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Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39 Sanyue Mai a, Xiuhua Qu b, Ping Li a, Qingjun Ma a, Xuan Liu a, **, Cheng Cao a, * a b
Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850, China General Navy Hospital of PLA, 6 Fucheng Rd, Haidian District, Beijing 100037, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2016 Accepted 22 March 2016 Available online xxx
RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-a and ESR2/ER-b. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERa and PRb in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. © 2016 Elsevier Inc. All rights reserved.
Keywords: c-Abl RBM39 Interact Phosphorylation Transcriptional coactivation
1. Introduction RBM39 (also called HCC1.4 or CAPERa) was first identified as an auto-antigen in a hepatocellular carcinoma patient [1,2] and as an inhibitor of v-Rel-mediated lymphocyte transformation [3]. RBM39, which was also shown to be a transcriptional co-activator of activating protein-1 (AP-1) and the estrogen receptor ERa, is involved in the regulation of the transcriptional responses of these proteins and promotes transcriptional initiation [1,4]. As a highly homologous protein to U2AF65, which is an essential serine-/ arginine-rich (SR-rich) splicing factor, RBM39 contains a long arginine- and serine-rich domain (RS domain) and three RNA-
Abbreviations: SH2, the Src homology 2; SH3, the Src homology 3; LC/MS/MS, liquid chromatography coupled with tandem mass spectrometry; AP-1, activating protein-1; SR-rich, serine-/arginine-rich; RS domain, arginine- and serine-rich domain; RRMs, RNA-recognition motifs; ROS, reactive oxygen species; PR, progesterone receptor; ER, estrogen receptor; GST, Glutathione S-transferase; MEF, mouse embryo fibroblasts; DKO, c-abl- and arg-depleted; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; ECL, electrochemiluminescence; NanoUPLC-MS/MS, Nano Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy; ESI, electrospray ionization; TOF, time-of-flight; MS, mass spectrometry; PBS, phosphate-buffered saline. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Liu), [email protected] (C. Cao).
recognition motifs (RRMs) and shares notable sequence similarity with U2AF65 but lacks a U2AF ligand motif [5]. Immunofluorescence microscopy has shown that RBM39 is located in a speckled network in the nucleus and co-localizes with splicing factor SC35 and uridine-rich small nuclear RNAs. RBM39 may play a role in hormone-dependent transcriptional activation and function as a splicing cofactor for steroid receptors [4,6,7]. The mammalian Abl nonreceptor tyrosine kinase (c-Abl), a modular protein that is widely expressed in adult and fetal tissues, has multiple functions [8,9]. The N-terminal portion of c-Abl is composed of Src homology (SH) 2, SH3, and kinase domains [10,11]. The existence of C-terminal DNA binding motifs and nuclear localization signals in c-Abl enables the protein to shuttle between the cytoplasmic and nuclear compartments [12,13], increasing its exposure to additional Abl kinase substrates. c-Abl is important for multiple cellular processes, including cell proliferation, apoptosis, differentiation, survival, adhesion, and migration, as well as inflammation and stress responses [8,9,14]. It is well established that the effects of c-Abl are mediated by proteineprotein interactions and the phosphorylation of substrate proteins by tyrosine kinase activity. c-Abl functions in both proapoptotic and antiapoptotic pathways, particularly under conditions of irradiation, DNA damage and other stressors, such as reactive oxygen species (ROS) [8,9,14e16]. Although the phenomenon that steroid hormone receptors
http://dx.doi.org/10.1016/j.bbrc.2016.03.108 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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recruit coregulators such as RBM39 to modulate their transcriptional response and facilitate transcriptional initiation has been well documented, the regulation of RBM39 by tyrosine kinases is largely unknown. In this report, we showed that c-Abl interacts with and phosphorylates RBM39. In addition, the transcriptional coactivation activity of RBM39 for ERa and PRb was promoted by cAbl. 2. Materials and methods 2.1. Vectors Flag-tagged vectors expressing RBM39, RBM39(Y95F), RBM39(Y99F), RBM39(Y95FY99F), RBM39(Y95FY99FY475F) and RBM39(Y95FY99FY475FY505F) were constructed by cloning the genes into the pcDNA3-based Flag vector (Thermo Fisher, CA, USA). The Myc-tagged c-Abl and c-Abl(K290R) plasmids were constructed by cloning these genes into the pCMV-Myc vector (BD Biosciences Clontech, CA, USA). Flag-ERa and Flag-PRb were kindly provided by Drs. Qinong Ye and Xuemin Zhang from the Academy of Military Medical Sciences, China. The luciferase reporter plasmids were constructed by cloning DNA fragments by PCR amplification with primers containing synthetic recombination sites in the pGL3-E2b-Luc vector (Promega, WI, USA). PRE-E1b-LUC was constructed by inserting the 50 -TGTACAGGATGTTCTAAGTCGATGCACTGTACAGGATGTTCT-30 sequence, which contains two progesterone receptor (PR) response elements, into the KpnI/Nhel sites of pGL3-E1b-LUC, and ERE-E1b-LUC was constructed by inserting the 50 -AGGTCACAGTGACCTAAGTCGATGCACAGGTCACAGTGACCT-30 sequence, which contains two estrogen receptor (ER) response elements, into the same sites of pGL3-E1b-LUC [4,17]. Glutathione S-transferase (GST) fusion proteins were generated by expressing pGEX4T-2based vectors (Amersham Biosciences, NJ, USA) in Escherichia coli (E.coli) BL21 (DE3). All constructs were confirmed by sequencing analysis. 