cytoplasmic distribution

Biochemical and Biophysical Research Communications 404 (2011) 865–869

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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution Yuan-I Chang a, Sheng-Chieh Hsu a, Gar-Yang Chau b,c, Chi-Ying F. Huang a,d, Jung-Sung Sung e, Wei-Kai Hua a, Wey-Jinq Lin a,f,⇑ a

Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan Department of Surgery, Taipei Veterans General Hospital, Taipei 112, Taiwan c Department of Surgery, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan d Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan e Taipei City Hospital, Yang-Ming Branch, Taipei 111, Taiwan f Department of Education and Research, Taipei City Hospital, Taipei 103, Taiwan b

a r t i c l e

i n f o

Article history: Received 6 December 2010 Available online 22 December 2010 Keywords: Protein arginine methylation Protein arginine methyltransferase 1 Heterogeneous nuclear ribonucleoprotein K Cytoplasmic/nuclear distribution

a b s t r a c t Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates, here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280–307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280–307, but not a.a. 308–327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction The heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein is a scaffold protein that integrates and relays intracellular signals. By interacting with DNA, RNA and proteins, hnRNP K participate in a variety of cellular functions including chromatin remodeling, transcription, RNA processing and export, translation, and signal transduction [1,2]. HnRNP K has been found to locate at various intracellular compartments with a predominant localization in the nucleus.

Abbreviations: hnRNP K, heterogeneous nuclear ribonucleoprotein K; PRMT1, protein arginine methyltransferase 1; RGG box, glycine- and arginine-rich box; DICE, differentiation control element; LOX, 15-lipoxygenase; MAPKs, mitogenactivated protein kinases; Erk, extracellular-signal-regulated kinase; JNK, c-Jun N-terminal kinase; 3H-AdoMet, S-adenosyl-L-[methyl-3H] methionine; AdOx, adenosine dialdehyde. ⇑ Corresponding author at: Institute of Biopharmaceutical Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Taipei 112, Taiwan. Fax: +886 2 2825 0883. E-mail address: [email protected] (W.-J. Lin). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.12.076

The hnRNP K proteins are subject to various posttranslational modifications which extend its functional diversity. For example, phosphorylation of hnRNP K by c-Src at Tyr 458, is important for its binding to, and translational regulation of, the mRNA of reticulocyte 15-lipoxygenase (LOX) mRNA [3]. In addition, ERK and JNK have both been shown to phosphorylate on Ser 284 and 353 of hnRNP K [4,5]. However, phosphorylation by ERK, but not JNK, results in cytoplasmic accumulation of hnRNP K that facilitates its involvement in translational suppression [4], due possibly to the additional phosphorylation on Ser 116 and 216 by JNK [5]. It is conceivable that individual phosphorylation event regulates functions of hnRNP K in an integrative and collaborative way. Emerging evidence indicates that protein arginine methylation is a pivotal posttranslational modification involved in numerous cellular functions [6,7]. HnRNP K proteins, like other heterogeneous nuclear ribonucleoproteins (hnRNPs), are methylated on multiple arginine residues within the glycine- and arginine-rich RGG motif (also known as GAR motif) [8–13]. Methylation of hnRNP K induced by UV irradiation has been shown to play a role in stimulating p53 transcriptional activity required for DNA

