Insulin exerts powerful influences on the biosynthesis of many enzymes and other proteins as part of its normal role in maintaining body fuel homeostasis. In some cases, this effect has been localized to changes in the transcription rates of certain genes. In one such case, insulin rapidly stimulated the transcription of the c-fos proto-oncogene within 10 min of hormone exposure. This proto-oncogene encodes a nuclear phosphoprotein that may serve as a trans-acting regulator of the expression of other genes. In this way, the induction of c-fos by insulin might be the first or an early step in a sequential series of biosynthetic or transcriptional responses to insulin. A sequence within the 5'-flanking region of the c-fos gene which is necessary for induction of the gene by insulin and active tumor- promoting phorbol esters has recently been identified. This region was previously identified as being responsible for serum induction of c-fos in fibroblasts, and has been labelled the serum response element (SRE). Four point mutations within the SRE abolished the ability of insulin and phorbol esters to induce the transfected gene, and also abolished the specific binding of a nuclear protein which bound to the normal SRE. The overall goals of the proposed studies are to determine the molecular steps which are involved in the induction of c-fos by insulin, and perhaps other agents, such as phorbol ester, which induce the gene by a different proximal mechanism. To do this, the SRE binding protein will be purified from an insulin-responsive tissue such as rat liver, in which c-fos is induced during liver regeneration. The purified protein will be used to immunize rabbits and mice for the production of antibodies, and proteolytic peptides will be sequenced; results of these studies will be used in the molecular cloning of a cDNA for the protein. The antibodies and the specific localization of the protein on two dimensional gels will be used to determine whether insulin and other stimuli modify the protein in any way, either directly or by altering its association with other proteins. Further studies will determine whether these modifications affect c-fos transcription. Other insulin-responsive regions of the gene will also be sought, and similar techniques applied to the search for other insulin-modified proteins which might interact with these sequences. The ultimate aim of these studies is eventually to link activation of the insulin receptor with this early transcriptional response by a known series of biochemical reactions.
