Rasmussen R K, Ji H, Eddes J S, Moritz R L, Reid G E, Simpson R J, Dorow D S
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
Electrophoresis. 1997 Mar-Apr;18(3-4):588-98. doi: 10.1002/elps.1150180342.
MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of approximately 31,500 and approximately 34,000, bound consistently to the MLK2N protein. To establish accurately the Mt / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (approximately 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI)-mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: http:(/)/ www.ludwig.edu.au/www/jpsl/jpslhome.htm l).
MLK2是蛋白激酶混合谱系激酶（MLK）家族的成员，由Dorow等人首次报道（《欧洲生物化学杂志》，1993年，213卷，701 - 710页），它包含几个不同的结构域，包括一个src同源3（SH3）结构域、一个激酶催化结构域、一个包含两个亮氨酸拉链基序的独特结构域、一个多碱性序列和一个cdc42/rac相互作用结合基序。在其他系统中，这些结构域中的每一个都已被证明在细胞信号转导调节中与特定类型的蛋白质相互作用相关。为了研究MLK2在招募特定底物中的作用，我们构建了一个重组cDNA，其编码MLK2的N端100个氨基酸（MLK2N），包括SH3结构域（第23 - 77位氨基酸），并与谷胱甘肽S - 转移酶融合。这种融合蛋白在大肠杆菌中表达，通过谷胱甘肽 - 琼脂糖亲和色谱法纯化，并用于亲和方法从35S标记的MDA - MB231人乳腺肿瘤细胞裂解物中分离MLK2 - SH3结构域结合蛋白。对结合蛋白的电泳分析表明，两种低丰度蛋白，分子量（Mr）约为31,500和约34,000，始终与MLK2N蛋白结合。为了准确确定这些MLK2 - SH3结构域结合蛋白的分子量/等电点（pI）位点，在二维电泳（2 - DE）主凝胶中鉴定了许多丰富的蛋白作为三角测量标记点。通过以下方法鉴定蛋白：（i）在电印迹到聚偏二氟乙烯（PVDF）膜上后进行直接埃德曼降解；（ii）对凝胶内蛋白酶解产生的肽进行埃德曼降解，并通过快速（约12分钟）微径柱（内径2.1 mm）反相高效液相色谱（HPLC）进行分离；（iii）质谱方法，包括肽质量指纹图谱和利用毛细管（内径0.2 - 0.3 mm）柱色谱的电喷雾（ESI）质谱（MS） - MS；或（iv）免疫印迹分析。利用这些信息，已经构建了一个用于人乳腺癌细胞系MDA - MB231的初步二维电泳蛋白质数据库，包含21种已鉴定的蛋白质，可通过万维网访问（网址：http://www.ludwig.edu.au/www/jpsl/jpslhome.htm l）。
