Identifycation of MYB77 transcription factor as a novel substrate of MPK3/6 in Vitro - Nguyen Xuan Canh

TAP CHI SINH HOC 2015, 37(1se): 27-32 DOI: 10.15625/0866-7160/v37n1se. IDENTIFYCATION OF MYB77 TRANSCRIPTION FACTOR AS A NOVEL SUBSTRATE OF MPK3/6 IN VITRO Nguyen Xuan Canh*, Dang Xuan Nghiem, Pham Tuan Anh, Bui Manh Hung Vietnam National University of Agriculture, Ha Noi, *nxcanh@vnua.edu.vn ABSTRACT: Mitogen-activated protein kinase (MPK) cascades have emerged as a major signal transduction mechanism that connects diverse receptors or sensors to cellular and nuclear responses in eukaryotes. Although function and activation of some MPKs in response to stimuli have been studied in details, but substrates of these kinases were still poorly understood. Here, we provide that Arabidopsis MYB77 transcription factor, a R2R3-MYB protein, is a substrate of MPK3/6 in vitro. The MPK3, MPK6 and MYB77 proteins from Arabidopsis were expressed in E. coli. Using in vitro phosphorylation assays we have identified that MYB77 was phosphorylated by recombinant MPK3/6. To identify the phosphorylation sites of MYB77 by MPK, purified MYB77 protein was phosphorylated by MPK, desalted and passed over TiO2 for improved phosphopeptide detection. This sample has been used to analysis for mass spectrometry. Seven phosphopeptides of MYB77 contained predictive MPKs phosphorylation sites (S-P motif) were analyzed by mass spectrometry. As results we identified four putative phosphorylation sites originated from 7 phosphopeptide peaks. Our results suggest that MYB77 is a novel substrate of MPK3/6 containing four phosphorylation sites in Arabidopsis. Keywords: Arabidopsis, MYB77, MPK3/6, phosphorylation, substrate, transcription factor. INTRODUCTION Environmental stresses activate several mitogen-activated protein kinases including MPK3 and MPK6 in Arabidopsis [3, 10, 14]. The activated MPKs can phosphorylate target proteins to result in changes in the activities, interactions or stabilities of transcription factors and other signaling components [10]. To understand the mechanism of the MPK signaling pathway, the characterization of the upstream and downstream components is essential. Previously, some substrates of Arabidopsis MPKs were identified and characterized such as MKS1, ACS2/6, PHOS32/34, and EIN3 [1, 5, 6, 9, 15]. However, most of these potential substrates have not been extensively characterized at the molecular and biochemical levels. In Arabidopsis, the R2R3-type MYB transcription factors contain two imperfect repeats and comprise a 125-member gene family. The large number of MYB transcription factors is involved in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone response in plants [11, 13]. Although MYB transcription factors have not previously been implicated in auxin signaling, one previous report provided that the expression of some members of this family is upregulated by auxin. Recent research showed that AtMYB77 is involved in controlling lateral root growth and development through auxin signal transduction. Auxin induces MAPK activation in roots of Arabidopsis seedlings [12]. Moreover, a previous report suggests that a MAPK signaling cascade is activated during the adventitious rooting process induced by auxin [3]. Using 10 different activated MPKs to probe high-density protein microarrays, MYB77 has been determined as substrate of MPK10 [9]. Our results provide here that MYB77 is a substrate of MPKs containing six phosphorylation sites. These results suggest that MPKs may function to control lateral root growth and development through MYB77. MATERIALS AND METHODS Expression and purification of recombinant proteins in E. coli The full-length MPK3 and MPK6 cDNA were fused to Histidine (His) in pQE-30 expression vector to generate His-MPK3 and His-MPK6. MYB77 transcription factor cDNA was fused to Glutathione S-transferase (GST) in pGEX-5X-1 expression vector to generate GST-MYB77. All constructs were expressed in E. coli strain BL21 (for GST-fusion protein) or E. coli strain M15 (for His-fusion protein). The expression of recombinant proteins in bacteria was induced by 1 mM isopropylthio-β-galactoside at 30°C for 3h. For protein extraction, cells were collected by centrifugation and then sonicated in a lysis buffer (50 mM Tris-HCl, pH 7.5; 1.37 M NaCl; 27 mM KCl; 2 mM PMSF; 0.1% Triton X-100 for the GST-fusion protein and 50 mM NaH2PO4; 300 mM NaCl; 10 mM imidazole; 2 mM PMSF; 0.1% Triton X-100 for the His-fusion protein). The His-MPK3/6 and GST-MYB77 recombinant fusion proteins were purified by Ni-NTA agarose (Qiagen) and Glutathione Sepharose (GE Healthcare) according to the manufacturer’s instructions, respectively. Kinase assay The in