Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

Abstract

Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to plateletderived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation. In fibroblasts, expression of a PKL mutant lacking the principal tyrosine phosphorylation sites resulted in loss of wound-induced cell polarization as well as directional migration. PKL phosphorylation was necessary for PDGF-stimulated PKL binding to the focal adhesion protein paxillin and expression of paxillin or PKL mutants defective in their respective binding motifs recapitulated the polarization defects. RNA interference or expression of phosphorylation mutants of PKL resulted in disregulation of PDGF-stimulated Rac1 and PAK activities, reduction of Cdc42 and Erk signaling, as well as mislocalization of ␤PIX. Together these studies position PKL as an integral component of growth factor and cell adhesion cross-talk signaling, controlling the development of front-rear cell polarity and directional cell migration. INTRODUCTIONDirected cell migration requires front-rear cell polarization and plays a fundamental role during development, the innate immune response and wound repair as well as in tumor cell metastasis (Lauffenburger and Horwitz, 1996;Ridley et al., 2003). Chemoattractant (e.g., growth factor) gradients in combination with extracellular matrix (ECM) cues provide the external stimuli necessary to establish and maintain the asymmetric shape of polarized cells, through the regulation of integrin-based cell adhesion and reorganization of the actin and microtubule cytoskeleton (Etienne-Manneville and Hall, 2002, 2008;Ridley et al., 2003). For example, coordinated signaling between PDGF receptors and integrins in fibroblasts and vascular cells facilitates directional cell migration during organogenesis, blood vessel patterning, and wound healing (Heldin and Westermark, 1999;Duchek et al., 2001;Nagel et al., 2004;Wood et al., 2006).The nonreceptor tyrosine kinase FAK, in combination with the Src family kinases (SFKs), has emerged as a central effector, mediating cross-talk between activated growth factor receptor tyrosine kinases such as PDGF and engaged integrins Juliano, 2002). In turn, FAK/Src regulates focal adhesion turnover and cytoskeletal remodeling (Webb et al., 2004;Zaidel-Bar et al., 2007), through tyrosine phosphorylation of various key focal adhesion adapter proteins such as paxillin and p130Cas (Thomas and Brugge, 1997;Schlaepfer et al., 1999;Brown and Turner, 2004), as well as critical regulators of Rho GTPase signaling including the guanine nucleoti...

Paxillin functions as an oncogene in human gliomas by promoting cell migration and invasion

“…Cell adhesion to neighboring cells, the surrounding ECM or stroma has been indicated to be crucial during the progression via regulating tumor cell proliferation and survival as well as enabling tumor cell dissemination and metastasis 18,19. Growing evidence show that the paxillin family of intracellular scaffold proteins may play roles in regulating the cellular migration machinery by coordinating multiple signals from growth factors, integrins, and cell surface receptors 20,21.…”

The cell adhesion-associated protein Git2 regulates morphogenetic movements during zebrafish embryonic development

“…ArfGAP GIT proteins have been implicated in the regulation of cell adhesion and motility in mammalian cell culture systems and knockout mice (Frank, et al, 2006; Mazaki et al, 2006; Pang et al, 2009, Yu et al, 2009). To examine the role of GIT in modulating dynamic cell behaviors in vivo , we chose to use the transparent zebrafish embryo as a model, which is ideal for live imaging studies.…”

“…Additionally, growth factors such as PDGF (Montero et al, 2003) and S1P (Kai et al, 2008) are deposited within the ECM and provide guidance cues to direct the migration of germ layer progenitor cells (Keller, 2005). Observations from cell culture studies indicate that GIT2 is also downstream of growth factor receptor activation (Brown et al, 2005; Yin et al, 2005; Yu et al, 2009) and thus, in vivo it is well positioned to mediate cell-matrix adhesion and growth factor cross-talk and thereby facilitate directional cell migration. Indeed, gene deletion of GIT in other species such as mice, worms and flies also leads to defective cell migration and guidance (Mazaki et al, 2006; Lucanic and Cheng, 2008; Bahri et al, 2009; Pang et al, 2009).…”

“…GIT proteins are substrates for non receptor tyrosine kinases FAK and Src. GIT2 tyrosine phosphorylation is necessary for directing cell migration upon PDGF growth factor signaling in fibroblasts (Brown et al, 2005, Yu et al, 2009), while GIT1 is similarly required for EGF-dependent vascular smooth muscle cell migration (Yin et al, 2005). Gene ablation in mice suggests differential physiological roles for GIT1 and GIT2, with GIT1 playing a critical role in lung and vasculature development (Pang et al, 2009) and GIT2 being important for neutrophil chemotaxis in association with the immune response (Mazaki et al, 2006).…”

The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation

“…However, most studies to date have linked Cat to changes in cell morphology and migration (10,(17)(18)(19)(20)(21)(22)(23). The involvement of Cat in cell migration has stemmed largely from findings demonstrating that its overexpression in fibroblasts leads to the loss of focal complexes (18).…”