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damage response [14]. In addition, methylation of hnRNP K by protein arginine methyltransferase 1 (PRMT1) inhibited its interaction with c-Src [13]. Mass spectrometric analysis has identified five major (Arg256, Arg258, Arg268, Arg296, and Arg299) and two minor (Arg303 and Arg287) methylarginines in the RGG motif of hnRNP K [8,12,13]. When arginine residues in the five major sites are simultaneously changed to glycines, named 5RG mutant, hnRNP K can no longer be methylated by PRMT1 in vitro [13]. In addition, hnRNP K is not methylated in PRMT1-deficient ES cells [13]. These results suggest that PRMT1 is the primary methyltransferase for arginine modification of these sites in hnRNP K, biochemical analysis is, however, essential to provide direct evidence. The possibility that PRMT1 provides a primed site for the subsequent methylation by other PRMTs is not completely excluded. To examine the methylation efficiency of potential PRMT1 sites and the possibility of cooperative methylation of these sites, we made several deletions in the RGG motif between residues 246 and 327 and directly assayed for their methylation by PRMT1. Kinetic analysis indicated that sequences between 280 and 307 harbored the most efficient PRMT1 substrate sites. Our results also indicated no cooperative methylation between the methylation regions assayed. Importantly, nuclear hnRNP K was dramatically decreased under a hypomethylated state and after deletion of the methylation regions. Together we have identified the preferred PRMT1 methylation region of hnRNP K by direct methylation assay and indicated a role of methylation in regulating the intracellular distribution of hnRNP K. 2. Materials and methods 2.1. Materials Monoclonal anti-FLAGÒ M5 and anti-HA.11 antibodies were purchased from Sigma–Aldrich and Covance Research Products Inc., respectively. S-adenosyl-L-[methyl-3H] methionine (3H-AdoMet, 63.6 Ci/mmol, NET-155H) and fluorographic enhancer, EN3-HANCE, were from Perkin Elmer Life Sciences Inc.

fetal bovine serum. Transfection of HEK293 and HeLa cells were performed by calcium phosphate precipitation and by using Lipofectamine™ 2000 Reagent (Invitrogen Corporation), respectively. HA-PRMT1-expressing HEK293 cells were harvested 24–36 h after transfection, resuspended in extraction buffer (50 mM Tris– HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 20 lg/ml leupeptin, 20 lg/ml aprotinin, 20 lg/ml pepstatin, and 1 mM PMSF), and homogenized on ice with a glass tissue grinder. Homogenates were centrifuged at 10,000g for 30 min at 4 °C, and supernatants were used for immunoprecipitation [15]. 2.5. Immunoprecipitation The anti-HA.11 antibodies (1 lg) were incubated with antimouse IgG agarose beads (20 ll) (Sigma–Aldrich) for 2 h at 4 °C. Cell homogenates (1 mg) from HA-PRMT1-expressing HEK293 cells were added and incubated for 2 h at 4 °C. At the end of incubation, beads were washed twice with PBS containing 0.05% of Tween-20. The immunoprecipitated PRMT1 was used immediately as an enzyme source for methylation assay [15]. 2.6. Methylation assay Methylation of recombinant hnRNP K protein substrates was carried out with GST-PRMT1, cell homogenates or immunoprecipitated PRMT1 as an enzyme source. The methylation assays were performed in the presence of 1.65 lCi of S-adenosyl-L-[methyl-3H] methionine (3H-AdoMet) and 25 mM Tris–HCl, pH 8.0, in a final volume of 30 ll for 30 min at 30 °C. Reactions were stopped by the addition of SDS sample buffer. Samples were subjected to SDS polyacrylamide gel electrophoresis (SDS–PAGE). After staining and de-staining, gels were soaked in the fluorographic enhancer, EN3HANCE, according to manufacturer’s instructions. The methyl incorporation was visualized by fluorography. To quantify methyl incorporation, proteins of interest were excised from the gels and tritium incorporation was measured by liquid scintillation counting. 2.7. Immunostaining

2.2. Plasmids The pET23a-Trx-hnRNP K plasmid [12,15] was used as a template for polymerase chain reaction (PCR) to obtain various hnRNP K deletion mutants for expression of His-tagged thioredoxin (Trx)fused recombinant proteins. The hnRNP K DNA fragments were also subcloned into pFLAG-CMV-2 plasmids for mammalian expression. The accuracy of all constructs was verified by DNA sequencing. The pPCDNA3HA2-PRMT1 plasmid was used for ectopic expression of HA-PRMT1 in HEK293 cells [15,16].