2.2. Cell culture and transfections MCF-7, 293T, and wild type mouse embryo fibroblasts (MEFs), as well as c-abl- and arg-depleted (DKO) MEFs, were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. For immunoprecipitation and immunoblot assays, transient transfections were performed using VigoFect (Vigorous Biotechnology, Beijing, China) when the cells reached 60e80% confluency. The cells were also treated with STI571 (Novartis, Basel, Switzerland), as indicated in the text. For the siRNA experiments, cells were transfected with 30 pmol of siRNA (GenePharma, Shanghai, China) using Lipofectamine RNAiMAX (Thermo Fisher, CA, USA) when they reached 60e80% confluency. The cells were incubated for another 2 days at 37 C following transfection, after which total RNA was extracted. The siRNA sequences targeting RBM39 were as follows: RBM39-siRNA: GCAGCAATGGCAAACAATT. For the luciferase assays, the cell culture and transfection method with doses have been described previously [4]. 2.3. Immunoprecipitation and immunoblot analysis Cell lysates were prepared in lysis buffer (50 mM TriseHCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 10 mg/ml pepstatin A) containing 1% (v/v) Nonidet P-40. Soluble proteins were subjected to immunoprecipitation with anti-Flag
(Sigma, MO, USA), anti-Myc (Sigma, MO, USA), or anti-RBM39 (Bethyl Laboratories, AL, USA) antibodies. Normal rabbit or mouse IgG (Sigma, MO, USA) was used as a control. An aliquot of total lysate (5%, v/v) was also included as a control. Immunoblot analysis was performed with anti-Myc (Sigma, MO, USA), anti-Flag (Sigma, MO, USA), anti-c-Abl (Santa Cruz, CA, USA), anti-RBM39 (Bethyl Laboratories, AL, USA) and anti-P-Tyr (Upstate Biotechnology, CA, USA) antibodies. Antigen/antibody complexes were visualized with chemiluminescence (ECL, GE Healthcare, Buckinghamshire, UK). A protein marker (Thermo Fisher, CA, USA) was used as a molecular weight standard. 2.4. Protein-binding assays GST fusion proteins were generated by expressing pGEX4T-2based vectors in E. coli BL21 (DE3) and were purified by affinity chromatography using glutathione-Sepharose beads (Amersham Biosciences, NJ, USA). In GST pull-down experiments, cell lysates were incubated with 2 mg of purified GST, GST-c-Abl SH2, GST-c-Abl SH3 or GST-RBM39 immobilized on the beads (GE Healthcare, Buckinghamshire, UK) for 2 h at 4 C. The resulting protein complexes were washed with lysis buffer and then subjected to analysis using SDS-PAGE, followed by immunoblotting with anti-Flag antibody and Coomassie blue staining. 2.5. LC/MS/MS After the gel was stained with Coomassie Blue, the protein band was excised from the gel. Dried gel pieces were incubated with trypsin. The sample was desalted through pre-column desalination 5 min before the sample was injected. The peptide mixtures were extracted with acetonitrile/water (1:1, v/v) in a 0.1% formic acid solution. The peptides were then separated by gradient elution with a C18 capillary column. Nano Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (NanoUPLC-MS/MS) analysis was carried out on an electrospray ionization-quadrupoleoa-time-of-flight (ESI-quadrupole-oa-TOF) tandem mass spectrometer (SYNAPT G2-S, Waters, MA, USA). One full mass spectrometry (MS) scan (350e1500 Da) coupled to a secondary spectrum scan (50e2000 Da) was followed by MS/MS that automatically measured peptides using data-dependent analysis mode. We analyzed the mass spectrometry data in the original document using PLGS2.4 software (Waters, MA, USA). The PKL format of the data files was generated using MS spectrum, and we next searched the NCBInr database using Mascot software (Matrix Science, SC, Columbia). 2.6. Luciferase reporter assays MCF-7 cells were plated in 24-well plates and were cultured for 24 h in DMEM without phenol red supplemented with 5% charcoalstripped FBS in 5% CO2 before transfection. Cells grown in 24-well dishes to 60%e70% confluency were transiently transfected with 100 ng of the reporter vector along with 5 ng of the steroid receptor and the indicated amount of RBM39 and c-Abl using Lipofectamine 2000 (Thermo Fisher, CA, USA). An appropriate amount of empty expression vector was used in the assays without the cofactor, and a total of 1 mg/well of DNA was added [4]. Cells were then washed with phosphate-buffered saline (PBS) and were maintained in DMEM without phenol red plus 5% charcoal-stripped serum. The media were also supplemented with the appropriate ligand: 108 M Pg (Sigma, MO, USA), 109 M E2 (Sigma, MO, USA), or vehicle (ethanol) alone (Sigma, MO, USA) [4]. After an incubation time of 36 h, the cells were harvested, and the lysates were assayed for luciferase activity using the Dual Luciferase Reporter Assay
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
S. Mai et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
