Glycogen Synthase Kinase 3- and Extracellular Signal-Regulated Kinase-Dependent Phosphorylation of Paxillin Regulates Cytoskeletal Rearrangement

Abstract

Paxillin is a 68-kDa focal adhesion-associated protein that plays an important role in controlling cell spreading and migration. Phosphorylation of paxillin regulates its biological activity and thus has warranted investigation. Serine 126 and serine 130 were previously identified as two major extracellular signal-regulated kinase (ERK)-dependent phosphorylation sites in Raf-transformed fibroblasts. Here serine 126 is identified as a phosphorylation site induced by lipopolysaccharide (LPS) stimulation of RAW264.7 cells. A number of other stimuli, including adhesion and colony-stimulating factor, induce serine 126 phosphorylation in RAW264.7 cells, and nerve growth factor (NGF) treatment induces serine 126 phosphorylation in PC12 cells. The kinase responsible for phosphorylation of this site is identified as glycogen synthase kinase 3 (GSK-3). Interestingly, this GSK-3-dependent phosphorylation is regulated via an ERK-dependent priming mechanism, i.e., phosphorylation of serine 130. Phosphorylation of S126/S130 was required to promote spreading in paxillin null cells, and LPS-induced spreading of RAW264.7 cells was inhibited by expression of the paxillin S126A/S130A mutant. Furthermore, this mutant also retarded NGF-induced PC12 cell neurite outgrowth. Hence, phosphorylation of paxillin on serines 126 and 130, which is mediated by an ERK/GSK-3 dual-kinase mechanism, plays an important role in cytoskeletal rearrangement.Paxillin is a 68-kDa focal adhesion-associated protein that functions as a scaffolding protein assembling signaling molecules into complex downstream of integrins (6, 34). It plays an important role in regulating cell spreading and migration. The paxillin knockout mouse exhibits embryonic lethality, which suggests that paxillin plays an essential role in development (18). Paxillin contains five LD motifs in the N-terminal half of the molecule. These peptide motifs mediate protein-protein interactions and bind a number of proteins, including focal adhesion kinase (FAK) and vinculin (6,40). Four LIM domains are found in the C-terminal half of paxillin, two of which are required for the discrete localization of paxillin to focal adhesions (3).Multiple stimuli induce phosphorylation of paxillin, including growth factors, integrin-dependent cell adhesion to extracellular matrix, and other ligands (6, 34). Two major tyrosine phosphorylation sites, Y31 and Y118, have been identified in the N-terminal half of paxillin (35). Phosphorylation of these sites modulates docking of SH2 domain-containing proteins, such as CRK, and is important for regulation of cell motility (32, 43). In addition to tyrosine phosphorylation sites, serine and threonine phosphorylation sites have been identified in paxillin. Serine residues 188 and 190 are phosphorylated following integrin ligation (1). Threonines 398 and 403 in LIM2 and serines 457 and 481 in LIM3 are phosphorylated following cell adhesion and stimulation with angiotensin II (4, 5). Phosphorylation of these LIM domain residues regulates focal adhesion localization ...

ERK2 but not ERK1 mediates HGF-induced motility in non small cell lung carcinoma cell lines

“…4D). Serine 126 has been described to be phosphorylated by glycogen synthase kinase 3 (GSK3) upon priming of the adjacent serine 130 by ERK (Cai et al, 2006). Consistent with this, HGFmediated phosphorylation of serine 126 was abrogated both by incubation with the MEK1 inhibitor U0126 or the GSK3 inhibitor IX (Fig.…”

“…So far, the specific contribution of ERK1 and ERK2 has not been studied with respect to the HGF-signalling cascade. Here we show that HGF-induced motility is specifically mediated by ERK2 in several cell lines, and this finding correlated with the observation that ERK2 but not ERK1 was required for the HGF-induced phosphorylation of the focal adhesion scaffold protein paxillin on serine 126, a site which has been described to be phosphorylated by glycogen synthase kinase (GSK) 3 upon priming of the adjacent serine 130 by ERKs (Cai et al, 2006). The important role of this phosphorylation in HGF-mediated migration was demonstrated by the finding that the siRNA-mediated depletion of endogenous paxillin results in a decrease in HGF-mediated migration, an effect which can be rescued by wild type paxillin but not a mutant in which serine 126 and the adjacent serine 130 are mutated to alanine.…”

Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics

“…Reciprocally, FAK inhibits GSK3 by promoting its serine-phosphorylation which may allow FAK to escape the inhibitory phosphorylation by GSK3 in some situations [37]. In contrast, other evidence indicates that GSK3 is able to promote cell spreading in some situations, for example, by phosphorylating paxillin [38]. GSK3 also binds the adhesion-regulating protein h-prune, such that GSK3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration [39].…”

A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions

“…Integrin activation, as well as growth factor stimulation, was shown to lead to the phosphorylation of paxillin on both tyrosine and serine residues (Bellis et al, 1997;Schaller and Schaefer, 2001). Serine phosphorylation was reported to have a role in FA targeting of paxillin (Brown et al, 1998), in cell spreading (Cai et al, 2006), and recently was implicated in regulating adhesion assembly via recruitment of a GIT-PIX-PAK complex (Nayal et al, 2006). Phosphorylation of two tyrosyl residues, namely Y31 and Y118, is induced upon integrin activation during epithelial-tomesenchymal transition and has been shown to enhance the migration of some cell types (Iwasaki et al, 2002;Petit et al, 2000), while inhibiting migration in others .…”