The goal of this study is to isolate and characterize substrates specific for the protein product of the src-related proto-oncogene, c-fgr. c-fgr substrates are hypothesized to be exclusively found in cells of the myeloid lineage, especially in the late monocyte/macrophage stage since normal c- fgr expression is restricted to these cells. One myeloid cell line in particular, 9.1.1, has several characteristics of a monocyte but surprisingly does not express any c-fgr. We will take advantage of this phenotype by introducing a c-fgr expression vector into 9.1.1 in the expectation that c-fgr substrates will now be phosphorylated. Newly phosphorylated proteins will be detected by comparison of the pattern of phosphotyrosine-containing proteins in the cells expressing c-fgr with the parental cell line by Western blot analysis using an anti-phosphotyrosine antibody. To further substantiate that a substrate is specifically phosphorylated by c-fgr, another src-related gene that is primarily expressed in myeloid cells, c-hck, will be separately introduced into 9.1.1. A subset of phosphotyrosine-containing proteins should be detected in 9.1.1 expressing c-fgr but should not be evident in 9.1.1 overexpressing c-hck nor in 9.1.1 alone. Tentative substrates that meet additional criteria predicted for c-fgr specific substrates will be purified to obtain partial amino acid sequence which will be used for isolation of the complementary DNA (cDNA) for the gene that encodes each putative substrate. The sequence of each cDNA will be determined in hopes that the identity or function of each protein will be elucidated. Because of the close homology between c-fgr and the other src-related genes, it is hoped that identification of c-fgr specific substrates will suggest what the particular substrates of other members of the family might be. Also, determination of the normal cellular role of these proto- oncogenes may discern how mutated forms are involved in inducing certain cancers.
