Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase.

Immunofluorescence studies with protein phosphatase-1 (PP1) isoforms-specific antibodies detected PP1delta, but not alpha or gamma1, at focal adhesions. PP1delta also co-immunoprecipitated with the focal adhesion kinase (FAK) and the alphav-integrin. In the present study glutathione S-transferase (GST)-PP1delta pulled-down FAK from fibroblasts extract and the interaction domain localized between residues 159 ...
and 295 of delta. The association was confirmed by the ability to GST-FAK-related non-kinase (FRNK) to pull-down PP1delta 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 2 h from the mitotic release (when 85-90% of the cells remained round) and decreased to basal level by 8 h (when cells were all polygonal). At the same time FAK underwent dephosphorylation, which was completed by 4 h. FAK obtained from cells at 1.5 h 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 8 h was PP1delta. The results suggest that FAK dephosphorylation by PP1delta occurs in cells released from mitosis, and confirmed the specific association of PP1delta, as detected previously in adherent cells.
Mesh Terms:
Animals, Cell Cycle, Cells, Cultured, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, Glutathione Transferase, Hela Cells, Humans, Mitosis, Muscle, Skeletal, Phosphoprotein Phosphatases, Phosphorylation, Protein Phosphatase 1, Protein-Tyrosine Kinases, Rabbits, Rats, Rats, Inbred F344, Recombinant Fusion Proteins
Biochem. J.
Date: Sep. 01, 2001
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