Cells were transfected with various pFLAG-CMV-2-hnRNP K plasmids for 48 h and fixed with 4% paraformaldehyde. The FLAG-tagged proteins were immunostained with anti-FLAGÒ M5 antibodies (1:500) and visualized with rhodamine-conjugated anti-mouse IgG antibodies (1:400). Hoechst 33258 (1 lg/ml) was used to stain nuclei. In some experiments, the cells were treated with a methyltransferase inhibitor, adenosine dialdehyde (AdOx, 20 lM), for 24 h prior to fixation. The cytoplasm/nucleus distribution of various hnRNP K was quantified at least 300 cells for each experiment under a fluorescent microscope.

2.3. Preparation of the recombinant proteins 2.8. Statistical analysis Recombinant GST-PRMT1 proteins and thioredoxin-fused hnRNP K proteins were expressed and purified as described [12,17]. Briefly, hnRNP K proteins were eluted from Ni+-NTA agarose (Qiagen) with elution buffer (0.5 M imidazole, 500 mM NaCl, and 20 mM Tris–HCl, pH 7.9), subjected to dialysis to remove imidazole and stored in a buffer containing 50 mM NaCl and 20 mM Tris–HCl, pH 7.9 [12,15]. 2.4. Cell culture, transfection and protein extraction HEK293 cells were cultured in MEM medium supplemented with 10% fetal bovine serum and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%

The Student’s t-test was used for statistical analysis. Values are presented as means ± s.e.m. All experiments were performed at least three times. p < 0.05 was considered statistically significant. 3. Results Various deletion mutations in the glycine- and arginine-rich RGG motif (residues 246–327) were generated to facilitate further examination of methylation of hnRNP K (Fig. 1A). Methylation of hnRNP K predominantly occurred in the RGG box as deletion of the entire RGG box (hnRNP K DRGG) completely eliminated methylation by GST-PRMT1 (Fig. 1B). This is in consistent with

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Fig. 1. Arginine methylation preferences of glycine- and arginine-rich sequences within heterogeneous nuclear ribonucleoprotein K proteins by PRMT1. Panel A is the schematic diagrams of various hnRNP K mutants. Deletion of the glycine- and arginine-rich motif (K DRGG) completely abolished methyl incorporation (B). Methyl incorporation into wild type and various mutant hnRNP K proteins catalyzed by GST-PRMT1 (C), cell lysates containing HA-PRMT1 (D), and immunoprecipitated HA-PRMT1 (E) was assayed. Methyl incorporation was visualized by fluorography. The hnRNP K proteins were excised from gels and tritium incorporation was quantified by liquid scintillation counting. All experiments were performed at least three times and data are presented as means ± s.e.m.; ⁄⁄⁄p < 0.005; N.D. means no difference.

the report that the 5RG mutant hnRNP K protein in which five arginines (256, 258, 268, 296, and 299) in the RGG box are simultaneously changed to glycines cannot be methylated by recombinant PRMT1 [13]. In order to reveal the methylation preference in the RGG box, we then made three deletion mutants (Fig. 1A) and carried out direct methylation reactions catalyzed by PRMT1. Deletion of residues 280–307 (hnRNP K D280–307) produced the greatest impact on methylation by GST-PRMT1, a decrease of 60% (Fig. 1C). Deletion of residues 246–279 (hnRNP K D246–279) also, although to a less extent, significantly reduced methylation, by about 30% (Fig. 1C). According to previous reports, there are two predicted PRMT1 sites (296 and 299) in hnRNP K D280–307 and three predicted sites (256, 258 and 268) in hnRNP K D246–279. Our results provided results demonstrating that arginine residues in the 280–307 region were preferred substrates than those in the 246–279 region. In addition, our results suggested that arginine residues (e.g. 296 and 299) could be methylated directly by PRMT1 without the existence of sequences in the 246–279 region. Similarly, arginine residues 256, 258 and 268 could be methylated directly by PRMT1 without the existence of sequences in the 280– 307 region. PRMT1 methylated the hnRNP K D308–327 mutant to a level similar to wild-type hnRNP K indicating that the two RGG motifs (residues 316–318 and 325–327) in this region were not substrate sites of GST-PRMT1 (Fig. 1C). Similar results were observed when methylation reactions were carried out using cell lysates containing HA-PRMT1 (Fig. 1D) or using HA-PRMT1 immunoprecipitated from HEK293 cells as enzyme sources (Fig. 1E), indicating that the catalytic property of recombinant GST-PRMT1

