Fry M J, Panayotou G, Dhand R, Ruiz-Larrea F, Gout I, Nguyen O, Courtneidge S A, Waterfield M D
Receptor Studies Group, Ludwig Institute for Cancer Research (Middlesex Branch), London, U.K.
Biochem J. 1992 Dec 1;288 ( Pt 2)(Pt 2):383-93. doi: 10.1042/bj2880383.
Specific phosphorylated tyrosine residues in the kinase insert region of the human platelet-derived-growth-factor beta-receptor mediate the formation of multienzyme complexes with this receptor. When phosphorylated, tyrosine residue 751 within the kinase insert region mediates binding of PtdIns 3-kinase to this receptor. A 17-amino-acid peptide containing this tyrosine residue was synthesized, phosphorylated by using epidermal-growth-factor receptor and then coupled to an Actigel matrix. The tyrosine-751 phosphopeptide column is used here as a final affinity step in the purification of the PtdIns 3-kinase from bovine brain to apparent homogeneity. The active resin-bound PtdIns 3-kinase is composed of two polypeptides, p110 and p85, which are elutable with SDS-containing buffers and detectable by silver staining of polyacrylamide gels. The 85 kDa protein is shown to be identical with the recently cloned p85 alpha. Phosphotyrosine is demonstrated to be an essential part of the structure required for binding of both of these proteins and PtdIns 3-kinase activity to this peptide. The active PtdIns 3-kinase complex from bovine brain, but not recombinant p85 subunits, shows specificity for binding to phosphopeptides containing a YXXM consensus sequence. Neither PtdIns 3-kinase activity, nor the complex of p85 and 110 kDa proteins, binds to several other phosphopeptide affinity columns lacking this sequence motif. The selectivity of binding of baculovirus-expressed free p85 alpha subunit of bovine brain PtdIns 3-kinase, the closely related protein p85 beta and purified bovine brain PtdIns 3-kinase to these and other phosphopeptide columns is examined.
人血小板衍生生长因子β受体激酶插入区中特定的磷酸化酪氨酸残基介导了与该受体多酶复合物的形成。激酶插入区内的酪氨酸残基751磷酸化后，介导磷脂酰肌醇3激酶（PtdIns 3-激酶）与该受体的结合。合成了一个含有此酪氨酸残基的17氨基酸肽段，用表皮生长因子受体进行磷酸化，然后偶联到Actigel基质上。在此，酪氨酸-751磷酸肽柱用作从牛脑中纯化PtdIns 3-激酶至表观均一性的最后一步亲和步骤。活性树脂结合型PtdIns 3-激酶由两种多肽p110和p85组成，它们可用含SDS的缓冲液洗脱，并可通过聚丙烯酰胺凝胶银染检测到。已证明85 kDa蛋白与最近克隆的p85α相同。磷酸酪氨酸被证明是这两种蛋白与PtdIns 3-激酶活性结合至该肽段所需结构的重要组成部分。来自牛脑的活性PtdIns 3-激酶复合物，但不是重组p85亚基，显示出对含有YXXM共有序列的磷酸肽结合的特异性。PtdIns 3-激酶活性以及p85和110 kDa蛋白的复合物均不与缺乏该序列基序的其他几种磷酸肽亲和柱结合。研究了杆状病毒表达的牛脑PtdIns 3-激酶游离p85α亚基、密切相关的蛋白p85β和纯化的牛脑PtdIns 3-激酶与这些及其他磷酸肽柱结合的选择性。
