Phosphorylation of Tyrosine Residues 31 and 118 on Paxillin Regulates Cell Migration through an Association with Crk in Nbt-II Cells

Abstract

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin–Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin–Crk complex in the collagen-induced cell motility.

Involvement of focal adhesion kinase in cellular invasion of head and neck squamous cell carcinomas via regulation of MMP-2 expression

“…The initial assembly of integrin-associated focal adhesions requires FAK autophosphorylation on Tyr 397 and complex formation with Src. The FAK-Src signalling complex functions to recruit and phosphorylate various FAK-associated proteins including paxillin (Bellis et al, 1995;Schaller et al, 1995) which participates in the coordination of integrin-mediated cell motility (Klemke et al, 1998;Petit et al, 2000). We show here that FRNK and siRNA against FAK inhibited Tyr 397 phosphorylation of FAK and Tyr 118 phosphorylation of paxillin in HNSCC-derived cells, which is accompanied by decreased cell motility.…”

TIMP-2 upregulates RECK expression via dephosphorylation of paxillin tyrosine residues 31 and 118

“…In response to motility signals, paxillin undergoes tyrosine phosphorylation primarily at two sites, Tyr-31/ 118 (Nakamura et al, 2000;Petit et al, 2000), and Src kinase has been implicated in regulating tyrosine phosphorylation of paxillin (Thomas et al, 1995;Richardson et al, 1997). As TIMP-2 suppresses cell migration (Ahn et al, 2004;Oh et al, 2004) and we showed in this study that TIMP-2 signaling downregulates Src kinase activity (Figure 1b), the effects of TIMP-2 on paxillin phosphorylation was examined.…”

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

“…Our previous work in human Caco-2 intestinal epithelial cells, a cell line which progressively differentiates as it grows past confluence and is thus a widely used human intestinal epithelial cell in vitro model system, suggests that Crk binding to Srcphosphorylated p130 Cas is an important regulator of intestinal epithelial cell spreading and sheet migration on collagen IV (Sanders and Basson, 2005), one of the major components of the intestinal epithelial basement membrane in vivo. This work also suggests that Crk binding to paxillin, which plays an important role in regulating migration in some cell systems (Petit et al, 2000;Lamorte et al, 2003), is not as important for regulation of these processes in Caco-2 cells on collagen IV.…”