vitro phosphorylation was performed in kinase buffer (25 mM Tris–HCl, pH 7.5, 1 mM DTT, 20 mM MgCl2, 2 mM MnCl2, 50 mM ATP). His-MPK3/4/6 fusion proteins (1 mg) were mixed with GST (1 mg), Myelin basic protein (MBP) (1 mg), GST-MYB77 (2 mg) in 20 ml of kinase reaction. GST and MBP proteins were used as negative and positive substrates, respectively. The reactions were initiated by adding 1 mCi [g32P] ATP and incubated at 30°C for 30 min. The reactions were stopped by boiling for 5 min and then loading to 12% SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250 and then analyzed by exposure to an autoradiograph film. Mass spectrometry Phosphorylated protein bands were cut out of the gel and then digested by modified trypsin (Promega). The phosphorylated peptides were purified by using TiO2 micro-columns. The tryptic peptides were dissolved in a buffer containing 80% acetonitrile and 5% trifluoroacetic acid, and then passed through a TiO2 microcolumn. The phosphopeptides were eluted with NH4OH (pH 10.5), purified by Poros Oligo R3 reversed-phase material (Applied Biosystems), and eluted using 2,5-dihydroxybenzoic acid solution (20 mg/ml in 50% acetonitrile/0.1% trifluoroacetic acid/1% ortho-phosphoric acid) [4, 7, 8]. MALDI-TOF analysis was performed using a Voyager-DE STR mass spectrometer (PerSeptive Biosystems Inc.). Mass spectra were obtained in the reflectron/delayed extraction mode. Monoisotopic peptide masses were analyzed using the MoverZ software [1]. RESULTS AND DISCUSSION Phosphorylation of MYB77 by MPK3 and MPK6 in vitro To investigate whether MYB77 is phosphorylated by MPK3/6, in vitro kinase assays were performed. Full-length of MYB77 transcription factor fused to glutathione S-transferase (GST) in pGEX5X-1 vector, full-length of MPK3/6 fused to His-tag (His) in pQE30 vector. Proteins were expressed in E. coli and purified on glutathionine-Sepharose for GST-MYB77 protein or Ni-NTA agarose for His-MPK3/6. Phosphorylation reactions were incubated in kinase buffer with [g32P]ATP at 30°C for 45 min. All steps were performed as described in methods. Reacted samples were fractionated through SDS-PAGE and subjected to autoradiography. GST and MBP (Myelin Basic Protein) were prepared as negative and positive control for MPKs substrates, respectively. As shown in Figure 1, His-MPK3/6 was autophosphorylated suggesting that the purified MPKs from E. coli have high kinase activity without any activations by activated MAP2Ks. Interestingly, GST-MYB77 as well as MBP proteins were phosphorylated by MPKs, but GST alone (~25 kDa) was not. (figure 1). These results collectively suggest that MYB77 is a substrate of MPK3/6 in vitro. Identifycation of MPKs phosphorylation sites of MYB77 protein The identification of the phosphorylation sites in kinase substrates is necessary for the verification of the phosphorylation and also for revealing the physiological function of the substrates’ phosphorylation. MPKs usually phosphorylate their substrates on serine or threonine residues that are followed by a proline residue (S/T-P motif). MYB77 protein obtained 301 residues, among them there are six ST sites and one TP site (figure 2). Figure 1. In vitro phospharylation of MYB77 by MPK3 (A) and MPK6 (B) One mg of the His-tagged MPKs (MPK3, MPK6) was mixed with 2 mg of GST–MYB77 fusion protein or 1 mg of GST or MBP in the kinase reaction buffer. After incubation at 30°C for 30 min, proteins were separated on 12% SDS-PAGE, stained with Coomassie brilliant blue (CBB-staining) and subjected to autoradiography (Autoradiograph). Protein molecular size markers are shown on the left. Positions of His-MPKs, GST-MYB77, GST and MBP are shown on the right. Figure 2. Amino acid sequence and domain structure of the MYB77 protein Two imperfect repeats (R1 and R2), each consisting of approximately 53 residues, exist in the N-terminal region. R1 and R2 domains are underlined. MYB77 protein also contained seven MPKs putative phosphorylation sites, which are serine or threonine followed by proline residues (S/T-P motif), that motifs indicated in yellow. Numbers represent residue positions. To map the phosphorylation sites of MYB77 by MPKs, GST-MYB77 protein was purified and phosphorylated by MPKs and analyzed by mass spectrometry. Phosphorylated and non-phosphorylated proteins were separated using gel electrophoresis. The proteins were excised from the gel and then digested with trypsin. The sequential elution from the IMAC phosphor-proteomics strategy was used to separate mono-phosphorylated peptides from multi-phosphorylated peptides. In this strategy, the IMAC flow through and washing solutions were collected and applied to TiO2 micro-columns for the enrichment of