toward hnRNP K proteins is similar to that of PRMT1 expressed in mammalian cells. To further examine the kinetics of methylation reactions of these mutants, we measured methylation reactions over a time course of 60 min (Fig. 2A and B). The hnRNP K D280–307 mutant displayed the lowest rate of methylation, indicating that this region harbored the most efficient substrate sites (Fig. 2B). In addition, the methylation rate of hnRNP K D246–279 mutant was also affected, although to a less extent, indicating this sequence was less efficient as PRMT1 substrates (Fig. 2B). The rate of methyl incorporation of the hnRNP K D308–327 mutant was similar to the wild type (Fig. 2B), indicating sequences in this deleted region did not influence the methylation efficiency. Taken together, our results suggested that there are multiple methylation sites between residues 246 and 307, with the most efficient substrate sites residing in the region between residues 280 and 307. HnRNP K proteins shuttle between nucleus and cytoplasm to participate in various cellular functions [1,2]. However, the molecular events that regulate its intracellular distribution are still not fully elucidated. Methylation by PRMT1 in other hnRNPs has been shown to regulate their intracellular distributions [11,18]. We thus investigated the potential role of protein methylation in its intracellular distribution. The hnRNP K protein, when ectopically expressed in HeLa cells (Fig. 3A and B), was predominantly, about 80%, localized in nucleus. Treatment with adenosine dialdehyde (AdOx), an inhibitor of S-adenosylhomocysteine hydrolase which indirectly suppresses methylation due to accumulation of products, resulted in a cytoplasm/nucleus distribution with about

Y.-I. Chang et al. / Biochemical and Biophysical Research Communications 404 (2011) 865–869

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Fig. 2. The kinetics of methylation of wild-type and mutant hnRNP Ks by GST-PRMT1. The kinetics of methylation of wild-type and the various mutant hnRNP K proteins (1 lg) by GST-PRMT1 (0.5 lg) was measured over a time period of 60 min. Methyl incorporation was visualized by fluorography (A) and quantified by liquid scintillation counting (B). All experiments were performed at least three times and data are presented as means ± s.e.m.; ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.005 as compared with the tritium incorporation of wild-type hnRNP K at the same time point.

35–40% in the nucleus (Fig. 3A), indicating that methylation might play a role in nuclear retention of hnRNP K proteins. The RGGdeleted mutant (hnRNP K DRGG) caused an even greater decrease of nuclear hnRNP K to about 10% (Fig. 3A). Similar results were observed in HEK293 cells (data not shown). Various deletion mutants of hnRNP K were then tested to identify the role of different sequence regions in the RGG box. Deletion of the major methylation region, either residues 246–279 or 280–307, caused a dramatic decrease in the level of nuclear hnRNP K, from 80% to about 25% (Fig. 3B). In addition to methylation, the RGG box is also phosphorylated in multiple sites [1] and contains many protein/protein interaction motifs [1]. Our results suggested that methylation is one of the multiple mechanisms regulating nuclear localization of hnRNP K. 4. Discussions HnRNP K has multiple methylarginine residues in the RGG motif (also known as GAR motif) [8,12,13]. The preference of PRMT1 toward individual arginine residue in the RGG motif is an interesting question. In this study, we use direct methylation assay and show that sequence regions 246–279 and 280–307 harbor PRMT1 methylation substrates and the most preferred sites locate in 280– 308. Two (Arg296 and Arg299) of the five major methylarginines previously identified by mass spectrometry are located in the region 280–307 [8,12,13] and the other three (Arg256, Arg258 and Arg268) are located in the region 246–279 [13]. Sequence comparison reveals that the R296GGR299GG cluster of hnRNP K is flanked by multiple proline residues, which are missing in residues 246–279. Whether these prolines play a role in substrate preference is yet to be investigated. Although simultaneous mutation of five arginines (Arg256, Arg258, Arg268, Arg296, and Arg299) abrogates methylation of hnRNP K proteins by PRMT1 [13], the possibility of sequential methylation events among these sites or whether PRMT1 methylation is a prerequisite for methylation by other PRMTs, as it has been

