Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

Abstract

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/ threonine kinases. The activities of the LIM2-and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to "inside-out" integrin-mediated signal transduction.

Serine phosphorylation of paxillin by heregulin-β1: role of p38 mitogen activated protein kinase

“…Recently serine phosphorylation of paxillin LIM domains was shown to play an important role in localization of paxillin to focal adhesions (Brown et al, 1998). In addition to serine phosphorylation, recruitment of paxillin to focal adhesions in growth factorstimulated cells also requires tyrosine phosphorylation (Abedi et al, 1995;Abedi and Zachary, 1997;Leventhal et al, 1997;Matsumoto et al, 1994;Hiregowdara et al, 1997).…”

“…In addition to the p38 MAPK -dependent pathway, data from the literature suggest that LIM2 and LIM3 domains of paxillin could serve as substrates for uncharacterized serine/threonine kinases (Brown et al, 1998). However, mutations in serine residues in LIM domains had no phenotypic e ect on paxillin localization to focal points (Brown et al, 1998), and thus, are unlikely to be involved in the observed HRGmediated serine phosphorylation of paxillin as it was accompanied by a distinct redistribution from focal adhesions to perinuclear areas (Figure 2).…”

“…However, mutations in serine residues in LIM domains had no phenotypic e ect on paxillin localization to focal points (Brown et al, 1998), and thus, are unlikely to be involved in the observed HRGmediated serine phosphorylation of paxillin as it was accompanied by a distinct redistribution from focal adhesions to perinuclear areas (Figure 2). In summary, regardless of the mechanism by which p38 MAPK controls activation of paxillin, our ®ndings have clearly demonstrated for the ®rst time a role of p38 MAPKdependent signal transduction pathway(s) in serine phosphorylation and disassembly of the paxillin from the focal complexes during HRG-induced cell shape changes and cell-scattering.…”

“…Since the focal adhesion complexes are characterized by high levels of tyrosine phosphorylation, antiphosphotyrosine mAb PY20 has also been used to localize the focal contacts (Brown et al, 1998). Since mAb PY20 does not stain ®laments like a-actinin, and in general exhibits very low background, we also used this antibody to localize focal adhesions.…”

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…The N-terminus (amino acids 1-325) of paxillin is defined by the most extensively characterized of its two structural domains, the LD domain [99]. The LD domain consists of five separate motifs that share sequence homology in their binding domain [66].…”