A PHOSPHATIDYLINOSITOL-3 KINASE BINDS TO PLATELET-DERIVED GROWTH-FACTOR RECEPTORS THROUGH A SPECIFIC RECEPTOR SEQUENCE CONTAINING PHOSPHOTYROSINE
A PHOSPHATIDYLINOSITOL-3 KINASE BINDS TO PLATELET-DERIVED GROWTH-FACTOR RECEPTORS THROUGH A SPECIFIC RECEPTOR SEQUENCE CONTAINING PHOSPHOTYROSINE
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DOI：
10.1128/mcb.11.2.1125
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发表时间：
1991-02-01
影响因子：
5.3
通讯作者：
WILLIAMS, LT
中科院分区：
文献类型：
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作者：
ESCOBEDO, JA;KAPLAN, DR;WILLIAMS, LT
血小板衍生生长因子(PDGF)刺激PDGF受体的自磷酸化，并促进该受体与几个细胞质分子的结合，包括磷脂酰肌醇-3激酶(PI3激酶)。在这项研究中，我们在体外检测了PI3激酶与免疫沉淀自磷酸化的PDGF受体之间的关系。细胞裂解产物中的PI3激酶与野生型受体结合，但不与缺失了激酶插入区域的突变受体结合。一种表面大小为85 kDa的蛋白质与受体结合，这与之前的观察结果一致，即这种大小的蛋白质与PI3激酶活性有关。此外，110 kDa和74 kDa的蛋白质与磷酸化的受体结合。去磷酸化的受体失去了结合PI3激酶活性和85 kDa蛋白的能力。一个由20个氨基酸组成的多肽，由一个包含受体(酪氨酸719)和邻近酪氨酸(Y708)的自磷酸化位点的插入区域的序列组成，可阻断PI3激酶与受体的结合，但只有当多肽在酪氨酸残基上被磷酸化时才能阻止。即使当PI3蛋白在酪氨酸上被磷酸化时，杂乱的多肽也不能阻止PI3蛋白与受体的结合。这些酪氨酸磷酸化的多肽不会阻止磷脂酶C-伽马或GTP酶激活蛋白与受体的结合。在单独的实验中(受体印迹)，可溶性的放射性标记受体与存在于十二烷基硫酸钠中的85 kDa蛋白质结合-聚丙烯酰胺凝胶电泳-转移到硝酸纤维素纸上的分级3T3细胞裂解物。这种结合被阻止PI3激酶活性与固定化受体结合的相同酪氨酸磷酸化多肽所阻断。这些发现表明，PDGF受体通过激酶插入区的特定序列直接与85 kDa的蛋白和PI3激酶活性结合。110 kDa的蛋白与受体的结合也涉及这些序列，表明该蛋白可能是PI3激酶的提交。磷酸酪氨酸是这些蛋白质与PDGF受体相互作用所必需的基本结构。
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 involves these sequences, suggesting that this protein may be a submit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.