The nitric oxide (NO)cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction pathway is important for the regulation of many physiological and pathophysiological processes, e.g., in the cardiovascular system it is a major determinant of smooth muscle cell, endothelial cell and platelet functions and is implicated in the development of hypertension and atherosclerosis. However, compared to cAMP-dependent protein kinase, little is known about the downstream effects of G-kinase activation. The investigators found that G-kinase regulates gene expression: in response to NO or cGMP, the kinase translocates to the nucleus, induces phosphorylation of CREB-related proteins and activates the fos, junB and TNF-alpha promoters. Preliminary data suggest that G-kinase transactivates these promoters through similar cis-acting elements recognized by transcription factors of the AP-1 (Fos-Jun), CRE- and CCAAT-enhancer binding protein families and that subcellular localization and biological activity of the kinase is regulated by specific anchoring proteins. The speciic aims of this proposal are: (1) to define DNA sequences of NO/cGMP-response elements (NGREs): (II) to identify transcription factors targeted by NO/cGMP/G-kinase; and (III) to identify key substrates and cytoplasmic or nuclear anchoring proteins which bind to G-kinase. NGREs will be defined by deletion and site-directed mutagenesis of putative enhancer elements in the fos and TNF-alpha promoters. Transcription factors targeted by NO/cGMP will be identified in transactivation studies using Ga14-fusion products and dominant negative mutants of candidate transcription factors as well as in DNA binding studies and DNA affinity chromatography using oligodeoxynucleotides encoding NGREs. Protein interaction cloning will be employed to identify proteins interacting with G-kinase and the effect of these proteins on subcellular localization and function of G-kinase will be tested. The widespread importance of NO as a signaling molecule has been recognized recently; the studies proposed in this grant application should provide new insights into the mechanism of action of NO and cGMP in mammalian cells. Since pharmacological manipulation of the NO/cGMP signal transduction pathway offers therapeutic potential for a wide variety of human diseases, a better understanding of the long-term downstream effects of NO and cGMP is urgently needed.
