Corvera S, Roach P J, DePaoli-Roach A A, Czech M P
Department of Biochemistry, University of Massachusetts Medical Center, Worcester 01605.
J Biol Chem. 1988 Mar 5;263(7):3116-22.
Insulin caused a rapid, dose-dependent increase in the binding of 125I-insulin-like growth factor-II (IGF-II) to the surface of cultured H-35 hepatoma cells. The [32P]phosphate content of the IGF-II receptors, immunoprecipitated from extracts of H-35 cell monolayers previously incubated with [32P]phosphate for 24 h, was decreased after brief exposure of the cells to insulin. Analysis of tryptic digests of labeled IGF-II receptors by bidimensional peptide mapping revealed that the decrease in the content of [32P]phosphate occurred to varying degrees on three tryptic phosphopeptides. Thin layer electrophoresis of an acid hydrolysate of isolated IGF-II receptors revealed the presence of [32P] phosphoserine and [32P]phosphothreonine. Insulin treatment of cells caused a decrease in the labeled phosphoserine and phosphothreonine content of IGF-II receptors. The ability of a number of highly purified protein kinases (cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, and casein kinase II) to catalyze the phosphorylation of purified IGF-II receptors was examined. Casein kinase II was the only kinase capable of catalyzing the phosphorylation of the IGF-II receptor on serine and threonine residues under the conditions of our assay. Bidimensional peptide mapping revealed that the kinase catalyzed phosphorylation of the IGF-II receptor on a tryptic phosphopeptide which comigrated with the main tryptic phosphopeptide found in receptors obtained from cells labeled in vivo with [32P]phosphate. IGF-II receptors isolated by immunoadsorption from insulin-treated H-35 cells were phosphorylated in vitro by casein kinase II to a greater extent than the receptors isolated from control cells. Similarly, IGF-II receptors from plasma membranes obtained from insulin-treated adipocytes were phosphorylated by casein kinase II to a greater extent than the receptors from control adipocyte plasma membranes. Thus, the insulin-regulated phosphorylation sites on the IGF-II receptor appear to serve as substrates in vivo for casein kinase II or an enzyme with similar substrate specificity.
胰岛素导致125I-胰岛素样生长因子-II（IGF-II）与培养的H-35肝癌细胞表面的结合迅速且呈剂量依赖性增加。从先前用[32P]磷酸盐孵育24小时的H-35细胞单层提取物中免疫沉淀的IGF-II受体的[32P]磷酸盐含量，在细胞短暂暴露于胰岛素后降低。通过双向肽图谱分析标记的IGF-II受体的胰蛋白酶消化物表明，[32P]磷酸盐含量的降低在三种胰蛋白酶磷酸肽上程度不同。分离的IGF-II受体的酸水解产物的薄层电泳显示存在[32P]磷酸丝氨酸和[32P]磷酸苏氨酸。胰岛素处理细胞导致IGF-II受体的标记磷酸丝氨酸和磷酸苏氨酸含量降低。检测了多种高度纯化的蛋白激酶（cAMP依赖性蛋白激酶、蛋白激酶C、磷酸化酶激酶和酪蛋白激酶II）催化纯化的IGF-II受体磷酸化的能力。在我们的测定条件下，酪蛋白激酶II是唯一能够催化IGF-II受体丝氨酸和苏氨酸残基磷酸化的激酶。双向肽图谱显示，该激酶催化IGF-II受体在一种胰蛋白酶磷酸肽上的磷酸化，该磷酸肽与在用[32P]磷酸盐体内标记的细胞获得的受体中发现的主要胰蛋白酶磷酸肽共迁移。通过免疫吸附从胰岛素处理的H-35细胞中分离的IGF-II受体在体外被酪蛋白激酶II磷酸化的程度比从对照细胞中分离的受体更高。同样，从胰岛素处理的脂肪细胞获得的质膜中的IGF-II受体被酪蛋白激酶II磷酸化的程度比对照脂肪细胞质膜中的受体更高。因此，IGF-II受体上的胰岛素调节磷酸化位点在体内似乎作为酪蛋白激酶II或具有相似底物特异性的酶的底物。
