Attenuation of Adhesion-dependent Signaling and Cell Spreading in Transformed Fibroblasts Lacking Protein Tyrosine Phosphatase-1B

Abstract

Previous biochemical evidence has yielded conflicting models for the role of protein tyrosine phosphatase-1B (PTP-1B) in the regulation of integrin signaling. Thus, to establish the physiological relevance for such a role, we employed a genetic approach by generating embryonic fibroblasts from PTP-1B knockout mice. Both primary fibroblasts and their derived cell lines were used in this study. Immortalization of wild-type primary cells with the SV40 Large T antigen resulted in a dramatic increase in the endogenous expression of PTP-1B, suggesting a role during transformation. Moreover, the absence of PTP-1B in the transformed cell lines led to a more pronounced effect on different pathways of fibronectin-mediated signaling compared with the untransformed state. Specifically, p130Cas phosphorylation, Erk activation as well as cell spreading were delayed in PTP-1B-deficient cells, compared with their wild-type counterparts. Interestingly, this attenuation in integrin-mediated events closely resembles that of Src-deficient fibroblasts. Indeed, PTP-1B deficient, transformed fibroblasts held in suspension do exhibit a hyperphosphorylation of the inhibitory site (Tyr-527) of Src, compared with their wild-type counterparts. These results establish PTP-1B as a positive physiological regulator of integrin signaling in transformed cells, acting upstream of Src Tyr-527 dephosphorylation that leads to several adhesion-dependent events.

The protein-tyrosine phosphatase DEP-1 modulates growth factor-stimulated cell migration and cell–matrix adhesion

“…It is tempting to speculate that Src activation occurs through DEP-1-mediated dephosphorylation of the Src C-terminal inhibitory phosphorylation site. Src kinase activation via such a mechanism has previously been shown for RPTPa and PTP-1B (Harder et al, 1998;Cheng et al, 2001).…”

“…Also, DEP-1-wt-expressing cells exhibited a flatter morphology and were more difficult to trypsinize than their nonexpressing counterparts (M Kovalenko, E Jandt; unpublished observation). Effects of several PTPs on cell-matrix adhesion have been described earlier, including positive effects of RPTPa (Harder et al, 1998) and PTP-1B (Cheng et al, 2001). These findings led us to test whether DEP-1 has an effect on cell-matrix adhesion.…”

“…Positive effects of RPTPa and PTP-1B on cellmatrix adhesion have previously been associated with PTP-mediated Src activation (Harder et al, 1998;Cheng et al, 2001). We, therefore, investigated the effect of DEP-1 variants on Src activity in the context of cellmatrix adhesion.…”

“…Cells were prepared as described by Cheng et al (2001). Briefly, cells were trypsinized, washed twice with DMEM containing 0.1% NCS and 0.2% BSA, and 1 Â 10 7 cells were diluted in 10 ml of the same medium.…”

The protein tyrosine phosphatase HePTP regulates nuclear translocation of ERK2 and can modulate megakaryocytic differentiation of K562 cells

“…In this context it is interesting to note that PTP1B −/− mouse fibroblasts immortalized with the SV40 Large T antigen show alterations in adhesion and integrin signaling, while PTP1B −/− primary fibroblasts exhibit only very subtle changes in this pathway compared to wild-type cells. 36 In conclusion, we propose that one important function of HePTP is the regulation of ERK1/2 nuclear translocation, rather than regulation of basal ERK activity. Furthermore, it appears that different aspects of K562 cell megakaryocytic differentiation have quite distinct requirements with regard to ERK activity, and that the outcome of ERK2 activation can be modulated by HePTP protein levels.…”