Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1.
TIMAP (TGF-beta1 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-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/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-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta 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-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.
Li
Laiji
L
Department of Medicine, University of Alberta, Edmonton, Alberta T5R 0B7, Canada.
Kozlowski
Kathy
K
Wegner
Binytha
B
Rashid
Tahsin
T
Yeung
Titus
T
Holmes
Charles
C
Ballermann
Barbara J
BJ
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism*
D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits; D000071679:Glycogen Synthase Kinase 3 beta; D017868:Cyclic AMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1