Abstract
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 Ser104, Thr202, Thr207, Ser358, and Ser380. 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 Thr202 or Thr207 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 Thr202 or Thr207 convert RIP140 into a more potent repressor, which is less responsive to a disturbance in the MAPK system. Furthermore, constitutive phosphorylation at both Thr202 and Thr207 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 Thr202 and Thr207 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.
Highlights
Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that contains several autonomous repressive domains (RDs)
We identified mitogen-activated protein kinase (MAPK)-mediated phosphorylation as the critical pathway for RD1-mediated repressive activity, which was caused by enhanced recruitment of histone deacetylases (HDACs) by phosphorylated RIP140
The protein phosphorylation sites prediction software NetPhos that assisted in the analysis of RIP140 showed both Thr202 and Thr207 to be potential sites for phosphorylation, we conclude that Thr202
Summary
Construction of Expression Vectors—Constructs of RIP140 full length/N-terminal fused to GAL4-BD [20], RIP140 full length fused to His-epitope [28], FLAG-epitope [22], and GAL4-tk-luciferase reporter [23] have been described previously. One gel was subjected to Western blot analysis probed with anti-FLAG antibody followed by horseradish peroxidase-conjugated secondary antibody and was detected with ECL (GE Healthcare), which constituted the input Another gel was fixed, dried, and exposed to a phosphoimager screen (Molecular Dynamics) overnight to detect labeled HDAC protein and visualized by autoradiography. Intensity of labeled HDAC was normalized to input His Pull Down—The expression vector for His-RIP140 was expressed and purified from insect cells in presence of phosphatase inhibitor that ensured phosphorylated RIP140 [28]. The fold relative luciferase activity was calculated by normalizing RLU activity found in the experimental samples to the RLU activity found in the pBD-GAL4-RIP140 full-length/N-terminal WT and pAD-GAL4HDAC3 co-transfection.
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