Protein phosphorylation is a key process in the transduction of hormonal signals and in the regulation of cellular metabolism. In addition, protein phosphorylation has been tightly associated with the control of gene expression and cell proliferation, although the details of this relationship have yet to be defined. To investigate the action of protein phosphorylation on nuclear events, we have focused our research on the modulation of gene expression by cAMP and phorbol myristate acetate (PMA), compounds which activate the cAMP-dependent protein kinase and the Ca2+-, phospholipid-dependent protein kinase, respectively. We selected the urokinase-type plasminogen activator (uPA) gene for detailed study because: 1) cAMP and PMA stimulate a rapid and substantial increase in uPA production in a number of cell types; and 2) uPA (one of two mammalian enzymes known which convert plasminogen to plasmin) is of considerable interest with respect to the role of the enzyme in tissue remodeling and cell migration. We have shown that cAMP and PMA increase cellular uPA production by activation of uPA gene transcription. To permit further detailed analysis of this process, we have isolated and sequenced the mouse uPA gene. In the proposed studies, we wish to define, in molecular terms, the features of the uPA gene critical for cAMP- and PMA-modulation and to explore the relationship of these sequences to protein kinases and putative regulatory proteins such as topoisomerases. To accomplish this we will employ uPA-mRNA and transcription assays currently used in this laboratory to assess the function and modulation of uPA gene-derivatives introduced into cultured cells. Regulatory sequences delineated in these studies will be compared to the sites of cAMP- and PMA-dependent changes in chromatin structure and topoisomerase interaction. The involvement of topoisomerase in cAMP- and PMA-modulation of gene activity will be explored by measuring the influence of topoisomerase inhibitors and by direct analysis of topoisomerase interaction with the uPA gene both in vitro and in vivo. The proposed studies will provide valuable insights into regulation of gene expression by cAMP and PMA, and may suggest general principles for the modulation of nuclear events by second messengers and protein phosphorylation. In addition, the general understanding of uPA gene expression developed during these studies may form the basis for precisely defining the role of uPA in processes such as invasive tumor growth and metastasis.
