title: Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP.
abstract: TIMAP is an endothelial-cell predominant member of the MYPT family of PP1c regulatory subunits. This study explored the TIMAP-PP1c interaction and substrate specificity in vitro. TIMAP associated with all three PP1c isoforms, but endogenous endothelial cell TIMAP preferentially co-immunoprecipitated with PP1cβ. Structural modeling of the TIMAP/PP1c complex predicts that the PP1c C-terminus is buried in the TIMAP ankyrin cluster, and that the PP1c active site remains accessible. Consistent with this model, C-terminal PP1c phosphorylation by cdk2-cyclinA was masked by TIMAP, and PP1c bound TIMAP when the active site was occupied by the inhibitor microcystin. TIMAP inhibited PP1c activity toward phosphorylase a in a concentration-dependent manner, with half-maximal inhibition in the 0.4-1.2 nM range, an effect modulated by the length, and by Ser333/Ser337 phosphomimic mutations of the TIMAP C-terminus. TIMAP-bound PP1cβ effectively dephosphorylated MLC2 and TIMAP itself. By contrast, TIMAP inhibited the PP1cβ activity toward the putative substrate LAMR1, and instead masked LAMR1 PKA- and PKC-phosphorylation sites. This is direct evidence that MLC2 is a TIMAP/PP1c substrate. The data also indicate that TIMAP can modify protein phosphorylation independent of its function as a PP1c regulatory subunit, namely by masking phosphorylation sites of binding partners like PP1c and LAMR1.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
authors.forename: Micheal J
authors.initials: MJ
authors.lastname: Shopik
authors.forename: Laiji
authors.initials: L
authors.lastname: Li
authors.forename: Hue-Anh
authors.initials: HA
authors.lastname: Luu
authors.forename: Marya
authors.initials: M
authors.lastname: Obeidat
authors.forename: Charles F B
authors.initials: CF
authors.lastname: Holmes
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Ballermann
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D017474:Receptors, Laminin / Q000378:metabolism; D012269:Ribosomal Proteins
chemical_list: D008565:Membrane Proteins; C459597:PPP1R16B protein, human; C510624:RPSA protein, human; D017474:Receptors, Laminin; D012269:Ribosomal Proteins; D054645:Protein Phosphatase 1