Immunofluorescence studies with protein phosphatase-1 (PP1) isoforms-specific antibodies detected PP1δ, but not α or γ1, at focal adhesions. PP1δ also co-immunoprecipitated with the focal adhesion kinase (FAK) and the αv-integrin. In the present study glutathione S-transferase (GST)–PP1δ pulled-down FAK from fibroblasts extract and the interaction domain localized between residues 159 and 295 of δ. The association was confirmed by the ability to GST–FAK-related non-kinase (FRNK) to pull-down PP1δ from fibroblasts extract. GST–FRNK also pulled-down purified muscle PP1 catalytic subunit, thus indicating direct interaction between FAK and PP1. FAK displays consensus sequences for phosphorylation by cell division cycle kinase-2–cyclin B, and might be a PP1 substrate. In fact, FAK immunoprecipitated from metabolically-labelled mitotic HeLa cells without tyrosine phosphatase inhibitors was phosphorylated on Ser only and was dephosphorylated in vitro by purified muscle PP1, with loss of phospho-Ser. No PP1 was associated with FAK immunoprecipitated from mitotic HeLa cells. However, progressively more PP1 activity was assayed in FAK-immunoprecipitates obtained from cells released from mitosis. The associated activity was maximal at 2h from the mitotic release (when 85–90% of the cells remained round) and decreased to basal level by 8h (when cells were all polygonal). At the same time FAK underwent dephosphorylation, which was completed by 4h. FAK obtained from cells at 1.5h was Ser-phosphorylated, and underwent dephosphorylation during in vitro incubation, with loss of phospho-Ser, indicating the presence of active FAK-bound phosphatase. The only FAK-associated PP1 isoform between 1 and 8h was PP1δ. The results suggest that FAK dephosphorylation by PP1δ occurs in cells released from mitosis, and confirmed the specific association of PP1δ, as detected previously in adherent cells.
Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase
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Marco FRESU, Mariarita BIANCHI, J. Thomas PARSONS, Emma VILLA-MORUZZI; Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase. Biochem J 1 September 2001; 358 (2): 407–414. doi: https://doi.org/10.1042/bj3580407
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