PKA has been implicated in cancer through its role in cell growth and differentiation. My current research interests involve using protein crystallography and fl uorescence polarization to investigate the inhibition of cAMP-dependant protein kinase A (PKA) by the beta type regulatory subunits.

Type I beta PKA upregulation has been found in breast cancer. Although, due to ubiquitous expression of PKA in various tissues and its involvement in normal cellular processes, PKA inhibitors are impractical as drug candidates. Therefore, an alternative approach is targeting the cAMP binding sites of the tissue specific beta type regulatory subunits in search of isoform specific agonists and antagonists.

Recently, I solved a crystal structure of a single cAMP binding domain of the type II beta regulatory subunit bound to PKA. This structure was obtained in the presence of a non-hydrolyzable ATP analogue, AMP-PNP, and shows a transition state of the active kinase before the transfer of the phosphate. Currently, crystallization trials are in progress for type II beta complexes with both cAMP binding domains, primarily in the presence of AMP-PNP. Also, I have expressed and purified the type II beta regulatory subunit, and I am currently scaling up for crystallization trials. These larger structures will give information on interdomain communication during cAMP activation and isoform specific differences in PKA regulatory subunits. Fluorescence polarization will be the primary technique to investigate the kinetics of inhibition and activation of the beta type PKA holoenzyme complexes.

In collaboration with a postdoctoral fellow in the lab, we are developing a system using a peptide competition assay. This assay will be used to screen both for antagonists and agonists, as well as be used to determine the effects on kinase inhibition of each domain of the regulatory subunit. The combination of structura l information and high throughput screening will be a powerful tool in development of novel isoform-specific kinase drugs.

PUBLICATIONS (resulting from this training)

Esposito V, Sjoberg T, Das R, Brown S, Taylor SS, Melacini G. (2006) NMR assignment of the cAMP-binding domain A of the PKA regulatory subunit. J Biomol NMR. 36:S5:64

Wu J, Brown S, von Daake S, Taylor SS. PKA Type II alpha Holoenzyme Reveals a Combinatorial Strategy for Isoform Diver sity. Science 2007 318:274-79.