Yu K T, Khalaf N, Czech M P
Department of Biochemistry, University of Massachusetts Medical School, Worcester 01605.
J Biol Chem. 1987 Dec 5;262(34):16677-85.
The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.
当使用肯普肽作为底物时，胰岛素处理的脂肪细胞的胞质部分显示蛋白激酶活性增加了2倍。胰岛素刺激的激酶活性的检测严重依赖于细胞匀浆缓冲液中磷酸酶抑制剂（如氟化物和钒酸盐）的存在。胞质蛋白激酶活性对胰岛素表现出高敏感性（ED50 = 2×10⁻¹⁰ M）和快速反应（2分钟后达到最大值）。对胞质激酶的动力学分析表明，胰岛素增加了肯普肽磷酸化的Vmax和ATP利用率，而不影响该酶对底物或核苷酸的亲和力。在阴离子交换柱和凝胶过滤柱上进行层析时，胰岛素刺激的胞质激酶活性与环磷酸腺苷依赖性蛋白激酶分离，并以单一峰形式迁移，表观分子量为50,000 - 60,000。部分纯化的激酶优先利用组蛋白、肯普肽、多功能钙调蛋白依赖性蛋白激酶底物肽、ATP柠檬酸裂解酶和乙酰辅酶A羧化酶作为底物，但不催化核糖体蛋白S6、酪蛋白、卵黄高磷蛋白、磷酸化酶b、糖原合酶、抑制剂II以及酪蛋白激酶II、蛋白激酶C和环磷酸鸟苷依赖性蛋白激酶的底物肽的磷酸化。对32P标记底物的磷酸氨基酸分析表明，胰岛素刺激的胞质激酶主要是丝氨酸特异性的。胰岛素激活的胞质激酶更喜欢Mn²⁺而不是Mg²⁺，并且不依赖于Ca²⁺。与核糖体蛋白S6激酶和蛋白酶激活的激酶II不同，胰岛素敏感的胞质激酶对氟化物不敏感。综上所述，这些结果表明一种新的胞质蛋白激酶活性被胰岛素激活。
