| Abscisic acid (ABA), one of the most important phytohormones, plays an important role as a signaling molecule in plant growth, development and adaptation to the environment. Mitogen-activated protein kinase (MAPK) signaling is prevalent among eukaryotic cells, which is induced by stresses, cytokines, plant hormones, growth factors and so on, playing a vital role in plant stress signal. Many studies have shown that ABA signal transduction is associated with the MAPK cascade pathway. MAPK is one of crucial downstream components in the signal transduction pathway of ABA. Therefore, it is important to identify MAPKs in ABA signal transduction and networks to reveal the cellular and molecular mechanism of ABA signalIn previous studies, our lab reported that a 46 kDa MAPK (p46MAPK) was involved in ABA-induced antioxidant defense in leaves of maize (Zea mays) plants (Zhang et al.,2006 Plant physiology; 2007 New phytologist). However, the identity of the p46MAPK in maize is not clear, and the alignment to other MAPKs is not known. It is important to reveal the identity and characterization for further research at molecular level. Firstly, we isolated and purified p46MAPK. Secondly, the identity of p46MAPK was identified by MS and bioinformatics assay. Lastly, the characterization of p46MAPK was studied.1. Purification of p46MAPK. In this study, partial purification of p46 MAPK was performed by monitoring its activity with an in-gel kinase assay and in-solution kinase assay using myelin basic protein (MBP), which is the best fit substrate for MAPKs. All the steps were carried out at 4℃unless stated otherwise. All chromatographic runs were carried out on an AKTA Purifier 100 system (GE Healthcare) and AKTA Prime System (GE Healthcare). The p46MAPK induced by ABA was partially purified by the 30%(NH4)2SO4 precipitation followed by 100,000g ultracentrifugation and chromatography on 40 ml Q-Sepharose FF,15 ml Phenyl-Sepharose FF,6 ml Resource Q,1 ml Mono QTM 5/50 GL, 3.5 ml Poly-L-lysine-agarose, and 120 ml Superdex 75 prep grade columns. After SDS-PAGE analysis and sliver staining, the enzymes from Superdex 75 prep grade column showed Only 45.4 kDa protein band is near to molecular weight of p46MAPK. And only one band of kinase activity was detected by in-gel kinase assays with MBP as a substrate. These results suggest that this polypeptide was the 46 kDa ABA-activated MAPK. We also purified the kinase from the ABA-treated maize leaves and water treated maize leaves at the same time, which further determined the purified kinase was the ABA-induced p46MAPK. Taking the activity at the first purification step as 100%, the kinase was purified more than 10,000 fold, with an overall yield of 1.1. The specific activity of the kinase was 20,420 pmol min mg-1.2. Identification of p46MAPK. Development in mass spectrometry (MS) technology has dramatically accelerated the application of proteomics in recent years. Biological mass spectrometry has become one of the most important technology platforms for protein identification. It is an important part of continuing study on isolated and purified protein. The partial purified protein was excised and sent to the National Center of Biomedical Analysis, Academy of Military Medical Sciences (Beijing, China) for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF-MS) analyses. Proteins were identified using MS/MS ion search of Mascot search engine (http://www.matrixscience.com, Matrix Science, London, England) and Viridiplantae (Green Plants) protein database (NCBI,20071116). It was identified that the p46MAPK was ZmMPK5 (gi|4239889) in maize. The search yielded a top score of 207 (protein scores greater than 69 are significant; P<0.05). Furthermore, the selected tryptic peptide (m/z 1779.841) sequenced by MS/MS revealed an amino acid sequence of TTSETDFMTEYVVTR, corresponding to residues 218-232 of ZmMPK5. In order to further confirm the result, the polyclonal antibody that recognizes the C-terminal region of ZmMPK5 was raised in rabbits, and the immunoprecipitation kinase assay confirmed the results. These results clearly indicate that the ABA-activated p46MAPK is ZmMPK5. 3. Bioinformatics assay of ZmMPK5. The related characterizations of ZmMPK5 is analyzed by bioinformatics assay, including molecular weight, isoelectric point, protein subcellular localization, alignment and phylogenetic trees analysis of the ZmMPK5 with other homologous MAPKs. The nominal mass of the kinase and isoelectric point are 44.9 kD and 5.39, respectively. ZmMPK5 is localized in the nucleus by SubLoc v1.0 database. Based on the sequence alignment a phylogenetic tree was constructed, which indicates that ZmMPK5 can be grouped into subgroup A, and ZmMPK5 is most homologous to OsMPK6, AtMPK6, NtSIPK and StMPK1. Furthermore, the similarity with the function of the protein known before is analyzed, and its function is also speculated.4. Characterization of ZmMPK5. Using of biochemistry, pharmacology, cell biology and other experimental methods, we studied the related characterization of p46MAPK (ZmMPK5). The results show that the molecular mass of the kinase is found to be 45.74 kDa by gel-filtration. The kinase showes activity in the range of 20 to 50℃in temperature, 2.5 to 15 mM MgCl2, and broad pH 5.0 to 9.0, with optimal activity at pH 8.0,35℃, and 10 mM MgCl2. For this kinase, the Km for myelin basic protein (MBP) substrate and ATP are 0.13μgμL-1 and 23μM, respectively. MBP is the preferred substrate, of which the threonine residue is phosphorylated. Immunoprecipitation gel kinase analysis further confirm that ABA-and H2O2-induced 46 kD MAPK is ZmMPK5.100μM SA,100μM ETH,10% PEG, 250 mM NaCl,500μM CdCl2, low temperature, wounding and UV are able to mediate the expression of ZmMPK5 and activation of ZmMPK5.In ZmMPK5:YFP-expressing Zea mays protoplasts, it was localized predominantly in the nucleus. Successful identification and related analysis of the nature of p46MAPK enable us to further define the function of the ZmMPK5 in ABA signal.
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