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 G 1 -to-Sphase 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. Members of the retinoblastoma protein (pRb) family, also known as "pocket proteins," are key regulators of cell cycle progression by serving as gatekeepers of the G 1 -to-S-phase transition (1, 2). Indeed, inactivating mutations of pRb function have been observed in a wide array of cancers. Of the three family members (pRb/p105, pRB2/p130, and pRBL1/p107), pRb/p105 has been most extensively studied for its definitive role in tumor suppression and its ability to bind and inhibit the neoplastic transforming transcriptional activator E2F-1 (3-6). In normal quiescent cells and early-G 1 cells, most pRb is in its active hypophosphorylated form and suppresses the activity of E2F-1 through protein-protein interactions via its pocket domain and C-terminal domain (pRb C term ) (3, 7-10). Upon mitogen stimulation, pRb is spatially and temporally phosphorylated throughout the G 1 phase, initially by the cyclin D/cyclin-dependent kinase 4 (Cdk4) or cyclin D/Cdk6 complex and later by the cyclin E/Cdk2 or cyclin A/Cdk2 complex (11-14). Phosphorylation of conserved serine (S) and threonine (T) residues located in the pRb pocket domain and pRb C term results in dissociation of the E2F-1 transactivation domain and E2F-1/DP1 coiled-coil/marked box (CM) domain, respectively (9, 13, 15, 16). Release of E2F-1/DP1 permits E2F-1-mediated transactivation of S-phase-specific genes and induction of DNA replication for cell cycle progression (17). Accordingly, others have reported that although the pRb pocket domain is necessary for growth suppression, pRb C term is required for both maximum E2F-1 ...

Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome

“…In the first, DRBD18(R→K), we mutated the three identified methylarginines to lysines, thereby creating a hypomethylated DRBD18 while preserving the residues’ positive charge. In the second methylmutant, DRBD18(R→F), we replaced the methylarginines with phenylalanine, which studies have shown can act as a methylarginine mimic ( 53 – 55 ). Because the methylarginines in DRBD18 are located in a long unstructured region of the protein (Figure 1 ), it is unlikely that mutations in this region will dramatically alter DRBD18 structure.…”

A Code of Mono-phosphorylation Modulates the Function of RB

“…Additionally, several methyl-transferases methylate RB at K810 (Carr et al, 2011;Cho et al, 2012), K873 (Munro et al, 2010), and K860 (Saddic et al, 2010) regulating interactions that control RB's transcriptional activity and function in G1/S-checkpoint and DNA-damage response. Recently, arginine RB methylation at R775, R787, and R798 reported to reduce RB ability to retain E2F1 activity (Kim et al, 2015). Adding to this picture are potential roles for RB ubiquitination (Miwa et al, 2006) and SUMOylation (Ledl et al, 2005;Meng et al, 2016).…”

RB1: a prototype tumor suppressor and an enigma

“…The idea that pRB's binding activity is tailored by phosphorylation is only part of the story. Evidence that pRB is both acetylated and methylated in specific contexts (Chan et al 2001;Nguyen et al 2004;Leduc et al 2006;Munro et al 2010;Saddic et al 2010), evidence of interplay between different post-translational modifications (Carr et al 2011;Macdonald and Dick 2012;Munro et al 2012;Kim et al 2015), and evidence that pRB interacts with specific partners in response to cellular cues (MacLellan et al 2000;Miyake et al 2000;Dick and Dyson 2003;Nguyen et al 2004;Carr et al 2014) all suggest additional levels of diversity. Collectively, these observations raise several intriguing possibilities: (1) There may be multiple pools of pRB that perform different functions.…”