System (Promega, WI, USA) with a TD-20/20 spectrophotometer (Promega, WI, USA). The assay was performed according to the manufacturer’s instructions (Promega, WI, USA). All samples for the luciferase assays were normalized for Renilla activity using a cotransfected control expression vector, as described previously [18]. The results were expressed as the average ± SD of three independent experiments. 3. Results 3.1. c-Abl interacts with RBM39 Using yeast-two hybrid analysis, we previously noted that c-Abl interacts with RBM39 (data not shown). To substantiate the interaction between c-Abl and RBM39, lysates from wild-type MEFs were immunoprecipitated with the anti-RBM39 antibody and were probed for anti-c-Abl and anti-RBM39. Immunoblotting showed that c-Abl was present in the anti-RBM39 immunoprecipitate but was not pulled down with the rabbit IgG antibody in MEFs (Fig. 1A). To confirm the interaction between c-Abl and RBM39, 293T cells were co-transfected with plasmids expressing Flag-RBM39 and Myc-c-Abl. Anti-Myc immunoprecipitation with the anti-Flag antibody showed a significant interaction between Flag-RBM39
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and Myc-c-Abl (Fig. 1B). In a reciprocal experiment, Myc-c-Abl was found in the anti-Flag immunoprecipitates prepared from cells expressing Flag-RBM39 and Myc-c-Abl (Fig. 1C). As controls, Myc-c-Abl was not found in the anti-Flag immunoprecipitates in cells expressing Myc-Abl and the Flag vector (Fig. 1C). These findings indicated that c-Abl constitutively interacts with RBM39. To further characterize the interaction between c-Abl and RBM39, lysates from 293T cells were incubated with GST or GSTRBM39 fusion proteins. We demonstrated that c-Abl binds to GST-RBM39 but not to GST (Fig. 1D). To investigate the domain in cAbl that interacts with RBM39, Lysates from 239 cells expressing Flag-RBM39 were incubated with GST-c-Abl SH2, GST-c-Abl SH3 or GST conjugated agarose beads, and the absorbates were analyzed by immunoblotting with anti-Flag antibody. Our results showed that RBM39 bound to both the SH2 and SH3 domains of c-Abl (Fig. 1E). These findings confirmed that RBM39 interacts with the SH2 and SH3 domains of c-Abl. 3.2. RBM39 is phosphorylated by c-Abl The interaction between RBM39 and c-Abl suggests that RBM39 is a substrate for the tyrosine kinase. To demonstrate that RBM39 is phosphorylated by c-Abl, 293T cells were transfected with Flag-
Fig. 1. c-Abl interacts with RBM39. A. Lysates from mouse embryo fibroblast cells were subjected to immunoprecipitation with anti-RBM39 antibody or IgG, fractionated by SDSPAGE, and subsequently analyzed by immunoblotting with anti-c-Abl or anti-RBM39. B. 293T cells were co-transfected with Flag-RBM39 and Myc-c-Abl or Myc vector. The anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-Flag or anti-Myc antibody. C. Lysates prepared from 293T cells co-transfected with Myc-c-Abl and Flag-RBM39 or Flag vector were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody as above. D. Lysates from 293T cells transfected with Flag-c-Abl were incubated with GST or GST-RBM39 conjugated agarose beads for 2 h. The absorbates were analyzed by immunoblotting with anti-Flag (upper panel). Loading of the GST proteins was assessed by Coomassie Blue Staining (lower panel). E. Lysates from 293T cells transfected with Flag-RBM39 were incubated with GST-c-Abl SH2, GST-c-Abl SH3 or GST conjugated agarose beads for 2 h. The absorbates were analyzed by immunoblotting with anti-Flag (upper panel). Staining with Coomassie blue confirmed the presence of GST-c-Abl SH2, GST-c-Abl SH3 and GST fusion protein (lower panel).
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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RBM39, Myc-c-Abl, or kinase inactive Myc-c-Abl(K290R). Anti-Flag immunoprecipitates were analyzed by immunoblotting with an anti-phosphotyrosine antibody (P-Tyr). The results showed that RBM39 was phosphorylated by c-Abl but not by kinase inactive cAbl(K290R). In addition, phosphorylation was significantly suppressed by the c-Abl-selective inhibitor STI571 (Fig. 2A). Notably, RBM39 was phosphorylated by c-Abl but not by abl related gene Arg (Fig. 2B).
To identify the phosphorylation sites in RBM39, we digested Flag-RBM39, which was purified with the anti-Flag antibody from cells expressing Flag-RBM39 and Myc-c-Abl, with trypsin and analyzed the peptides by LC/MS/MS. The results suggested that Y95 and Y99 were phosphorylated (Fig. 2C). We further mutated Y95 and (or) Y99 to a similar amino-acid residue phenylalanine (F) and immunoblotted the anti-Flag immunoprecipitates with anti-P-Tyr antibody. The results showed that
Fig. 2. c-Abl phosphorylates RBM39. A. 293T cells were co-transfected with Flag-RBM39 and Myc-c-Abl, Myc-c-Abl(K290R) or Myc vector, and treated with or without STI571 for 24 h. The anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-PTyr or anti-Flag. B. Lysates from 293T cells co-transfected with Flag-RBM39 and Myc-Abl, MycArg or Myc vector were subjected to immunoprecipitation with anti-Flag and were subsequently analyzed by immunoblotting with anti-PTyr or anti-Flag. C. 293T cells were cotransfected with Flag-RBM39 and Myc-c-Abl, and the anti-Flag immunoprecipitates were fractionated by SDS-PAGE. The gel was stained by coomassive blue, and the band containing Flag-RBM39 was excised and subjected to LC/MS/MS analysis. Mass spectra of the YpRSPYpSGPK peptide was shown. Yp indicates a phospho-Tyr residue. D. 293T cells were cotransfected with myc-c-Abl and Flag-RBM39 or Flag-RBM39 mutants expressing plasmids. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr, antiFlag or anti-Myc. E. 293T cells were cotransfected with myc-c-Abl and Flag-RBM39 or Flag-RBM39 mutants expressing plasmids. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag.
