Phosphorylation of TIMAP by Glycogen Synthase Kinase-3β Activates Its Associated Protein Phosphatase 1

Abstract

TIMAP (TGF-␤1 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 [ 32 P]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-3␤. Site-specific Ser to Ala substitution identified amino acid residues Ser 333 /Ser 337 as the likely PKA/GSK-3␤ phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAP V64A/F66A ) abolished PP1c binding and TIMAPassociated PP1c activity. TIMAP V64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3␤ stimulated phosphorylation of TIMAP V64A/F66A , but not wild-type TIMAP, suggesting that the PKA/GSK-3␤ 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-3␤-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.PP1c 2 belongs to the family of serine/threonine phosphatases (1) that includes PP2A, PP4-PP7, and calcineurin (PP2B).Ser/Thr phosphatases counteract essentially every signal involving Ser/Thr kinases, and like the kinases their activity is tightly regulated. Because of extreme sequence conservation (Ͼ90%) among PP1c isoforms, substrate-and location-specific actions are not inherent in PP1c itself but dictated by more than 50 PP1c regulatory (targeting) subunits (1). Among these, the myosin phosphatase-targeting (MYPT) subunits regulate muscle and non-muscle cell contraction through dephosphorylation of regulatory myosin light chains (2). Most PP1c regulatory subunits share the PP1c binding motif RVXF, but their structures are otherwise diverse, serving in each case highly specific and localized functions (1).The function of PP1c targeting subunits is regulated by changes in expression, by allosteric effects of substrates, and by phosphorylation (1). For example, phosphorylation at or near the RVXF motif of NIPP-1 reduces PP1c binding, and phosphorylation elsewhere in the molecule can increase or decrease PP1c activity (3). The activity of...

TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells

“…TIMAP is a member of the MYPT family of PP1c-targeting subunits (33,48). The question therefore arises whether the effects of TIMAP silencing observed here can be attributed to reduced TIMAP/PP1c phosphatase activity.…”

“…Endogenous TIMAP was immunoprecipitated from glomerular EC as previously described (33). Briefly, EC monolayers were washed once with ice-cold PBS and immediately harvested in cold immunoprecipitation lysis buffer, described earlier (33). The soluble cell fraction was incubated with control chicken anti-IgY antibody or chicken anti-TIMAP antibody at 4°C for 2 h followed by incubation with goat anti-chicken IgG-coated beads (Aves) overnight at 4°C.…”

“…Endogenous TIMAP was immunoprecipitated from glomerular EC as previously described (33). Briefly, EC monolayers were washed once with ice-cold PBS and immediately harvested in cold immunoprecipitation lysis buffer, described earlier (33).…”

TIMAP is a positive regulator of pulmonary endothelial barrier function

“…Unlike nuclear targets, at least one membrane-associated target of TIMAP was already characterized (16,19). We studied membrane localization of TIMAP and showed an increased amount of membrane-associated TIMAP after S1P treatment.…”

“…Therefore, it was suggested that TIMAP, as a PP1 targeting subunit, regulates LAMR1 phosphorylation (16). Furthermore, TIMAP-associated PP1c regulation of filopodia formation was shown in glomerular endothelial (GEN) cells (19).…”