A Protein Arginine N-Methyltransferase 1 (PRMT1) and 2 Heteromeric Interaction Increases PRMT1 Enzymatic Activity

Abstract

Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene expression by methylating arginine residues within target proteins. PRMT1 is responsible for most cellular arginine methylation activity and can work independently or in collaboration with other PRMTs. In this study, we demonstrate a direct interaction between PRMT1 and PRMT2 using co-immunoprecipitation, bimolecular fluorescence complementation, and enzymatic assays. As a result of this interaction, PRMT2 stimulated PRMT1 activity, affecting its apparent V(max) and K(M) values in vitro and increasing the production of methylarginines in cells. Active site mutations and regional deletions from PRMT1 and -2 were also investigated, which demonstrated that complex formation required full-length, active PRMT1. Although the inhibition of methylation by adenosine dialdehyde prevented the interaction between PRMT1 and -2, it did not prevent the interaction between PRMT1 and a truncation mutant of PRMT2 lacking its Src homology 3 (SH3) domain. This result suggests that the SH3 domain may mediate an interaction between PRMT1 and -2 in a methylation-dependent fashion. On the basis of our findings, we propose that PRMT1 serves as the major methyltransferase in cells by forming higher-order oligomers with itself, PRMT2, and possibly other PRMTs.

Arginine methylation in yeast proteins during stationary-phase growth and heat shock

“…Figure 2b displays the MS 2 of sDMA (top) and the MS 3 derived from the primary product ions 158.1 m/z (middle) and 172.0 m/z (bottom). As observed previously, the MS 2 for sDMA and aDMA are similar except for the unique product ions 46 m/z for aDMA and 172.1 m/z for sDMA (Rappsilber et al 2003;Brame et al 2004;Gehrig et al 2004;Lakowski and Frankel 2009;Lakowski et al 2010b;Pak et al 2011). The MS 3 spectrum of the primary product ion 172.2 m/z is diagnostic for sDMA (Fig.…”

“…An MRM assay was used for the simultaneous quantification of aDMA, [Cθ1D 3 , Cθ2D 3 ]aDMA, sDMA, [Cθ1D 3 , Cθ3D 3 ]sDMA, histidine, and MMA (total) (Supplemental Table 1). The methods for detection of undeuterated methylarginines and histidine were described previously (Lakowski and Frankel 2009;Pak et al 2011). The method of quantification of δMMA and ηMMA using MRM 3 is described elsewhere (Lakowski et al 2013).…”

The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex

“…Moreover, not only have PRMTs been shown to homooligomerize, but some studies support the possibility that PRMTs in mammals and plants have the ability to form heteromers. For example, mammalian PRMT2 can interact with PRMT1 both in vivo and in vitro, T. brucei PRMT Is an Enzyme-Prozyme Pair FEBRUARY 10, 2017 • VOLUME 292 • NUMBER 6 although the extent to which this occurs in vivo is unknown (60). Interestingly, recombinant PRMT2 enhanced the activity of recombinant PRMT1 up to 15-fold even when PRMT2 harbored inactivating mutations, suggesting a possible regulatory role for PRMT2.…”

Protein Arginine N‐Methyltransferase Substrate Preferences for Different Nη‐Substituted Arginyl Peptides

“…The active-site residue D51 is thought to play a role in a catalytic His-Asp dyad for deprotonation of the substrate arginine guanidino group, and E153 is positioned to form hydrogen bonds to the arginine guanidino group. [19,20] D51R and E153Q have previously been shown to abolish enzyme activity, [19,[21][22][23][24] and it has been proposed that D51 and H293 might form a structurally important dyad. [23] We found that D51N PRMT1 was less active toward the peptide series as a whole than wild-type PRMT1, yet this enzyme variant was most active toward R1-OH and R1-CH 3 .…”

“…[15] 14 C Methylation and tricine gel separation of the R1 peptide series: Enzymes were prepared as previously described. [15,22,25,36] The PRMT1 D51N variant was created from pET-28(+)-PRMT1 using QuikChange (Agilent Technologies) and primers D51N-F (5'-CCA CGA GGA GAT GCT AAA GAA TGA G-3') and D51N-R (5'-CGT GAG GGT TCG CAC CTC ATT CTT TA-3'). R1 peptide (250 mm) was incubated in methylation buffer (HEPES (50 mm pH 8.0), NaCl (10 mm), dithiothreitol (DTT, 1 mm)) [25] with S-adenosyl-l-[methyl- ), and PRMT1, PRMT4, PRMT6, PRMT1 E153Q, or PRMT1 D51N (2 mm) for 16 h at 37 8C.…”