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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Fig. 3. c-Abl regulates the coactivation activity of RBM39 in steroid hormone receptor-mediated transcription. A. and B. 293T cells were co-transfected with ERE-Luc (or PRE-Luc), ERa (or PRb), RBM39 or RBM39 mutants and c-Abl. Cells were treated with or without E2 (or Pg) as indicated. 36 h later, cell lysates were subjected to the luciferase reporter assay. Average ± SD of 3 independent experiments were presented. C. and D. Cells were co-transfected with ERE-Luc (or PRE-Luc), ERa (or PR b), c-Abl and RBM39 or RBM39 RNAi. Cells were treated with or without E2 (or Pg) as indicated. 36 h later, cell lysates were subjected to the luciferase reporter assay. Average ± SD of 3 independent experiments were presented.
tyrosine phosphorylation was moderately decreased but not eliminated (Fig. 2D, E), which suggested that there were other tyrosine residues on RBM39 that were phosphorylated by c-Abl. The tyrosine residues on RBM39 were then individually mutated according to the characteristic YxxP motif of the c-Abl phosphorylation sites. Further mutation of Y475 and Y505 into F resulted in substantial abrogation of tyrosine phosphorylation (Fig. 2E). These results indicated that tyrosine phosphorylation of RBM39 primarily occurs on Y95 and Y99. Moreover, Y475 and Y505 may also be phosphorylated, although these results were not confirmed by LC/MS/MS. 3.3. c-Abl promotes the transcriptional coactivation activity of RBM39 for ERa and PRb RBM39 was previously shown to act as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-a and ESR2/ER-b [1]. The capability of the RBM39 cofactor to interact with both ERa and PRb and then stimulate ER- and PR-mediated transcriptional activity in mammalian cells was tested [4]. To investigate the effect of c-Abl mediated phosphorylation on RBM39, cells
were transfected with ERE-E1b-luciferase reporter (or PRE-E1bluc) with RBM39 or RBM39(Y95FY99F) in the presence of ERa (or PRb) and c-Abl. As expected, expression of luciferase directed by the promoter containing ER (or PR) responsive element was promoted by RBM39 when the cells were treated with E2 (or Pg), and RBM39(Y95FY99F) had a significant compressed effect compared to the wild-type (Fig. 3A and Fig. 3B), in the presence of c-Abl. These results showed that c-Abl mediated phosphorylation boasted the transcriptional coactivation activity of RBM39 on the ERa and PRb, and Y95, Y99 together with other phosphorylation sites contributed to the activation of RBM39 as a coactivator. Further, wild type (Scramble), RBM39 overexpressing or RBM39 knock down cells were transfected with ER-Luc (PR-Luc) and c-Abl or vector, c-Abl had a much higher effect in RBM39 overexpressing cells than in RBM39 knockdown cells (Fig. 3C and D), which suggested c-Abl regulated the expression of ER (or PR) responsive genes mainly (if not only) through RBM39 interaction. 4. Discussion RBM39 was previously identified as an auto-antigen in a
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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hepatocellular carcinoma patient [1,2] and as an inhibitor of v-Relmediated lymphocyte transformation [3]. RBM39 was also shown to be a transcriptional co-activator of AP-1 and ERa [1,4]. A highly homologous protein to U2AF65, which is an essential SR-rich splicing factor, RBM39 contains a long RS domain and three RRMs. RBM39 shares notable sequence similarity with U2AF65 but lacks a U2AF ligand motif [5]. The functions of many proteins are influenced by their interactions with other proteins. The present study confirmed that RBM39 interacts with c-Abl, SH2 and SH3 domains of c-Abl can bind to RBM39. In addition, phosphorylation of RBM39 by the kinase has been demonstrated in cells. RBM39 was previously shown to act as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-a and ESR2/ER-b [1]. The ability of the RBM39 cofactor to interact with both ERa and PRb and stimulate ER- and PR-mediated transcriptional activity has been reported [4]. However, tyrosine phosphorylation has not been reported to participate in the regulation of RBM39 activity for transcriptional coactivation. Here we showed that Tyr95/99 and other sites were phosphorylated by c-Abl, and the phosphorylation enhanced the transcriptional coactivation activity of RBM39 for ERa and PRb transcriptional factors. Considered that approximately 80% of all breast cancers are ER-positive and approximately 65% of these breast cancers are also PR-positive, our report may provide a sight into the role of c-Abl in breast cancer. In addition to its role in transcriptional coactivation activity, RBM39 is similar to the RNA splicing factor U2AF65, which preferentially binds to the polypyrimidine tract immediately downstream of the branchepoint sequence at the 30 splice site and enhances the subsequent recruitment of the U2 small nuclear ribonucleoprotein to the branchpoint. RBM39 has been suggested to be a potential splicing factor because of the similar domain structures and sequences between U2AF65 and RBM39. Therefore, the identification of RBM39 as a transcriptional cofactor that stimulates transcription in a hormone-dependent and ligand-dependent manner and the presence of RRM and RS domains in RBM39 similar with the known splicing protein U2AF65 suggest that RBM39 can play a role in alternative splicing by recruiting specific cofactors to the promoter. In addition, previous reports have demonstrated that RBM39 directly interacts with transcription factors to stimulate transcription and mediate appropriate alternative splicing decisions in a promoter-dependent manner [4]. Further studies are necessary to determine whether c-Abl-mediated tyrosine phosphorylation of RBM39 contributes to the regulation of alternative splicing. Acknowledgments This work was supported by the National Natural Science Foundation of China [30871240, 31070674]. We thank American Journal Experts (AJE) for language help.
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.03.108.
