Fujita-Yamaguchi Y, Sacks D B, McDonald J M, Sahal D, Kathuria S
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Biochem J. 1989 Nov 1;263(3):813-22. doi: 10.1042/bj2630813.
Since the studies on tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro suggested that protamine and poly(L-lysine) may activate phosphorylation of the receptor beta subunit [Sacks & McDonald (1988) J. Biol. Chem. 263, 2377-2383], we examined the effects of a variety of basic polycations/proteins and polyamines on insulin receptor kinase activity. The insulin receptor purified from human placental membranes was incubated with each basic polycation/protein or polyamine and assayed for tyrosine-specific protein kinase activity by measuring 32P incorporation into the src-related peptide. At a concentration of 1 microM, poly(L-lysine) and poly(L-ornithine) markedly stimulated kinase activity, whereas poly(L-arginine) and histones H1 and H2B inhibited insulin receptor kinase. In contrast, at a concentration of 1 mM, three polyamines (spermine, spermidine and putrescine) did not alter kinase activity. Poly(L-lysine) and poly(L-ornithine) stimulated the insulin receptor kinase by 5-10-fold at concentrations of 0.1-1 microM. Protamine sulphate also showed a significant stimulatory effect at a concentration of 100 microM. Preincubation of the receptor with poly(L-lysine) or poly(L-ornithine) for 20-60 min resulted in maximal kinase activation. Poly(L-lysine), the most effective activator of the receptor kinase, was used to characterize further the mechanisms of the kinase activation. Poly(L-lysine) activates the insulin receptor kinase by increasing the Vmax. without changing the Km. Poly(L-lysine) markedly stimulates the kinase activity of insulin receptor preparations that have lost both basal kinase activity and the ability to be stimulated by insulin. Insulin and poly(L-lysine) also differed in their ability to stimulate the kinase activity of prephosphorylated receptors. Prephosphorylation of the receptors did not affect the stimulation of the kinase by insulin. In contrast, prephosphorylation of receptors resulted in a markedly enhanced ability of poly(L-lysine) to stimulate kinase activity. These studies suggest that the mechanisms by which poly(L-lysine) and insulin activate the kinase are different. In conjunction with other additional evidence, it is suggested that poly(L-lysine) interacts directly with the beta-subunit of the receptor, thereby activating the receptor kinase.
由于体外胰岛素受体激酶对钙调蛋白酪氨酸磷酸化的研究表明，鱼精蛋白和聚（L-赖氨酸）可能会激活受体β亚基的磷酸化[Sacks & McDonald (1988) J. Biol. Chem. 263, 2377 - 2383]，我们研究了多种碱性聚阳离子/蛋白质和多胺对胰岛素受体激酶活性的影响。将从人胎盘膜中纯化的胰岛素受体与每种碱性聚阳离子/蛋白质或多胺一起孵育，并通过测量32P掺入src相关肽来检测酪氨酸特异性蛋白激酶活性。在1 microM的浓度下，聚（L-赖氨酸）和聚（L-鸟氨酸）显著刺激激酶活性，而聚（L-精氨酸）以及组蛋白H1和H2B则抑制胰岛素受体激酶活性。相比之下，在1 mM的浓度下，三种多胺（精胺、亚精胺和腐胺）不会改变激酶活性。聚（L-赖氨酸）和聚（L-鸟氨酸）在0.1 - 1 microM的浓度下将胰岛素受体激酶活性刺激了5 - 10倍。硫酸鱼精蛋白在100 microM的浓度下也显示出显著的刺激作用。将受体与聚（L-赖氨酸）或聚（L-鸟氨酸）预孵育20 - 60分钟可导致激酶活性最大程度激活。聚（L-赖氨酸）是受体激酶最有效的激活剂，被用于进一步表征激酶激活的机制。聚（L-赖氨酸）通过增加Vmax而不改变Km来激活胰岛素受体激酶。聚（L-赖氨酸）显著刺激已经丧失基础激酶活性以及被胰岛素刺激能力的胰岛素受体制剂的激酶活性。胰岛素和聚（L-赖氨酸）在刺激预磷酸化受体的激酶活性方面也存在差异。受体的预磷酸化不影响胰岛素对激酶的刺激。相反，受体的预磷酸化导致聚（L-赖氨酸）刺激激酶活性的能力显著增强。这些研究表明聚（L-赖氨酸）和胰岛素激活激酶的机制不同。结合其他额外证据，表明聚（L-赖氨酸）直接与受体的β亚基相互作用，从而激活受体激酶。
