Hall F L, Braun R K, Mihara K, Fung Y K, Berndt N, Carbonaro-Hall D A, Vulliet P R
Division of Orthopaedic Surgery, Childrens Hospital of Los Angeles, California 90054-0700.
J Biol Chem. 1991 Sep 15;266(26):17430-40.
Site-specific analysis of tyrosine hydroxylase phosphorylation in rat pheochromocytoma led previously to the identification of a novel growth factor-sensitive serine/threonine protein kinase, designated proline-directed protein kinase (PDPK). In this article we describe further the activation, purification, subunit configuration, and biochemical characteristics of this cytoplasmic enzyme system. In human A431 epidermoid carcinoma cells PDPK activity was found to be stimulated by epidermal growth factor in a dose-dependent, time-dependent manner. The PDPK purified from the cytosol of mouse FM3A mammary carcinoma cells exhibited the same chromatographic behavior and biochemical properties as the tyrosine hydroxylase-associated enzyme purified originally from rat pheochromocytoma. The presence of p34cdc2 was ultimately detected in all active fractions of highly purified PDPK by Western blotting and immunoprecipitation; however, it was determined that this catalytic subunit is complexed with a 58-kDa regulatory subunit that is clearly distinct from that of the "growth-associated" M phase-specific histone H1 kinase (i.e. cyclin B). The 58 kDa regulatory subunit of PDPK was identified by direct immunoblotting as a mammalian A-type cyclin. Furthermore, the p58cyclin A subunit of PDPK was found to be phosphorylated on tyrosine residues in vivo and in vitro, the latter of which resulted in a significant increase in PDPK activity. Additional distinctions between this growth factor-sensitive PDPK (p34cdc2-p58cyclin A) and the M phase-specific histone H1 kinase (p34cdc2-p62cyclin B-p13suc1) are identified on the basis of chromatographic behavior, enzyme kinetics, and physicochemical properties. Based on these findings, it is proposed that PDPK represents a unique complex of the p34cdc2 protein kinase which is active in the cytoplasm of proliferative cells, is regulated differently from the M phase-specific histone H1 kinase by phosphorylation reactions, and is modulated selectively by growth factors.
先前对大鼠嗜铬细胞瘤中酪氨酸羟化酶磷酸化的位点特异性分析，鉴定出一种新型的生长因子敏感型丝氨酸/苏氨酸蛋白激酶，命名为脯氨酸定向蛋白激酶（PDPK）。在本文中，我们进一步描述了这种细胞质酶系统的激活、纯化、亚基结构和生化特性。在人A431表皮癌细胞中，发现PDPK活性受到表皮生长因子的剂量依赖性、时间依赖性刺激。从小鼠FM3A乳腺癌细胞胞质溶胶中纯化的PDPK，与最初从大鼠嗜铬细胞瘤中纯化的酪氨酸羟化酶相关酶表现出相同的色谱行为和生化特性。通过蛋白质印迹法和免疫沉淀法，最终在高度纯化的PDPK的所有活性组分中检测到p34cdc2的存在；然而，已确定该催化亚基与一个58 kDa的调节亚基复合，该调节亚基与“生长相关”的M期特异性组蛋白H1激酶（即细胞周期蛋白B）的调节亚基明显不同。通过直接免疫印迹法将PDPK的58 kDa调节亚基鉴定为哺乳动物A型细胞周期蛋白。此外，发现PDPK的p58细胞周期蛋白A亚基在体内和体外的酪氨酸残基上发生磷酸化，后者导致PDPK活性显著增加。基于色谱行为、酶动力学和物理化学性质，确定了这种生长因子敏感型PDPK（p34cdc2-p58细胞周期蛋白A）与M期特异性组蛋白H1激酶（p34cdc2-p62细胞周期蛋白B-p13suc1）之间的其他差异。基于这些发现，有人提出PDPK代表p34cdc2蛋白激酶的一种独特复合物，它在增殖细胞的细胞质中具有活性，通过磷酸化反应与M期特异性组蛋白H1激酶受到不同的调节，并受到生长因子的选择性调节。