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Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39 Sanyue Mai a, Xiuhua Qu b, Ping Li a, Qingjun Ma a, Xuan Liu a, **, Cheng Cao a, * a b
Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850, China General Navy Hospital of PLA, 6 Fucheng Rd, Haidian District, Beijing 100037, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2016 Accepted 22 March 2016 Available online xxx
RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-a and ESR2/ER-b. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERa and PRb in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. © 2016 Elsevier Inc. All rights reserved.
Keywords: c-Abl RBM39 Interact Phosphorylation Transcriptional coactivation
1. Introduction RBM39 (also called HCC1.4 or CAPERa) was first identified as an auto-antigen in a hepatocellular carcinoma patient [1,2] and as an inhibitor of v-Rel-mediated lymphocyte transformation [3]. RBM39, which was also shown to be a transcriptional co-activator of activating protein-1 (AP-1) and the estrogen receptor ERa, is involved in the regulation of the transcriptional responses of these proteins and promotes transcriptional initiation [1,4]. As a highly homologous protein to U2AF65, which is an essential serine-/ arginine-rich (SR-rich) splicing factor, RBM39 contains a long arginine- and serine-rich domain (RS domain) and three RNA-
Abbreviations: SH2, the Src homology 2; SH3, the Src homology 3; LC/MS/MS, liquid chromatography coupled with tandem mass spectrometry; AP-1, activating protein-1; SR-rich, serine-/arginine-rich; RS domain, arginine- and serine-rich domain; RRMs, RNA-recognition motifs; ROS, reactive oxygen species; PR, progesterone receptor; ER, estrogen receptor; GST, Glutathione S-transferase; MEF, mouse embryo fibroblasts; DKO, c-abl- and arg-depleted; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; ECL, electrochemiluminescence; NanoUPLC-MS/MS, Nano Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy; ESI, electrospray ionization; TOF, time-of-flight; MS, mass spectrometry; PBS, phosphate-buffered saline. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Liu), [email protected] (C. Cao).
recognition motifs (RRMs) and shares notable sequence similarity with U2AF65 but lacks a U2AF ligand motif [5]. Immunofluorescence microscopy has shown that RBM39 is located in a speckled network in the nucleus and co-localizes with splicing factor SC35 and uridine-rich small nuclear RNAs. RBM39 may play a role in hormone-dependent transcriptional activation and function as a splicing cofactor for steroid receptors [4,6,7]. The mammalian Abl nonreceptor tyrosine kinase (c-Abl), a modular protein that is widely expressed in adult and fetal tissues, has multiple functions [8,9]. The N-terminal portion of c-Abl is composed of Src homology (SH) 2, SH3, and kinase domains [10,11]. The existence of C-terminal DNA binding motifs and nuclear localization signals in c-Abl enables the protein to shuttle between the cytoplasmic and nuclear compartments [12,13], increasing its exposure to additional Abl kinase substrates. c-Abl is important for multiple cellular processes, including cell proliferation, apoptosis, differentiation, survival, adhesion, and migration, as well as inflammation and stress responses [8,9,14]. It is well established that the effects of c-Abl are mediated by proteineprotein interactions and the phosphorylation of substrate proteins by tyrosine kinase activity. c-Abl functions in both proapoptotic and antiapoptotic pathways, particularly under conditions of irradiation, DNA damage and other stressors, such as reactive oxygen species (ROS) [8,9,14e16]. Although the phenomenon that steroid hormone receptors
http://dx.doi.org/10.1016/j.bbrc.2016.03.108 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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recruit coregulators such as RBM39 to modulate their transcriptional response and facilitate transcriptional initiation has been well documented, the regulation of RBM39 by tyrosine kinases is largely unknown. In this report, we showed that c-Abl interacts with and phosphorylates RBM39. In addition, the transcriptional coactivation activity of RBM39 for ERa and PRb was promoted by cAbl. 2. Materials and methods 2.1. Vectors Flag-tagged vectors expressing RBM39, RBM39(Y95F), RBM39(Y99F), RBM39(Y95FY99F), RBM39(Y95FY99FY475F) and RBM39(Y95FY99FY475FY505F) were constructed by cloning the genes into the pcDNA3-based Flag vector (Thermo Fisher, CA, USA). The Myc-tagged c-Abl and c-Abl(K290R) plasmids were constructed by cloning these genes into the pCMV-Myc vector (BD Biosciences Clontech, CA, USA). Flag-ERa and Flag-PRb were kindly provided by Drs. Qinong Ye and Xuemin Zhang from the Academy of Military Medical Sciences, China. The luciferase reporter plasmids were constructed by cloning DNA fragments by PCR amplification with primers containing synthetic recombination sites in the pGL3-E2b-Luc vector (Promega, WI, USA). PRE-E1b-LUC was constructed by inserting the 50 -TGTACAGGATGTTCTAAGTCGATGCACTGTACAGGATGTTCT-30 sequence, which contains two progesterone receptor (PR) response elements, into the KpnI/Nhel sites of pGL3-E1b-LUC, and ERE-E1b-LUC was constructed by inserting the 50 -AGGTCACAGTGACCTAAGTCGATGCACAGGTCACAGTGACCT-30 sequence, which contains two estrogen receptor (ER) response elements, into the same sites of pGL3-E1b-LUC [4,17]. Glutathione S-transferase (GST) fusion proteins were generated by expressing pGEX4T-2based vectors (Amersham Biosciences, NJ, USA) in Escherichia coli (E.coli) BL21 (DE3). All constructs were confirmed by sequencing analysis. 