Research Plan: This proposal details experiments designed to determine the mechanisms involved in insulin- stimulated transcription of c-fos, by characterizing insulin's effects on the binding of transcriptional regulatory factors to the c-fos promoter. The sponsor's laboratory previously has shown that the serum response element (SRE) of the c-fos promoter is required for insulin-stimulated transcription of c-fos. It binds a number of nuclear proteins which include the serum response factor (SRF) and p62 Ternary Complex Factor (p62TCF). This laboratory has previously described a growth factor-inducible SRE/nuclear protein complex (Band 2) which appears to be the SRE/SRF/p62TCF complex. This complex is readily inducible by insulin in a variety of cell lines. In addition, a novel SRE/SRF complex binding protein (Band 3), also inducible by insulin and other growth factors, has recently been identified in the laboratory. In the specific aims section, experiments are proposed which will characterize the Band 3 complex. These experiments are designed to identity the Band 3 protein DNA binding site, and subsequently assess the effects of mutations which prevent Band 3 formation on insulin-induced c- fos transcription. A proposal for purification and cloning of the protein is detailed. The long range goal is to study insulin-induced modification of the Band 3 protein.
