Kato K, Ito H, Kamei K, Inaguma Y, Iwamoto I, Saga S
Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan.
J Biol Chem. 1998 Oct 23;273(43):28346-54. doi: 10.1074/jbc.273.43.28346.
The immunofluorescence localization of alphaB-crystallin in U373 MG human glioma cells with an antibody specific for alphaB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of alphaB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of alphaB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for alphaB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alphaB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in alphaB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.
用针对在丝氨酸45位点磷酸化的αB-晶体蛋白的特异性抗体，对U373 MG人胶质瘤细胞中的αB-晶体蛋白进行免疫荧光定位，结果显示在细胞周期的有丝分裂期细胞有强烈染色。在所有检测的细胞系以及小鼠胚胎组织切片中均检测到有丝分裂细胞中αB-晶体蛋白的磷酸化形式。通过等电聚焦或SDS-聚丙烯酰胺凝胶电泳以及随后对有丝分裂期收集的细胞提取物进行的蛋白质印迹分析，生化检测到丝氨酸45和丝氨酸19位点磷酸化的αB-晶体蛋白水平增加，但丝氨酸59位点磷酸化的αB-晶体蛋白水平未增加。当我们使用源自未磷酸化的αB2-晶体蛋白的氨基末端72个氨基酸的肽作为底物，估计有丝分裂的U373 MG细胞提取物中αB-晶体蛋白的磷酸化活性时，我们发现负责丝氨酸45和丝氨酸19磷酸化的活性显著增强，但负责丝氨酸59磷酸化的活性受到抑制。在存在花萼海绵诱癌素A的情况下，通过暴露于H2O2刺激的细胞提取物中，部分纯化了负责氨基末端72个氨基酸肽中丝氨酸45和丝氨酸59磷酸化的蛋白激酶。负责丝氨酸45和丝氨酸59磷酸化的活性分别在Superdex 200柱上对应于约40 kDa和60 kDa的级分中洗脱，而负责丝氨酸19磷酸化的激酶不稳定。p44/42丝裂原活化蛋白（MAP）激酶和MAP激酶活化蛋白（MAPKAP）激酶-2分别集中在丝氨酸45激酶级分和丝氨酸59激酶级分中。从兔肌肉中纯化的重组人p44 MAP激酶和MAPKAP激酶-2分别选择性地磷酸化丝氨酸45和丝氨酸59。丝氨酸45激酶级分和丝氨酸59激酶级分分别磷酸化髓鞘碱性蛋白和hsp27。这些结果表明，细胞中αB-晶体蛋白丝氨酸45和丝氨酸59的磷酸化分别由p44/42 MAP激酶和MAPKAP激酶-2催化，并且在细胞分裂期间，p44/42 MAP激酶对丝氨酸45的磷酸化增强，而MAPKAP激酶-2对丝氨酸59的磷酸化受到抑制。
