The regulatory (R) subunits of cAMP-dependent protein kinase (PKA) are multidomain proteins that mediate a variety of protein-protein interactions in the cAMP signaling pathway. Four isoforms of R are known: RIalpha, RIbeta, RIIalpha, and RIIbeta. My project focuses on the RIalpha subunit, which is associated with many diseases. RIalpha is up-regulated in breast cancers and in many other cancers and is also associated with RIalpha cardiac disorders such as myxomas and Carney complex disease. These cardiac disorders are nonmalignant tumors and have led to the prediction that RIalpha may be a tumor suppressor. RIalpha is also subject to nonsense-mediated mRNA decay (NMD) so that the message is destroyed rather than expressing mutant or deletion RIalpha mutants that are non-functional.

The RI subunits, unlike the RII subunits of PKA, contain an intracellular disulfide bond near the N-terminus. While the importance of this disulfide bond is still not clear, I found that the RIalpha subunit was often reduced in different cell types. We had assumed, based on our earlier expression studies in E. coli, that RIalpha was mostly in an oxidized state. A recent report also indicated that the oxidation state, as well as the location to particulate vs. soluble fractions in cells, was influenced by exposure to oxidants. Thus the oxidation of RIalpha may serve as a redox sensor. To confirm this, I used a 2-D gel system that discriminates disulfide-bonded proteins and have collaborated with a research scientist in the lab to confirm the disulfide bonding by mass spectrometry. This is a significant part of our effort to map the PKA proteome. The formation of stable disulfide bonds also allows us to examine quantitatively whether heterodimers are formed preferentially between RIalpha and RIbeta. This also may be significant for cancer cells where RIbeta expression is even higher than RIalpha. I will use this assay system to determine whether this is true in cells and whether this provides a mechanism for abolishing homodimers of RIalpha. I will also be characterizing a Carney complex associated mutant form of RIalpha to see if it forms aberrant complexes with other proteins that might account for its effect in causing cardiac myxomas.

To determine if mutant forms of RIalpha that are functionally defective will always display a dominant negative phenotype, I will express mutant forms of RIalpha that are defective in binding cAMP in cells and will monitor their effect on PKA activity by using FRET reporters that are sensitive to PKA activation. I will also use FRET reporters that detect cAMP. I will also co-express these mutant proteins in E. coli with wild type RIalpha using a polycistronic vector. In this way we can characterize the properties of the heterodimers. By combining these biochemical and cellular assays, we hope to gain insight into the role of RIalpha in disease.

PUBLICATIONS (resulting from this training)

Day ME, Koller A, Sastri M, Taylor SS. (2007) Deciphering the role of oxidation in regulatory subunits of protein kinase A. In Preparation.