Phosphorylation of TIMAP by glycogen synthase kinase-3β activates its associated protein phosphatase 1

Abstract

TIMAP (TGF-β1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P] orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3β. Site-specific Ser to Ala substitution identified amino acid residues Ser333/ Ser337 as the likely PKA/GSK-3β phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAP V64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3β stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3β site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3β-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.