同源异型框蛋白Msx1的两个新的磷酸化位点的鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.031

生物技术通报 ›› 2016, Vol. 32 ›› Issue (6): 211-218.doi: 10.13560/j.cnki.biotech.bull.1985.2016.06.031

同源异型框蛋白Msx1的两个新的磷酸化位点的鉴定

程庆灵, 王敬强,

复旦大学遗传工程国家重点实验室，上海 200438

收稿日期:2015-10-27 出版日期:2016-06-27 发布日期:2016-06-28
作者简介:程庆灵，女，硕士，研究方向：Msx1翻译后修饰在发育中的分子作用；E-mail：13210700132@fudan.edu.cn
基金资助:
国家自然科学基金资助项目(31471230)，上海市浦江人才计划资助(14PJ1401300)

Identification of Two Novel Phosphorylated Sites of Homeoprotein Msx1

CHENG Qing-ling, WANG Jing-qiang

State Key Laboratory of Genetic Engineering，Fudan University，Shanghai 200438

Received:2015-10-27 Published:2016-06-27 Online:2016-06-28

摘要/Abstract

摘要： 同源异型框蛋白Msx1的同系物Msx2中已鉴定出磷酸化修饰位点，并且发挥着重要的生物学功能。但是对Msx1的磷酸化修饰情况还不清楚，为了对Msx1的磷酸化修饰情况进行研究。利用生物信息学的方法对Msx1的磷酸化修饰位点进行预测，进一步利用免疫沉淀和质谱技术对其进行具体实验验证。C2C12细胞中的Msx1蛋白复合物经胰蛋白酶消化后，通过高效液相色谱和质谱技术进行分离和分析。结果发现两个新的磷酸化修饰位点，分别是152位的丝氨酸和160位的丝氨酸，而且两个磷酸化位点在人和小鼠中是高度保守的。进一步，制备针对这两个磷酸化位点的特异性磷酸化抗体。磷酸化位点的鉴定以及特异性抗体的获得为进一步研究Msx1的生物学功能提供了良好的条件。

关键词: 同源异型框蛋白, Msx1, 磷酸化修饰, 质谱鉴定

Abstract: Some phosphorylated sites in the homolog Msx2 of the homeoprotein Msx1 has been identified，which plays a crucial biological role. However，the phosphorylated sites in Msx1 are not known yet，thus in order to examine the Msx1 phosphorylated status，we applied bioinformatics analysis to predict phosphorylation sites in Msx1，further used immunoprecipitation and mass spectrometry to experimentally verify the phosphorylation sites in Msx1. Tryptic digests of Msx1 protein complexes purified from C2C12 cells were separated and analyzed by nLC-MS-MS. Two novel phosphorylation sites(Ser152 and Ser160)were identified，moreover，these two sites were highly conserved in mouse and human. Furthermore，the specifically-phosphorylated antibody was prepared targeting these two phosphorylation sites.

Key words: homeoprotein, Msx1, phosphorylation, mass spectrometric identification

程庆灵, 王敬强,. 同源异型框蛋白Msx1的两个新的磷酸化位点的鉴定[J]. 生物技术通报, 2016, 32(6): 211-218.

CHENG Qing-ling, WANG Jing-qiang. Identification of Two Novel Phosphorylated Sites of Homeoprotein Msx1[J]. Biotechnology Bulletin, 2016, 32(6): 211-218.

