Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

Abstract

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.

The rat tyrosine phosphatase η increases cell adhesion by activating c-Src through dephosphorylation of its inhibitory phosphotyrosine residue

“…This prompted us to investigate whether r-PTPZ interacts with c-Src, since c-Src activation is a feature of cell-substratum adhesion in various cells (Harder et al, 1998;Jones et al, 2002;Li et al, 2002). In addition, there is evidence that the increased cell adhesion mediated by c-Src activation involves tyrosine phosphatase activity (Arregui et al, 1998;Harder et al, 1998;Jandt et al, 2003). Here, we report that PTPZ interacts with c-Src in vitro and in vivo.…”

The interplay between Src and integrins in normal and tumor biology

“…For example, ptpaÀ/À cells and shp2À/À fibroblasts exhibit a delay in cell spreading on fibronectin (Oh et al, 1999;Su et al, 1999). In addition, overexpression of a dominant-negative mutant of PTP1B in mouse L cells impairs cell spreading on fibronectin (Arregui et al, 1998). As described above, a target for these PTPs is Src and the spreading phenotype when these phosphatases are inhibited is similar to the srcÀ/À phenotype.…”

“…Overexpression of a catalytically defective mutant of PTP1B in mouse L cells reduces Src activity when the cells are plated on fibronectin (Arregui et al, 1998). Furthermore, these cells also showed reduced levels of tyrosine phosphorylation of focal adhesion proteins that become tyrosine phosphorylated upon cell adhesion.…”

“…Furthermore, these cells also showed reduced levels of tyrosine phosphorylation of focal adhesion proteins that become tyrosine phosphorylated upon cell adhesion. PTP1B was also co-immunoprecipitated with b1 integrin and colocalized with integrins at focal adhesions (Arregui et al, 1998). PTP1B has also been purified and identified from breast cancer cells as a phosphatase that could dephosphorylate a peptide containing phosphotyrosine 527 (Bjorge et al, 2000).…”

Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice

“…19 PTP1B is an established regulator of epidermal growth factor receptor, 20 integrin, 21,22 and insulin receptor 23 signaling. Whole-body PTP1B knockout (KO) mice exhibit improved systemic insulin sensitivity, enhanced glucose tolerance, and resistance to high-fat dieteinduced obesity.…”