demonstrated with some phosphorylation events [3,19], remains intriguing. Our results indicate that these methylation events occur without a requirement for prior methylation in the other region of the protein, since hnRNP K D280–307 and hnRNP K D246–279 can be independently methylated by PRMT1 and the methyl incorporation into wild-type hnRNP K is equal to the sum of incorporation into hnRNP K D280–307 and hnRNP K D246–279. Our results are also supported by a previous report using peptides derived from hnRNP K [20]. The hnRNP K protein has been shown to shuttle between cytoplasm and nuclear [1,2]. In this study, we further show that deletion of either residues 246–279 or residues 280–307 impairs the intracellular distributions of hnRNP K in HeLa (Fig. 3), suggesting a role of arginine methylation in regulating the nuclear localization of hnRNP K. This is supported by the observation that a hypomethylated state (AdOx treatment) significantly reduced the nuclear localization of hnRNP K in both HeLa cells (Fig. 3) and HEK293 cells (data not shown). The catalytic activity of PRMT1 may have a direct contribution to the regulation of localization in these cells. However, hnRNP K has been shown to be predominantly in nucleus in PRMT1-null ES cells [13], suggesting that PRMT1 may not directly regulate the intracellular localization of hnRNP K in ES cells or other complementary methylation events exist in the context of PRMT1 deficiency. Arginine methylation has been shown to affect association of hnRNP K with its interaction proteins [13,14]. Whether methylation marks a signature for nuclear retention of hnRNP K through protein/protein interaction is of particular interest. Furthermore, deletion of the RGG box also causes a cytoplasmic localization of hnRNP K (Fig. 3A). Since these regions are overlapped with the KI domain (K interactive region, residues 240–337) and several phosphorylation sites [1], there are likely multiple events governing the intracellular distribution of hnRNP K proteins. Together, in this study, we have identified the preferred PRMT1 methylation region of hnRNP K by direct methylation assay and indicated a role of methylation in regulating the intracellular distribution of hnRNP K.

Y.-I. Chang et al. / Biochemical and Biophysical Research Communications 404 (2011) 865–869

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Taiwan (NSC 96-2311-B-010-009-MY3 to W.-J. Lin), Taipei City Hospital (96002-62-084 to W.J. Lin), University System of Taiwan (VGHUST95-P7-27 to W.-J. Lin), and a grant from Ministry of Education, Aim for the Top University Plan.

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Fig. 3. Intracellular distribution of hnRNP K proteins is affected by protein methylation. HeLa cells were transfected with wild-type pFLAG-CMV-2-hnRNP K or various mutants for 48 h (A and B). After fixation, FLAG-tagged proteins were stained with anti-FLAGÒ M5 antibodies (1:500) and visualized with rhodamineconjugated anti-mouse IgG antibodies (1:400). The cells were counterstained for DNA with Hoechst 33258 (1 lg/ml). In some experiments, the cells were treated with a methyltransferase inhibitor, AdOx (20 lM), for 24 h prior to fixation (A). The cytoplasm/nucleus distribution of various hnRNPs was quantified (A and B). All experiments were performed at least three times and data are presented as means ± s.e.m.

Acknowledgments We thank Drs. F.-F. Chen and M.-Y. Chen for their critical discussions. This research was supported by National Science Council,

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