Tobe K, Kadowaki T, Tamemoto H, Ueki K, Hara K, Koshio O, Momomura K, Gotoh Y, Nishida E, Akanuma Y
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
J Biol Chem. 1991 Dec 25;266(36):24793-803.
Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.
通过用从细胞外信号调节激酶1（ERK1）的部分cDNA预测的氨基酸序列衍生的化学合成肽免疫兔子，制备了两种位点特异性抗体，有人提出ERK1编码微管相关蛋白2（MAP2）激酶（Boulton，T.G.，Yancopoulos，G.D.，Gregory，J.S.，Slauer，C.，Moomaw，C.，Hsu，J.和Cobb，M.H.（1990）科学249，64 - 67）。在十二烷基硫酸钠（SDS）存在下进行免疫沉淀和蛋白质印迹分析，针对含有类似于胰岛素受体自身磷酸化位点之一的三个酪氨酸残基的肽的抗体（αY91）特异性识别42 kDa和44 kDa的蛋白质。另一方面，针对ERK1 cDNA基因产物的COOH末端部分（αC92）的肽的抗体识别44 kDa蛋白质比识别42 kDa蛋白质更有效。在不存在SDS的情况下进行免疫沉淀，αY91几乎不能识别这两种蛋白质，而αC92识别44 kDa蛋白质但不能识别42 kDa蛋白质。在SDS - 聚丙烯酰胺凝胶电泳后，在含髓磷脂碱性蛋白（MBP）的凝胶中进行激酶测定，结果显示胰岛素或12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）刺激的αY91免疫沉淀物中的MBP激酶活性在分子量42 kDa和44 kDa处共迁移。另一方面，αC92免疫沉淀物中受刺激的MBP激酶活性仅在分子量44 kDa处共迁移。胰岛素刺激凝胶中的MBP激酶活性以及这两种蛋白质的磷酸化超过10倍，在5分钟时达到最高水平。胰岛素和TPA通过从头苏氨酸和酪氨酸磷酸化快速刺激42 kDa和44 kDa蛋白质的磷酸化。分别对42 kDa和44 kDa蛋白质进行胰蛋白酶磷酸肽图谱分析，发现一个单一的主要磷酸肽含有磷酸苏氨酸和磷酸酪氨酸，这在胰岛素和TPA刺激的磷蛋白中是共同的。用蛋白磷酸酶2A处理这两种磷蛋白导致激酶活性完全丧失，并选择性地使磷酸苏氨酸去磷酸化。这些数据强烈表明这两种蛋白质与表观分子量为42 kDa的丝裂原活化蛋白（MAP）激酶高度相关（Ray，L.B.和Sturgill，T.W.（1988）美国国家科学院院刊85，3753 - 3757），并且这两种免疫相似但不同的MBP / MAP2激酶可能代表MBP / MAP2激酶的同工酶形式。这些数据还表明胰岛素和TPA通过非常相似的途径通过苏氨酸和酪氨酸残基的从头磷酸化激活MBP / MAP2激酶活性。