2.2. Cell culture and transfections MCF-7, 293T, and wild type mouse embryo fibroblasts (MEFs), as well as c-abl- and arg-depleted (DKO) MEFs, were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. For immunoprecipitation and immunoblot assays, transient transfections were performed using VigoFect (Vigorous Biotechnology, Beijing, China) when the cells reached 60e80% confluency. The cells were also treated with STI571 (Novartis, Basel, Switzerland), as indicated in the text. For the siRNA experiments, cells were transfected with 30 pmol of siRNA (GenePharma, Shanghai, China) using Lipofectamine RNAiMAX (Thermo Fisher, CA, USA) when they reached 60e80% confluency. The cells were incubated for another 2 days at 37 C following transfection, after which total RNA was extracted. The siRNA sequences targeting RBM39 were as follows: RBM39-siRNA: GCAGCAATGGCAAACAATT. For the luciferase assays, the cell culture and transfection method with doses have been described previously [4]. 2.3. Immunoprecipitation and immunoblot analysis Cell lysates were prepared in lysis buffer (50 mM TriseHCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 10 mg/ml pepstatin A) containing 1% (v/v) Nonidet P-40. Soluble proteins were subjected to immunoprecipitation with anti-Flag
(Sigma, MO, USA), anti-Myc (Sigma, MO, USA), or anti-RBM39 (Bethyl Laboratories, AL, USA) antibodies. Normal rabbit or mouse IgG (Sigma, MO, USA) was used as a control. An aliquot of total lysate (5%, v/v) was also included as a control. Immunoblot analysis was performed with anti-Myc (Sigma, MO, USA), anti-Flag (Sigma, MO, USA), anti-c-Abl (Santa Cruz, CA, USA), anti-RBM39 (Bethyl Laboratories, AL, USA) and anti-P-Tyr (Upstate Biotechnology, CA, USA) antibodies. Antigen/antibody complexes were visualized with chemiluminescence (ECL, GE Healthcare, Buckinghamshire, UK). A protein marker (Thermo Fisher, CA, USA) was used as a molecular weight standard. 2.4. Protein-binding assays GST fusion proteins were generated by expressing pGEX4T-2based vectors in E. coli BL21 (DE3) and were purified by affinity chromatography using glutathione-Sepharose beads (Amersham Biosciences, NJ, USA). In GST pull-down experiments, cell lysates were incubated with 2 mg of purified GST, GST-c-Abl SH2, GST-c-Abl SH3 or GST-RBM39 immobilized on the beads (GE Healthcare, Buckinghamshire, UK) for 2 h at 4 C. The resulting protein complexes were washed with lysis buffer and then subjected to analysis using SDS-PAGE, followed by immunoblotting with anti-Flag antibody and Coomassie blue staining. 2.5. LC/MS/MS After the gel was stained with Coomassie Blue, the protein band was excised from the gel. Dried gel pieces were incubated with trypsin. The sample was desalted through pre-column desalination 5 min before the sample was injected. The peptide mixtures were extracted with acetonitrile/water (1:1, v/v) in a 0.1% formic acid solution. The peptides were then separated by gradient elution with a C18 capillary column. Nano Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (NanoUPLC-MS/MS) analysis was carried out on an electrospray ionization-quadrupoleoa-time-of-flight (ESI-quadrupole-oa-TOF) tandem mass spectrometer (SYNAPT G2-S, Waters, MA, USA). One full mass spectrometry (MS) scan (350e1500 Da) coupled to a secondary spectrum scan (50e2000 Da) was followed by MS/MS that automatically measured peptides using data-dependent analysis mode. We analyzed the mass spectrometry data in the original document using PLGS2.4 software (Waters, MA, USA). The PKL format of the data files was generated using MS spectrum, and we next searched the NCBInr database using Mascot software (Matrix Science, SC, Columbia). 2.6. Luciferase reporter assays MCF-7 cells were plated in 24-well plates and were cultured for 24 h in DMEM without phenol red supplemented with 5% charcoalstripped FBS in 5% CO2 before transfection. Cells grown in 24-well dishes to 60%e70% confluency were transiently transfected with 100 ng of the reporter vector along with 5 ng of the steroid receptor and the indicated amount of RBM39 and c-Abl using Lipofectamine 2000 (Thermo Fisher, CA, USA). An appropriate amount of empty expression vector was used in the assays without the cofactor, and a total of 1 mg/well of DNA was added [4]. Cells were then washed with phosphate-buffered saline (PBS) and were maintained in DMEM without phenol red plus 5% charcoal-stripped serum. The media were also supplemented with the appropriate ligand: 108 M Pg (Sigma, MO, USA), 109 M E2 (Sigma, MO, USA), or vehicle (ethanol) alone (Sigma, MO, USA) [4]. After an incubation time of 36 h, the cells were harvested, and the lysates were assayed for luciferase activity using the Dual Luciferase Reporter Assay
