Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.
Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.
复制标题
DOI：
10.1016/s0021-9258(18)54708-6
复制
发表时间：
1991-11
期刊：
影响因子：
--
通讯作者：
J. Tavaré;B. Zhang;L. Ellis;R. Roth
中科院分区：
文献类型：
--
作者：
J. Tavaré;B. Zhang;L. Ellis;R. Roth
胰岛素促进多种细胞中酪氨酸、丝氨酸和苏氨酸残基上的胰岛素受体β-亚基磷酸化，所述细胞包括在用编码野生型受体蛋白的cDNA转染后瞬时表达人胰岛素受体的猿猴COS细胞。为了检查丝氨酸1305和1306以及苏氨酸1348作为这些细胞中胰岛素刺激的磷酸化位点的潜在作用，将这些残基（即丝氨酸1305和1306或苏氨酸1348）用中性（丙氨酸）或带负电荷（天冬氨酸）氨基酸替换。在COS细胞中瞬时表达这些突变体受体后，二维磷酸肽图谱显示，苏氨酸1348是主要的，如果不是唯一的，胰岛素刺激的苏氨酸磷酸化位点。相比之下，虽然丝氨酸1305和/或1306以胰岛素依赖性方式磷酸化，但这些位点在这些细胞中仅包含小比例的胰岛素受体丝氨酸磷酸化。用中性或带负电荷的氨基酸取代丝氨酸1305和1306或苏氨酸1348对完整细胞中这些突变受体的胰岛素刺激的酪氨酸自磷酸化没有影响。此外，胰岛素刺激的外源性蛋白酪氨酸激酶活性的突变体受体是不受影响的，作为评估后，无论是在完整的细胞中的受体磷酸化或免疫纯化的受体和它们的自体磷酸化在体外。
Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.