Brautigan D L, Brown M, Grindrod S, Chinigo G, Kruszewski A, Lukasik S M, Bushweller J H, Horal M, Keller S, Tamura S, Heimark D B, Price J, Larner A N, Larner J
Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Biochemistry. 2005 Aug 23;44(33):11067-73. doi: 10.1021/bi0508845.
Insulin-stimulated glucose disposal in skeletal muscle proceeds predominantly through a nonoxidative pathway with glycogen synthase as a rate-limiting enzyme, yet the mechanisms for insulin activation of glycogen synthase are not understood despite years of investigation. Isolation of putative insulin second messengers from beef liver yielded a pseudo-disaccharide consisting of pinitol (3-O-methyl-d-chiro-inositol) beta-1,4 linked to galactosamine chelated with Mn(2+) (called INS2). Here we show that chemically synthesized INS2 has biological activity that significantly enhances insulin reduction of hyperglycemia in streptozotocin diabetic rats. We used computer modeling to dock INS2 onto the known three-dimensional crystal structure of protein phosphatase 2C (PP2C). Modeling and FlexX/CScore energy minimization predicted a unique favorable site on PP2C for INS2 in a surface cleft adjacent to the catalytic center. Binding of INS2 is predicted to involve formation of multiple H-bonds, including one with residue Asp163. Wild-type PP2C activity assayed with a phosphopeptide substrate was potently stimulated in a dose-dependent manner by INS2. In contrast, the D163A mutant of PP2C was not activated by INS2. The D163A mutant and wild-type PP2C in the absence of INS2 had the same Mn(2+)-dependent phosphatase activity with p-nitrophenyl phosphate as a substrate, showing that this mutation did not disrupt the catalytic site. We propose that INS2 allosterically activates PP2C, fulfilling the role of a putative mediator mimetic of insulin signaling to promote protein dephosphorylation and metabolic responses.
胰岛素刺激的骨骼肌葡萄糖处置主要通过以糖原合酶为限速酶的非氧化途径进行，然而尽管经过多年研究，胰岛素激活糖原合酶的机制仍不清楚。从牛肝中分离出的假定胰岛素第二信使产生了一种假二糖，它由与螯合有Mn(2+)的半乳糖胺以β-1,4连接的皮诺醇（3-O-甲基-D-手性肌醇）组成（称为INS2）。在这里我们表明，化学合成的INS2具有生物活性，可显著增强链脲佐菌素糖尿病大鼠中胰岛素降低高血糖的作用。我们使用计算机建模将INS2对接至蛋白磷酸酶2C（PP2C）已知的三维晶体结构上。建模和FlexX/CScore能量最小化预测了PP2C上一个与催化中心相邻的表面裂隙中INS2的独特有利位点。预计INS2的结合涉及形成多个氢键，包括与残基Asp163形成的一个氢键。用磷酸肽底物测定的野生型PP2C活性受到INS2的剂量依赖性强烈刺激。相比之下，PP2C的D163A突变体未被INS2激活。在不存在INS2的情况下，D163A突变体和野生型PP2C对以对硝基苯磷酸为底物具有相同的Mn(2+)依赖性磷酸酶活性，表明该突变未破坏催化位点。我们提出INS2通过变构激活PP2C，履行假定的胰岛素信号转导介质模拟物的作用，以促进蛋白质去磷酸化和代谢反应。
