PRMT4-Mediated Arginine Methylation Negatively Regulates Retinoblastoma Tumor Suppressor Protein and Promotes E2F-1 Dissociation

Abstract

The retinoblastoma protein (pRb/p105) tumor suppressor plays a pivotal role in cell cycle regulation by blockage of the G₁-to-S-phase transition. pRb tumor suppressor activity is governed by a variety of posttranslational modifications, most notably phosphorylation by cyclin-dependent kinase (Cdk) complexes. Here we report a novel regulation of pRb through protein arginine methyltransferase 4 (PRMT4)-mediated arginine methylation, which parallels phosphorylation. PRMT4 specifically methylates pRb at the pRb C-terminal domain (pRb C^term) on arginine (R) residues R775, R787, and R798 in vitro and R787 in vivo. Arginine methylation is important for efficient pRb C^term phosphorylation, as manifested by the reduced phosphorylation of a methylation-impaired mutant, pRb (R3K). A methylmimetic form of pRb, pRb (R3F), disrupts the formation of the E2F-1/DP1-pRb complex in cells as well as in an isolated system. Finally, studies using a Gal4–E2F-1 reporter system show that pRb (R3F) expression reduces the ability of pRb to repress E2F-1 transcriptional activation, while pRb (R3K) expression further represses E2F-1 transcriptional activation relative to that for cells expressing wild-type pRb. Together, our results suggest that arginine methylation negatively regulates the tumor suppressor function of pRb during cell cycle control, in part by creating a better substrate for Cdk complex phosphorylation and disrupting the interaction of pRb with E2F-1.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00945-14.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes of Health (CA14779, to Y.I.) and by an American Cancer Society Research Scholar grant (RSG-13-383-01-MPC) to Y.I. This work was also partially supported by a grant from the Department of Defense (W81XWH1110575, to Y.I.).