Low M r Phosphotyrosine Protein Phosphatase Associates and Dephosphorylates p125 Focal Adhesion Kinase, Interfering with Cell Motility and Spreading

Abstract

Low M r phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M r phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M r phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60 src with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M r phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M r phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.Cell adhesion and motility are based on the organization and remodeling of macromolecular complexes called focal adhesions. Following integrin engagement and clustering, several proteins, such as tensin, paxillin, p190Rho-GAP, and p125 focal adhesion kinase (FAK), 1 accumulate in these specialized sites at the cytoplasmic face of plasma membrane (1-3). Autophosphorylation of FAK on Tyr-397 creates a binding site for pp60 src , which phosphorylates FAK further (4). This enables the recruitment of other structural or signaling proteins, including pp130CAS , Csk, talin, PI3-kinase, ␣-actinin, and Grb2, which triggers the activation of the extracellular signal-regulated kinase (ERK) cascade (5-7). FAK is implicated in the control of cell migration by modulating the turnover of focal adhesions and in the transmission of cell survival signals from the extracellular matrix (1-3). Cell motility and spreading are clearly affected by FAK expression as fibroblasts isolated from FAK knockout mice exhibit reduced motility and a more rounded shape with abundant focal contacts that cover much of the ventral surface (8 -10). Vice versa, overexpression of FAK stimulates cell migration in Chinese hamster ovary cells (11).The role of FAK phosphorylation in the above processes is not completely clarified yet; the catalytic activity of FAK and its autophosphorylation site are required to restore cell migration in FAK-deficient cells (12). Indeed, PTEN and PTP1B tyrosine phosphatases are negative regulators of FAK tyrosine phosphorylation, cell spreading, and migration (13,14). However, in contrast to ...

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