Escobedo J A, Kaplan D R, Kavanaugh W M, Turck C W, Williams L T
Cardiovascular Research Institute, University of California San Francisco 94143.
Mol Cell Biol. 1991 Feb;11(2):1125-32. doi: 10.1128/mcb.11.2.1125-1132.1991.
Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.
血小板衍生生长因子（PDGF）刺激PDGF受体的自身磷酸化以及该受体与几种细胞质分子的结合，其中包括磷脂酰肌醇-3激酶（PI3激酶）。在本研究中，我们在体外检测了PI3激酶与免疫沉淀的自身磷酸化PDGF受体的结合情况。来自细胞裂解物的PI3激酶与野生型受体结合，但不与缺失激酶插入区的突变型受体结合。一种表观大小为85 kDa的蛋白质与该受体结合，这与之前观察到的这种大小的蛋白质与PI3激酶活性相关的结果一致。此外，110 kDa和74 kDa的蛋白质也与磷酸化受体结合。去磷酸化的受体失去了结合PI3激酶活性以及85 kDa蛋白质的能力。由激酶插入区的一个序列组成的20个氨基酸的肽段，该序列包含受体的一个自身磷酸化位点（酪氨酸719）以及附近的一个酪氨酸（Y708），能阻断PI3激酶与受体的结合，但只有当该肽段在酪氨酸残基上被磷酸化时才行。该肽段的一个 scrambled 版本即使在酪氨酸上被磷酸化也不会阻断PI3激酶与受体的结合。这些酪氨酸磷酸化的肽段不会阻断磷脂酶C-γ或GTP酶激活蛋白与受体的结合。在单独的实验（受体印迹）中，可溶性放射性标记受体特异性地与存在于转移到硝酸纤维素纸上的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离的3T3细胞裂解物中的一种85 kDa蛋白质结合。这种结合被与阻止PI3激酶活性与固定化受体结合的相同酪氨酸磷酸化肽段所阻断。这些发现表明，PDGF受体通过激酶插入区中的特定序列直接与一种85 kDa蛋白质以及一种PI3激酶活性结合。一种110 kDa蛋白质与受体的结合也涉及这些序列，这表明该蛋白质可能是PI3激酶的一个亚基。磷酸酪氨酸是这些蛋白质与PDGF受体相互作用所需的必需结构。
