THE EGF RECEPTOR: STRUCTURE, FUNCTION, AND HOMOLOGY

使用未降解的、生化均一的、纯化的EGF受体激酶 在A431细胞大约90%的产率中，我们已经确定了几个 A431细胞或组织提取物中存在的首选底物：34 kDa 蛋白质(pp60src的底物，125 kDa蛋白质，94/92 kDa双链 来自人类胎盘和80和105 kDa孕激素受体亚基。 94 kDa蛋白和孕激素受体蛋白被纯化为 EGF受体激酶均一，Km为10-7M。他们将会是 与其他酪氨酸残基特异性检测的特异性范围 激酶包括纯化的pp60src、PDGF受体和胰岛素受体。如果 检测到磷酸盐受体活性，指纹分析将 揭示与EGF受体相同或不同的位点的磷酸化 激活点。 94 kDa胎盘底物抗体将用于1)识别EGF 存在94 kDa蛋白的受体阳性细胞系，2)检测 细胞内蛋白质中的P-酪氨酸，以及3)表皮生长因子对细胞内蛋白质的影响。 94 kDa蛋白酪氨酸残基的磷酸化。如果双重本地化 在胞浆和胞核中，EGF对这些细胞的影响被证实。 分配情况将会确定。将尝试确定 94 kDa蛋白的生物学作用(S)，它与 已知的类固醇受体。94 kDa的主要磷酸盐受体位点 蛋白质(和孕激素受体亚单位)将被测序为 胰多肽，以及其他蛋白酶还发现的侧翼序列 将进行测序，以测试共性并解释高度亲和力 这些底物中的一种是EGF受体激酶。由于蛋白激酶C和 CAMP依赖的蛋白激酶参与EGF受体磷酸化， 被它们磷酸化的EGF受体将在 EGF受体激酶底物，确定可能的改变 底物特异性、亲和力和/或速率。补充研究： TPA无反应变体将使无细胞系统与细胞系统相关 系统调查结果。 我们的发现指向了一种高度发达的蛋白激酶反应矩阵 它在细胞质水平上整合了激素的作用。长期的 目标是用高度纯化的方法建立酶途径 准备，将这些发现与细胞生物学结果相关联 旨在确定以下各项监管后果的调查 特异性受体和受体-底物的磷酸化反应。

Using undegraded, biochemically homogeneous EGF receptor kinase, purified in approximately 90% yield from A431 cells, we have identified several preferred substrates present in A431 cells or tissues extracts: a 34 kDa protein (a substrate of pp60src, a 125 kDa protein, a 94/92 kDa doublet from human placenta and the 80 and 105 kDa progesterone receptor subunits. The 94 kDa protein and progesterone receptor proteins are purified to homogeneity and have Km's of 10-7M for EGF receptor kinase. They will be tested for range of specificity with other tyrosine residue specific kinases including purified pp60src, PDGF receptor and insulin receptor. If phosphate acceptor activity is detected, fingerprinting analysis will reveal phosphorylation at sites identical to or different from EGF receptor kinase sites. Antibodies to 94 kDa placental substrate will be used to 1) identify EGF receptor positive cell lines in which 94 kDa protein is present, 2) test for P-tyrosine in the protein in cells, and 3) test effects of EGF on tyrosine residue phosphorylation in 94 kDa protein. If dual localization in cytosol and nucleus is confirmed, the effects of EGF on such distribution will be determined. Attempts will be made to identify the biological role(s) of 94 kDa protein, which shares many properties with known steroid receptors. The major phosphate acceptor site on 94 kDa protein (and progesterone receptor subunits) will be sequenced as the tryptic peptide, and flanking sequences revealed by other proteases also will be sequenced to test for commonality and explain the high affinities of these substrates for EGF recptor kinase. Since protein kinase C and cAMP-dependent protein kinase participate in EGF receptor phosphorylation, EGF receptors phosphorylated by them will be tested for kinase activity on EGF receptor kinase substrates, determining possible alterations in substrate specificity, affinity and/or rate. Complementary studies with TPA nonresponding variants will correlate cell-free system with cellular system findings. Our findings point to a highly developed matrix of protein kinase reactions which integrate hormone action at the cytoplasmic level. The long-term goal is establishment of the enzymic pathways using highly purified preparations, correlating such findings with results from cell biological investigations directed at identifying the regulatory consequences of specific receptor and receptor-substrate phosphorylation reactions.