Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP.

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 PP1cß. 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 PP1cß effectively dephosphorylated MLC2 and TIMAP itself. By contrast, TIMAP inhibited the PP1cß 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.

Protein phosphatase inhibitors isolated from Spongia irregularis collected in Papua New Guinea.

Irregularasulfate (1), a new nitrogen-containing sesterterpenoid, and the known sesterterpenoids hipposulfate C (2), halisulfate-7 (3), and igernellin (4), have been isolated from the marine sponge Spongia irregularis collected in Papua New Guinea. The structure of 1 was elucidated via analysis of its spectroscopic data. Sesterterpenoids 1, 2, and 3 are moderate inhibitors of the catalytic subunits of the mammalian Ser/Thr protein phosphatases calcineurin, PP-1, and PP-2A. The phosphate analogue of 3 and the thiophosphate analogue of 2 have been prepared from the corresponding natural products and evaluated for their ability to inhibit the phosphatase activity of calcineurin.