Regulation of co-repressive activity of and HDAC recruitment to RIP140 by site-specific phosphorylation

Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that contains several autonomous repressive domains (RDs). The N-terminal RD acts by recruiting histone deacetylases (HDACs). In a comprehensive proteomic analysis of RIP140 by MS, 11 phosphorylation sites of RIP140 are identified; among them five sites are located in the N-terminal RD including Ser(104), Thr(202), Thr(207), Ser(358), and Ser(380). The role of phosphorylation of RIP140 in regulating its biological activity and the underlying mechanism are examined using a site-directed mutagenesis approach. Mutations mimicking constitutive phosphorylation or dephosphorylation are introduced. The N-terminal RD phosphorylation, mediated by the mitogen-activated protein kinase (MAPK), enhances its repressive activity through increased recruitment of HDAC. Mutations mimicking constitutive dephosphorylation at Thr(202) or Thr(207) significantly impair its repressive activity and HDAC recruitment, whereas mutation at Ser358 only slightly affects its HDAC recruitment and the repressive activity. Consistently, mutations mimicking constitutive phosphorylation at either Thr(202) or Thr(207) convert RIP140 into a more potent repressor, which is less responsive to a disturbance in the MAPK system. Furthermore, constitutive phosphorylation at both Thr(202) and Thr(207) residues renders RIP140 fully repressive and strongly interacting with HDAC. The activity of this mutant is resistant to the MAPK inhibitor, indicating an essential role for Thr(202) and Thr(207) in MAPK-mediated modulation of RIP140 function. The study provides insights into the modulation of RIP140 biological activity through a specific cellular signaling pathway that augments phosphorylation at specific residues of RIP140 molecule and alters its cofactor recruitment.