Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

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. 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...

Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

“…The recruitment of GIT proteins to adhesion sites relies on their interaction with paxillin (18,25). In nonpolarized cells, GIT2 binds to paxillin upon phosphorylation by FAK and/or Src (26). Thus, it is more than likely that the proper localization of GIT2 to sealing zones of polarized osteoclasts is determined by Src-dependent phosphorylation.…”

Re-expression of the tumor suppressor NF2/merlin inhibits invasiveness in mesothelioma cells and negatively regulates FAK

“…Our findings indicate that merlin overexpression leads to disruption of FAK binding to its interacting partners Src and p85. The inhibitory effect of merlin on FAK activity could be either direct, as merlin has been previously shown to form a complex with FAK and NHERF (James et al, 2004), or indirect through inhibition of Rac/PAK signaling, which is known to cross-talk with FAK signaling (Hsia et al, 2003;Brown et al, 2005;Mitra et al, 2005). Indeed, we and others have previously linked merlin to Rac/PAK signaling, as PAK was found to be able to phosphorylate and inactivate merlin (Kissil et al, 2002;Xiao et al, 2002) and unphosphorylated merlin has been shown to inhibit PAK activity (Kissil et al, 2003;Hirokawa et al, 2004;Xiao et al, 2005) and Rac translocation to matrix adhesions (Okada et al, 2005).…”

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…Localization to focal adhesions is largely mediated by interaction of the C-terminal domain of GIT with paxillin [63][64][65]. Both GIT1 and GIT2 also bind constitutively to the Rac and Cdc42 GEF PIX (Pak-interacting exchange factor), and PIX in turn binds the serine-threonine kinase PAK (p21-activated kinase).…”