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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System (Promega, WI, USA) with a TD-20/20 spectrophotometer (Promega, WI, USA). The assay was performed according to the manufacturer’s instructions (Promega, WI, USA). All samples for the luciferase assays were normalized for Renilla activity using a cotransfected control expression vector, as described previously [18]. The results were expressed as the average ± SD of three independent experiments. 3. Results 3.1. c-Abl interacts with RBM39 Using yeast-two hybrid analysis, we previously noted that c-Abl interacts with RBM39 (data not shown). To substantiate the interaction between c-Abl and RBM39, lysates from wild-type MEFs were immunoprecipitated with the anti-RBM39 antibody and were probed for anti-c-Abl and anti-RBM39. Immunoblotting showed that c-Abl was present in the anti-RBM39 immunoprecipitate but was not pulled down with the rabbit IgG antibody in MEFs (Fig. 1A). To confirm the interaction between c-Abl and RBM39, 293T cells were co-transfected with plasmids expressing Flag-RBM39 and Myc-c-Abl. Anti-Myc immunoprecipitation with the anti-Flag antibody showed a significant interaction between Flag-RBM39
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and Myc-c-Abl (Fig. 1B). In a reciprocal experiment, Myc-c-Abl was found in the anti-Flag immunoprecipitates prepared from cells expressing Flag-RBM39 and Myc-c-Abl (Fig. 1C). As controls, Myc-c-Abl was not found in the anti-Flag immunoprecipitates in cells expressing Myc-Abl and the Flag vector (Fig. 1C). These findings indicated that c-Abl constitutively interacts with RBM39. To further characterize the interaction between c-Abl and RBM39, lysates from 293T cells were incubated with GST or GSTRBM39 fusion proteins. We demonstrated that c-Abl binds to GST-RBM39 but not to GST (Fig. 1D). To investigate the domain in cAbl that interacts with RBM39, Lysates from 239 cells expressing Flag-RBM39 were incubated with GST-c-Abl SH2, GST-c-Abl SH3 or GST conjugated agarose beads, and the absorbates were analyzed by immunoblotting with anti-Flag antibody. Our results showed that RBM39 bound to both the SH2 and SH3 domains of c-Abl (Fig. 1E). These findings confirmed that RBM39 interacts with the SH2 and SH3 domains of c-Abl. 3.2. RBM39 is phosphorylated by c-Abl The interaction between RBM39 and c-Abl suggests that RBM39 is a substrate for the tyrosine kinase. To demonstrate that RBM39 is phosphorylated by c-Abl, 293T cells were transfected with Flag-
Fig. 1. c-Abl interacts with RBM39. A. Lysates from mouse embryo fibroblast cells were subjected to immunoprecipitation with anti-RBM39 antibody or IgG, fractionated by SDSPAGE, and subsequently analyzed by immunoblotting with anti-c-Abl or anti-RBM39. B. 293T cells were co-transfected with Flag-RBM39 and Myc-c-Abl or Myc vector. The anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-Flag or anti-Myc antibody. C. Lysates prepared from 293T cells co-transfected with Myc-c-Abl and Flag-RBM39 or Flag vector were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody as above. D. Lysates from 293T cells transfected with Flag-c-Abl were incubated with GST or GST-RBM39 conjugated agarose beads for 2 h. The absorbates were analyzed by immunoblotting with anti-Flag (upper panel). Loading of the GST proteins was assessed by Coomassie Blue Staining (lower panel). E. Lysates from 293T cells transfected with Flag-RBM39 were incubated with GST-c-Abl SH2, GST-c-Abl SH3 or GST conjugated agarose beads for 2 h. The absorbates were analyzed by immunoblotting with anti-Flag (upper panel). Staining with Coomassie blue confirmed the presence of GST-c-Abl SH2, GST-c-Abl SH3 and GST fusion protein (lower panel).
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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RBM39, Myc-c-Abl, or kinase inactive Myc-c-Abl(K290R). Anti-Flag immunoprecipitates were analyzed by immunoblotting with an anti-phosphotyrosine antibody (P-Tyr). The results showed that RBM39 was phosphorylated by c-Abl but not by kinase inactive cAbl(K290R). In addition, phosphorylation was significantly suppressed by the c-Abl-selective inhibitor STI571 (Fig. 2A). Notably, RBM39 was phosphorylated by c-Abl but not by abl related gene Arg (Fig. 2B).
To identify the phosphorylation sites in RBM39, we digested Flag-RBM39, which was purified with the anti-Flag antibody from cells expressing Flag-RBM39 and Myc-c-Abl, with trypsin and analyzed the peptides by LC/MS/MS. The results suggested that Y95 and Y99 were phosphorylated (Fig. 2C). We further mutated Y95 and (or) Y99 to a similar amino-acid residue phenylalanine (F) and immunoblotted the anti-Flag immunoprecipitates with anti-P-Tyr antibody. The results showed that
Fig. 2. c-Abl phosphorylates RBM39. A. 293T cells were co-transfected with Flag-RBM39 and Myc-c-Abl, Myc-c-Abl(K290R) or Myc vector, and treated with or without STI571 for 24 h. The anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-PTyr or anti-Flag. B. Lysates from 293T cells co-transfected with Flag-RBM39 and Myc-Abl, MycArg or Myc vector were subjected to immunoprecipitation with anti-Flag and were subsequently analyzed by immunoblotting with anti-PTyr or anti-Flag. C. 293T cells were cotransfected with Flag-RBM39 and Myc-c-Abl, and the anti-Flag immunoprecipitates were fractionated by SDS-PAGE. The gel was stained by coomassive blue, and the band containing Flag-RBM39 was excised and subjected to LC/MS/MS analysis. Mass spectra of the YpRSPYpSGPK peptide was shown. Yp indicates a phospho-Tyr residue. D. 293T cells were cotransfected with myc-c-Abl and Flag-RBM39 or Flag-RBM39 mutants expressing plasmids. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr, antiFlag or anti-Myc. E. 293T cells were cotransfected with myc-c-Abl and Flag-RBM39 or Flag-RBM39 mutants expressing plasmids. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-P-Tyr or anti-Flag.
