Many hormones exert their effect through receptor-coupled pathways that result in production of the intracelllular second messenger cAMP. The diverse physiological responses induced by elevated cAMP levels are mediated by the cAMP-dependent protein kinase )PKA) through phosphorylation of metabolic enzymes, channel proteins, and transcriptional regulators. Selectivity of PKA action is achieved in part through compartmentalization of the kinase with preferred substrates. A family of anchoring proteins, called AKAPs, have been characterized that maintain PKA at particular subcellular sites and target the kinase for specific functional roles. In this application we propose to investigate nuclear targeting of PKA and its role in mediating the hormone-induced expression of genes. We have discovered a novel anchoring protein, called AKAP 95, that binds PKA and is exclusively localized to the nuclear matrix. The properties of this AKAP will be characterized as part of a long term goal to define the relationship between structural organization in the cell nucleus and the regulation of nuclear signaling pathways. Molecular and genetic approaches are proposed that will define the nuclear complexes forced by aKAP 95 and identify new proteins that may be structural components of the nuclear matrix. As AKAPs are known to be multifunction, experiments are proposed that will determine whether association of other kinases and phosphatases with the nuclear matrix occurs through AKAP 95. We have also discovered that AKAP 95 is a DNA binding protein and we will establish whether the protein preferentially binds a specific DNA sequence, and whether binding affects transcriptional activity. Targeting of PKA and other signaling enzymes to the nucleus may be important for normally-regulated gene expression. We will use inhibitor peptide and DNA constrictors to determine the effect of disrupting PKA anchoring on cAMP-induced expression of the prolactin gene in cultured rat pituitary tumor GH cells. Antisense oligonucleotides and constructs will be used to specifically reduce PKA binding states at the nuclear matrix to establish the role of AKAP 95-mediated targeting in regulating ranscriptional events. These studies have relevance to cancer, as malignant progression is often accompanied by disruption of both nuclear organization and signal transduction mechanisms.
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| Eide, T; Coghlan, V; Orstavik, S et al. (1998) Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95. Exp Cell Res 238:305-16 |
| Vo, N K; Gettemy, J M; Coghlan, V M (1998) Identification of cGMP-dependent protein kinase anchoring proteins (GKAPs). Biochem Biophys Res Commun 246:831-5 |
