Our objectives are to delineate the precise mechanism whereby nuclear cAMP-dependent protein kinase subunits (regulatory subunits RI and RII, catalytic subunit C) regulate the induction of specific gene products at the transcriptional level. The experimental systems in which these mechanisms will be probed are the cAMP stimulated rat C6 glioma cell in which cAMP causes transcriptional induction of LDH mRNA, and the rat H4IIE hepatoma cell line in which the induction of the phosphoenolpyruvate carboxykinase gene is regulated by cAMP and insulin. Preliminary studies indicate that the cAMP induction mechanism differs in the two cell lines with respect to cAMP-dependent protein kinase isozyme involvement. Specifically we propose to study (a) the effect of cAMP on the turnover of nuclear catalytic and regulatory subunits; (b) the structural modifications of chromatin caused by the subunits as phosphorylative modifications of histones and nonhistone nuclear proteins, and changes in the nuclease-sensitivity of chromatin; (c) since we have identified that the regulatory subunit RII possesses topoisomerase activity, we will define the significance of this finding by studying the effect of RII on chromatin structure (nuclease-sensitivity) and the transcriptional capacity of chromatin. Further studies deal with the regulation of RII topoisomerase activity in intact cells. Following cAMP and insulin stimulation, we will measure nuclear RII levels, nuclear RII topoisomerase activity and we will analyze the degree of phosphorylation, including phosphorylation sites, of RII. Finally, we propose to study the effect of the subunits on the structure and transcriptional capacity of minichromosomes reconstituted from cAMP-responsive structural gene sequences such as the LDH A subunit and phosphoenolpyruvate carboxykinase genes in a Xenopus oocyte extract and we will compare it with their effects on minichromosomes reconstituted with cAMP-unresponsive structural gene sequences. These studies will contribute significantly to our understanding of hormonally regulated gene expression during differentiation and development.
