Asymmetric Arginine Dimethylation of Heterogeneous Nuclear Ribonucleoprotein K by Protein-arginine Methyltransferase 1 Inhibits Its Interaction with c-Src

Abstract

). Whereas these five residues were quantitatively modified, Arg 303 was asymmetrically dimethylated in <33% of hnRNP K and Arg 287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNAbinding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.Arginine dimethylation is a common post-translational modification in eukaryotes (1-5). The enzymes responsible for this modification are the protein-arginine methyltransferases (PRMTs).3 They are classified in two groups (2). Type I enzymes promote the formation of asymmetricThe known mammalian type I enzymes are PRMT1, PRMT2, PRMT3, PRMT4/coactivator-associated arginine methyltransferase 1, PRMT6, and the recently discovered brain-specific PRMT8 (6 -11). Type II enzymes catalyze the symmetric N G ,NЈ G -dimethylation of arginine residues. PRMT5 and PRMT7 are the mammalian type II enzymes described so far (12)(13)(14). PRMT1, which is predominantly localized to the cytoplasm (15), is thought to account for the generation of ϳ85% of asymmetric dimethylarginine residues (16) Many proteins involved in RNA metabolism like hnRNP A1 (36), hnRNP A2 (37), Sam68 (38), and SAF-A (hnRNP U) (39) contain regions with clustered arginine residues in Arg-Gly-Gly motifs (RGG box) or RG repeats. These arginine residues are typically asymmetrically dimethylated. In addition, asymmetric arginine dimethylation has also been found in clustered RXR motifs (40) and other sequences. Therefore, the prediction of a methylated arginine is difficult. Furthermore, the enzyme responsible for the dimethylation of a particular protein is unknown in many cases, and the substrate specificities of the different methyltransferases remain poorly characterized. The exact knowledge of the methylated arginine residues and their quantitative distribution as well as the identification of the relevant methyltransferases is a prerequisite for the functional analysis of arginine methylation.HnRNP K belongs to the family of heterogeneous nuclear RNPs that participate in the processing of pre-mRNAs and in the export of mRNAs from the nucleus. An N-terminal bipartite nuclear-localization signal and an hnRNP K-specific nuclear shuttling signal confer the capacity for bi-directional transport across the nuclear envelop (41, 42). The cytoplasmic accumulation of hnRNP K is mediated by its Erk-dependent serine phosphorylation (43). In the cytoplasm hnRNP K functions in the post-tr...

Deciphering the Cross Talk between hnRNP K and c-Src: the c-Src Activation Domain in hnRNP K Is Distinct from a Second Interaction Site

“…hnRNP K contains three prolinerich motifs which have been shown to interact with the isolated SH3 domain of the Src kinase family members c-Src, Fyn, and Lyn in vitro (49,53,54). The arginine residues (R256, 258, 268, 296, 299) of hnRNP K, which are located between the prolinerich motifs, are asymmetrically dimethylated by the protein arginine methyltransferase 1 (PRMT1) (40). The methylation of those arginine residues inhibits the activation of c-Src by hnRNP K, suggesting that the proline-rich motifs are important for the activation of c-Src in vitro and in vivo (40).…”

“…The arginine residues (R256, 258, 268, 296, 299) of hnRNP K, which are located between the prolinerich motifs, are asymmetrically dimethylated by the protein arginine methyltransferase 1 (PRMT1) (40). The methylation of those arginine residues inhibits the activation of c-Src by hnRNP K, suggesting that the proline-rich motifs are important for the activation of c-Src in vitro and in vivo (40). Thus, hnRNP K acts as a scaffold protein that integrates signaling cascades by facilitating cross talk between ERK, c-Src, and PRMT1 with factors that mediate mRNA-directed processes.…”

Investigation of the Molecular Origins of Protein-arginine Methyltransferase I (PRMT1) Product Specificity Reveals a Role for Two Conserved Methionine Residues

“…Coupled with the observation that many PRMT targets have multiple arginines that are methylated (39,40), the diversity of potential protein products is large and suggests the possibility for a methylarginine code. The biological relevance of such a code has most recently been validated as part of the complex set of post-translational histone modifications affecting gene expression (2,6,7).…”

Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing

“…8). HnRNPs are known to undergo posttranslational modifications such as phosphorylation [49,50], methylation [51,52], and sumoylation [53]. In the case of hnRNP K, phosphorylation can regulate RNA binding [49].…”