THE EGF RECEPTOR: STRUCTURE, FUNCTION, AND HOMOLOGY

使用未降解的，生物化学均质的EGF受体激酶，纯化 在A431细胞的约90%产率中，我们已经鉴定了几种 A431细胞或组织提取物中存在的优选底物： 蛋白质（pp 60 src的底物，125 kDa蛋白质，94/92 kDa双联体 来自人胎盘和80和105 kDa孕酮受体亚单位。 纯化94 kDa蛋白和孕酮受体蛋白， 对EGF受体激酶Km为10- 7 M。 他们将 检测其他酪氨酸残基特异性的特异性范围 激酶包括纯化的pp 60 src、PDGF受体和胰岛素受体。 如果 检测到磷酸受体活性，指纹分析将 显示在与EGF受体相同或不同的位点磷酸化 激酶位点。 抗94 kDa胎盘基质的抗体将用于1）鉴定EGF 受体阳性细胞系，其中存在94 kDa蛋白，2）测试 3）EGF对细胞内蛋白质中P-酪氨酸的影响， 酪氨酸残基磷酸化的94 kDa蛋白质。 如果对偶定位 在细胞质和细胞核被证实，EGF对这种影响， 分配将确定。 将努力查明 94 kDa蛋白质的生物学作用，它与 已知的类固醇受体 94 kDa上的主要磷酸受体位点 蛋白质（和孕酮受体亚基）将被测序， 胰蛋白酶肽和侧翼序列揭示的其他蛋白酶也 将被测序以测试共性并解释高亲和力 EGF受体激酶的底物。 由于蛋白激酶C和 cAMP依赖性蛋白激酶参与EGF受体磷酸化， EGF受体的磷酸化将被测试激酶活性， 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无应答变体将无细胞系统与细胞免疫系统相关联。 系统发现。 我们的发现指向一个高度发达的蛋白激酶反应矩阵 其在细胞质水平整合激素作用。 长期 目的是建立酶途径，使用高度纯化的 制备，将这些发现与细胞生物学结果相关联， 调查旨在确定监管后果， 特异性受体和受体-底物磷酸化反应。

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.