参考文献

[1]Thingholm TE, Jensen ON, Larsen MR. Analytical strategies for Phosphoproteomics[J]. Proteomics, 2009, 9(6)：1451-1468.
[2]Duan JJ, Lozada AF, Gou CY, et al. Nicotine recruits glutamate receptors to postsynaptic sites[J]. Mol Cell Neurosci, 2015, 68：340-349.
[3]Linke D, Koudelka T, Becker A, Tholey A. Identification and relative quantification of phosphopeptides by a combination of multi-protease digestion and isobaric labeling[J]. Rapid Commun Mass Spectrom, 2015, 29(10)：919-926.
[4]Blazek M, Santisteban TS, Zengerle R, et al. Analysis of fast protein phosphorylation kinetics in single cells on a microfluidic chip[J]. Lab Chip, 2014, 15(3)：726-734.
[5]Luo R, Zhou C, Lin J, Yang D, et al. Identification of in vivo protein phosphorylation sites in human pathogen Schistosoma japonicum by a phosphoproteomic approach[J]. Proteomics, 2012, 75：868-877.
[6]Zhang B, Liu JY. Mass spectrometric identification of in vivo phosphorylation sites of differentially expressed proteins in elongating cotton fiber cells[J]. PLoS One, 2013, 8：e58758.
[7]Lelli KM, Noro B, Mann RS. Variable motif utilization in homeotic selector(Hox)-cofactor complex formation controls specificity[J]. Proc Natl Acad Sci USA, 2011, 108(52)：21122-21127.
[8]Dai J, Mou Z, Shen S, et al. Bioinformatic analysis of Msx1 and Msx2 involved in craniofacial development[J]. J Craniofac Surg, 2014, 25(1)：129-134.
[9]Zhang W, Qu HC, Zhang Y. Association of MSX1 and TGF-β1 genetic polymorphisms with hypodontia：meta-analysis[J]. Genet Mol Res, 2014, 13(4)：10007-10016.
[10]Nassif A, Senussi I, Meary F, et al. Msx1 role in craniofacial bone morphogenesis[J]. Bone, 2014, 66：96-104.
[11]Reddy NA, Adusumilli G, Devanna R, et al. MSX1 gene variant - its presence in tooth absence - a case control genetic study[J]. J Int Oral Health, 2013, 5(5)：20-26.
[12]Mundstock CA, Bortolini MC, Salzano FM, et al. MSX1 and PAX9 investigation in monozygotic twins with variable expression of tooth agenesis[J]. Twin Res Hum Genet, 2013, 16(6)：1112-1116.
[13]Souza LT1, Kowalski TW, Collares MV, et al. MSX1 gene and nonsyndromic oral clefts in a Southern Brazilian population[J]. Braz J Med Biol Res, 2013, 46(7)：555-558.
[14]Yilmaz A, Engeler R, Constantinescu S, et al. Ectopic expression of Msx2 in mammalian myotubes recapitulates aspects of amphibian muscle dedifferentiation[J]. Stem Cell Res, 2015, 15(3)：542-553.
[15]Seo YJ, Park JW, Kim YH, et al. Associations between the risk of tooth agenesis and single-nucleotide polymorphisms of MSX1 and PAX9 genes in nonsyndromic cleft patients[J]. Angle Orthod, 2013, 83(6)：1036-1042.
[16]Kim NY, Kim YH, Park JW, et al. Association between MSX1 SNPs and nonsyndromic cleft lip with or without cleft palate in the Korean population[J]. J Korean Med Sci, 2013, 28(4)：522-526.
[17]Xie H, Cherrington BD, Meadows JD, et al. Msx1 homeodomain protein represses the αGSU and GnRH receptor genes during gonadotrope development[J]. Mol Endocrinol, 2013, 27(3)：422-436.
[18]Rafighdoost H, Hashemi M, Narouei A, et al. Association between CDH1 and MSX1 gene polymorphisms and the risk of nonsyndromic cleft lip and/or cleft palate in a southeast Iranian population[J]. Cleft Palate Craniofac J, 2013, 50(5)：e98-e104.
[19]Wang J, Abate-Shen C. Transcriptional repression by the Msx1 homeoprotein is associated with global redistribution of the H3K27me3 repressive mark to the nuclear periphery[J]. Nucleus, 2012, 3(2)：155-161.
[20]Song YJ, Lee H. PIAS1 negatively regulates ubiquitination of Msx1 homeoprotein independent of its SUMO ligase activity[J]. Mol Cells, 2011, 32(3)：221-226.
[21]Wang J, Kumar RM, Biggs VJ, et al. The Msx1 homeoprotein recruits Polycomb to the nuclear periphery during development[J]. Dev Cell, 2011, 21(3)：575-588.
[22]Wang JQ, Abate-Shen C. The msx1 homeoprotein recruits G9a methyltransferase to repressed target genes in myoblast cells[J]. PLoS One, 2012, 7(5)：e37647.

同源异型框蛋白Msx1的两个新的磷酸化位点的鉴定

Identification of Two Novel Phosphorylated Sites of Homeoprotein Msx1

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 3

编辑推荐

Metrics

本文评价

[1]	梁星星, 王佳, 许文涛. 抗病毒核苷酸类似物磷酸化修饰研究进展[J]. 生物技术通报, 2022, 38(2): 218-226.
[2]	廖乐乐;娄世锋;曾翰庆;白凤霞;. 高纯度白介素-3与假单胞菌外毒素融合蛋白的表达、纯化及质谱鉴定[J]. , 2010, 0(06): 138-141.
[3]	刘建军;庄志雄;邓平建;赵锦;房师松;何建凡. 蛋白质组研究中的分析技术[J]. , 2002, 0(06): 23-26.