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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Fig. 3. c-Abl regulates the coactivation activity of RBM39 in steroid hormone receptor-mediated transcription. A. and B. 293T cells were co-transfected with ERE-Luc (or PRE-Luc), ERa (or PRb), RBM39 or RBM39 mutants and c-Abl. Cells were treated with or without E2 (or Pg) as indicated. 36 h later, cell lysates were subjected to the luciferase reporter assay. Average ± SD of 3 independent experiments were presented. C. and D. Cells were co-transfected with ERE-Luc (or PRE-Luc), ERa (or PR b), c-Abl and RBM39 or RBM39 RNAi. Cells were treated with or without E2 (or Pg) as indicated. 36 h later, cell lysates were subjected to the luciferase reporter assay. Average ± SD of 3 independent experiments were presented.
tyrosine phosphorylation was moderately decreased but not eliminated (Fig. 2D, E), which suggested that there were other tyrosine residues on RBM39 that were phosphorylated by c-Abl. The tyrosine residues on RBM39 were then individually mutated according to the characteristic YxxP motif of the c-Abl phosphorylation sites. Further mutation of Y475 and Y505 into F resulted in substantial abrogation of tyrosine phosphorylation (Fig. 2E). These results indicated that tyrosine phosphorylation of RBM39 primarily occurs on Y95 and Y99. Moreover, Y475 and Y505 may also be phosphorylated, although these results were not confirmed by LC/MS/MS. 3.3. c-Abl promotes the transcriptional coactivation activity of RBM39 for ERa and PRb RBM39 was previously shown to act as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-a and ESR2/ER-b [1]. The capability of the RBM39 cofactor to interact with both ERa and PRb and then stimulate ER- and PR-mediated transcriptional activity in mammalian cells was tested [4]. To investigate the effect of c-Abl mediated phosphorylation on RBM39, cells
were transfected with ERE-E1b-luciferase reporter (or PRE-E1bluc) with RBM39 or RBM39(Y95FY99F) in the presence of ERa (or PRb) and c-Abl. As expected, expression of luciferase directed by the promoter containing ER (or PR) responsive element was promoted by RBM39 when the cells were treated with E2 (or Pg), and RBM39(Y95FY99F) had a significant compressed effect compared to the wild-type (Fig. 3A and Fig. 3B), in the presence of c-Abl. These results showed that c-Abl mediated phosphorylation boasted the transcriptional coactivation activity of RBM39 on the ERa and PRb, and Y95, Y99 together with other phosphorylation sites contributed to the activation of RBM39 as a coactivator. Further, wild type (Scramble), RBM39 overexpressing or RBM39 knock down cells were transfected with ER-Luc (PR-Luc) and c-Abl or vector, c-Abl had a much higher effect in RBM39 overexpressing cells than in RBM39 knockdown cells (Fig. 3C and D), which suggested c-Abl regulated the expression of ER (or PR) responsive genes mainly (if not only) through RBM39 interaction. 4. Discussion RBM39 was previously identified as an auto-antigen in a
Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108
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hepatocellular carcinoma patient [1,2] and as an inhibitor of v-Relmediated lymphocyte transformation [3]. RBM39 was also shown to be a transcriptional co-activator of AP-1 and ERa [1,4]. A highly homologous protein to U2AF65, which is an essential SR-rich splicing factor, RBM39 contains a long RS domain and three RRMs. RBM39 shares notable sequence similarity with U2AF65 but lacks a U2AF ligand motif [5]. The functions of many proteins are influenced by their interactions with other proteins. The present study confirmed that RBM39 interacts with c-Abl, SH2 and SH3 domains of c-Abl can bind to RBM39. In addition, phosphorylation of RBM39 by the kinase has been demonstrated in cells. RBM39 was previously shown to act as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-a and ESR2/ER-b [1]. The ability of the RBM39 cofactor to interact with both ERa and PRb and stimulate ER- and PR-mediated transcriptional activity has been reported [4]. However, tyrosine phosphorylation has not been reported to participate in the regulation of RBM39 activity for transcriptional coactivation. Here we showed that Tyr95/99 and other sites were phosphorylated by c-Abl, and the phosphorylation enhanced the transcriptional coactivation activity of RBM39 for ERa and PRb transcriptional factors. Considered that approximately 80% of all breast cancers are ER-positive and approximately 65% of these breast cancers are also PR-positive, our report may provide a sight into the role of c-Abl in breast cancer. In addition to its role in transcriptional coactivation activity, RBM39 is similar to the RNA splicing factor U2AF65, which preferentially binds to the polypyrimidine tract immediately downstream of the branchepoint sequence at the 30 splice site and enhances the subsequent recruitment of the U2 small nuclear ribonucleoprotein to the branchpoint. RBM39 has been suggested to be a potential splicing factor because of the similar domain structures and sequences between U2AF65 and RBM39. Therefore, the identification of RBM39 as a transcriptional cofactor that stimulates transcription in a hormone-dependent and ligand-dependent manner and the presence of RRM and RS domains in RBM39 similar with the known splicing protein U2AF65 suggest that RBM39 can play a role in alternative splicing by recruiting specific cofactors to the promoter. In addition, previous reports have demonstrated that RBM39 directly interacts with transcription factors to stimulate transcription and mediate appropriate alternative splicing decisions in a promoter-dependent manner [4]. Further studies are necessary to determine whether c-Abl-mediated tyrosine phosphorylation of RBM39 contributes to the regulation of alternative splicing. Acknowledgments This work was supported by the National Natural Science Foundation of China [30871240, 31070674]. We thank American Journal Experts (AJE) for language help.
Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.03.108.
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Please cite this article in press as: S. Mai, et al., Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.108