FAK are targeted for phosphorylation, including the focal adhesion proteins p130Cas and paxillin (reviewed in Brown and Turner, 2004;Playford and Schaller, 2004;Mitra et al., 2005). Phosphorylated paxillin binding to the SH2/SH3 adaptor protein Crk is implicated in Rac activation and stimulation of cell motility (Petit et al., 2000;Lamorte et al., 2003;Valles et al., 2004), whereas binding of paxillin to the p120RasGAP SH2 domain may displace and allow for the activation of p190RhoGAP and subsequent decrease in RhoA activity (Iwasaki et al., 2002).The Cdc42, Rac1, and RhoA members of the Ras superfamily of p21 GTPases are central intermediaries in coordinating the defined temporal-spatial progression of signals emanating from initial integrin ligation, through acquisition of a polarized state, to initiation and maintenance of directed ce...

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.