Churcher Y, Moss S E
Department of Physiology, University College London, UK.
Biochem J. 1995 Jun 15;308 ( Pt 3)(Pt 3):777-83. doi: 10.1042/bj3080777.
We have previously shown that, during interleukin-2-driven G1/S transition in activated human T-lymphoblasts, a restricted group of cellular proteins become tyrosine phosphorylated de novo, and that p56lck is the major active tyrosine kinase at this stage of the cell cycle. We now report that okadaic acid, a potent and specific inhibitor of protein phosphatases type 1 and type 2A, inhibits S-phase entry, and that this occurs with the simultaneous disappearance of a 56 kDa tyrosine phosphoprotein. We show that this protein is the lck tyrosine kinase and that okadaic acid stimulates a mobility shift to 59 and 64 kDa forms. These two forms of lck were found to have decreased autocatalytic activity, as judged by immune-complex kinase assays. Two-dimensional phosphopeptide mapping and phosphoamino-acid analyses revealed that, in the presence of okadaic acid, lck becomes phosphorylated mainly on serine and to a lesser extent threonine, and that phosphorylation occurs at novel sites. These results show that the kinase activity of lck is at least partly regulated by protein phosphatases, and suggest a role for lck in directing growth-factor-mediated DNA synthesis during T-cell proliferation.
我们先前已经表明，在白细胞介素-2驱动的活化人T淋巴母细胞从G1期向S期转变的过程中，一组特定的细胞蛋白会重新发生酪氨酸磷酸化，并且p56lck是细胞周期这一阶段主要的活性酪氨酸激酶。我们现在报告，冈田酸（一种强效且特异性的蛋白磷酸酶1型和2A型抑制剂）会抑制进入S期，并且在这种情况下，一种56 kDa的酪氨酸磷酸化蛋白会同时消失。我们表明这种蛋白就是lck酪氨酸激酶，并且冈田酸会促使其迁移率转变为59 kDa和64 kDa的形式。通过免疫复合物激酶分析判断，发现这两种形式的lck的自身催化活性降低。二维磷酸肽图谱分析和磷酸氨基酸分析表明，在存在冈田酸的情况下，lck主要在丝氨酸上磷酸化，苏氨酸磷酸化程度较低，并且磷酸化发生在新的位点。这些结果表明，lck的激酶活性至少部分受蛋白磷酸酶调节，并提示lck在T细胞增殖过程中指导生长因子介导的DNA合成中发挥作用。