mono-phosphorylated peptides. The solutions eluted from the TiO2 micro-columns were diluted in formic acid and applied to Poros Oligo R3 desalting micro-columns. Finally, MALDI-TOF mass spectrometry was used to analyze the phosphorylated peptides. A total of five serine residues and one tyrosine residue became phosphorylated. These residues originated from seven phosphopeptide peaks not found in the non-phosphorylated control sample (figure 3 and table 1). Based on the mass spectrometry analysis, five serine (S) residues at positions 62, 148, 151, 165, 168 and tyrosine residues at position 279 of MYB77 were identified as putative phosphorylation sites of MPKs. Table 1. Phosphopeptides isolated from tryptic digestion of MYB77 after phosphorylated by MPKS. Amino acid residues that could potentially be phosphorylated by MPKs are in bold and underlines. Figure 3. MALDI-TOF mass spectrometry to identify MPKs phosphorylation sites on MYB77 A. MALDI-TOF mass spectrometry for peptide sequence SPEEDETIVTARAQF; B. MALDI-TOF mass spectrometry for peptide sequence RDCGVITPKVE; C. MALDI-TOF mass spectrometry for peptide sequence YMSPESPNGIDVSDSSTIPSPSSPVAQL; D. MALDI-TOF mass spectrometry for peptide sequence SAFAPVDTGLYMSPESPNGIDVSD. In this work, we have identified MYB77 as a novel substrate of MPK3 and MPK6. The role of MPK cascades in controlling hormones signaling and response to stresses have been documented in numerous studies. However, information of MPK substrates in plant regulating hormones is very restricted. Since the MYB77 plays important roles in lateral roots development as well as in modulation of auxin signal transduction, we think that it could act as a downstream regulator of auxin-related MPKs at least to MPK3 and MPK6. Consequently, we performed in vitro phosphorylation assays to test the whether MPK3/6 can phosphorylate MYB77. In accordance with hypothesis, recombinant MPK3/6 proteins strong phosphorylated GST-MYB77 protein as well as MBP, an artificial MPKs substrate (figure 1). In addition, MPKs generally phosphorylate their substrates on serine or threonine followed by proline residues (S/T-P motif). MYB77 protein contains six S-P motifs and one T-P site (figure 2). Among them serine at positions of 62, 148, 151, 165, 168 and tyrosine residue at position of 279 have been identified as putative MPK phosphorylation sites by MALDI-TOF mass spectrometry (figure 3 and table 1). Therefore, it is speculated that the activities of MYB77 may be regulated by the direct phosphorylation by MPKs. However, this possibility needs further investigation. Acknowledgements: This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106-NN.02-2013.30. A part of research was supported by Prof. Woo Sik Chung from Division of Life Science, Gyeongsang National University, Republic of Korea. REFERENCES Caspersen M. B., Qiu J. L., Zhang X., Andreasson E., Naested H., Mundy J., Svensson B., 2007. 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NGHIÊN CỨU XÁC ĐỊNH NHÂN TỐ PHIÊN MÃ MYB77 LÀ CƠ CHẤT CỦA ENZYM MPK3/6 TRONG ĐIỀU KIỆN IN VITRO Nguyễn Xuân Cảnh, Phạm Tuấn Anh, Đặng Xuân Nghiêm, Bùi Mạnh Hùng Học viện Nông nghiệp Việt Nam, Hà Nội TÓM TẮT Chuỗi các enzym thuộc nhóm Mitogen-activated protein kinase (MPK) được biết đến với vai trò hết sức quan trọng trong việc dẫn truyền tín hiệu trong tế bào sinh vật nhân thực. Có rất nhiều nghiên cứu phát hiện ra vai trò và chức năng của các enzym MPK trong việc đáp ứng lại với các yếu tố môi trường, tuy nhiên việc nghiên cứu phát hiện ra những cơ chất của nó tham gia vào quá trình này còn hạn chế. Trong nghiên cứu này chúng tôi cung cấp các bằng chứng để xác định được nhân tố phiên mã MYB77 thuộc họ protein MYB-R2R3 ở cây Arabidopsis là cơ chất của enzym MPK3 và MPK6 trong điều kiện in vitro. Các gen mã hóa cho protein MPK3, MPK6 và MYB77 được tách dòng từ cây Arabidopsis sau đó được biểu hiện trong vi khuẩn E. coli. Các protein tái tổ hợp sau đó được dùng cho phản ứng phosphoryl hóa sử dụng đồng vị phóng xạ P32, sau phản ứng chúng tôi nhận thấy protein MYB77 đã bị phosphoryl hóa cả bởi MPK3 và MPK6. Để cung cấp thêm thông tin về cơ chất MYB77 chúng tôi đã xác định các điểm phosphoryl hóa thông qua việc phân tích khối phổ. Protein MYB77 được phosphoryl hóa trong điều kiện in vitro bằng enzym MPK3 sử dụng nguồn phosphate (ATP) không đánh dấu phóng xạ. Protein này được phân cắt bằng enzym trypsin, các đoạn polipeptide sau đó được tinh sạch và làm giàu qua cột TiO2. Lượng polipeptide thu được sẽ được sử dụng cho phân tích khối phổ, kết quả xác định được 4 điểm phosphoryl hóa trong tổng số 7 đoạn polipeptide. Từ khóa: Arabidopsis, cơ chất, MYB77, MPK3/6, nhân tố phiên mã, phosphoryl hóa. Ngày nhận bài: 22-10-2014