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.
