Tyrosine phosphatase SHP‐2 regulates IL‐1 signaling in fibroblasts through focal adhesions

Abstract

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.

Tyrosine phosphatase PTPα regulates focal adhesion remodeling through Rac1 activation

“…1, A and B). This morphology of FA has been termed "supermature," denoting an area significantly larger than that of typical mature FA (10,16,22). Quantitative analysis confirmed that, even after 16 h of spreading, these supermature FA in PTP␣ Ϫ/Ϫ fibroblasts were significantly larger than those in PTP␣ wt cells (8.3 Ϯ 3.4 vs. 3.0 Ϯ 2.2 m long; Fig.…”

“…Using paxillin and ␣-actinin immunofluorescence, we examined the subcellular distribution of FA in primary murine fibroblasts derived from PTP␣ wt and PTP␣ Ϫ/Ϫ mice (46) spreading on fibronectin. Paxillin, which is present in filopodia and initiates FA formation (34), was used as a marker to study the early stages of FA formation and maturation, and ␣-actinin was used as a marker to characterize the later stages of FA maturation (22,51). Immunofluorescence staining of paxillin from PTP␣ wt and PTP␣ Ϫ/Ϫ fibroblasts revealed key differences in the structure and stability of peripheral FA (Fig.…”

“…Focal complexes display a punctuate appearance (32). As the rate of FA assembly and dissipation decreases near the edge of the lamellipodium, highly dynamic focal complexes mature into larger and more stable FA by the recruitment of additional proteins such as ␣-actinin (3,19,22). Mature FA facilitate the transmission of tensile forces to the substrate, and, during locomotion, these structures translocate toward the center of the cell, thus facilitating cell movement relative to the substrate (54).…”

Phosphorylation of SHP-2 Regulates Interactions between the Endoplasmic Reticulum and Focal Adhesions to Restrict Interleukin-1-induced Ca2+ Signaling

“…Notably, the ER proteins kinectin (23) and the IP 3 receptor (24) co-localize with focal adhesions. We have shown earlier that the ER-associated protein calnexin co-localizes with focal adhesions (25) and that SHP-2 is critical for IL-1-induced focal adhesion maturation (16), in part by regulating phosphorylation of phospholipase C␥1. Accordingly, we sought to determine what molecular determinants of SHP-2 are responsible for mediating IL-1 promotion of interactions between focal adhesions and the ER.…”

“…For example, SHP-2 helps to sustain ERK activity in response to platelet-derived growth factor (13), possibly by modulating signals arising from integrin-associated proteins (14). SHP-2 can also enhance IL-1-induced ERK activation; this process is tightly linked to integrin clustering in focal adhesions (15,16) and is mediated possibly by Src family-related kinase phosphorylation of SHPS-1 and recruitment of SHP-2 (13), or by interaction of SHP-2 with focal adhesion proteins such as paxillin and Gab1 (16). Further, SHP-2 regulates cell spreading on fibronectin, focal adhesion formation (17), and RhoA activity (18).…”