Regulatory subunit (RIα) of cAMP-dependent protein kinase A (PKA). Crosstalk between PKA and PKG

Cyclic AMP-dependent protein kinase (PKA) plays a major role in signal transduction and protein phosphorylation in mammalian cells. PKA is a complex protein that holds a core composed of two regulatory (R) and two catalytic (C) subunits; this is the inactive isoform when cAMP is absent. In the presence of cAMP the holoenzyme of PKA dissociates the dimer regulatory subunits from the catalytic subunits and the released catalytic subunits are then able to phosphorylate serine or threonine residues in cellular protein. In the presence of both cAMP and cGMP, both cyclic nucleotides will compete and bind to the R subunit of PKA. In this paper we are proposing that there is a phosphorylation site, Ser 99 on the R subunit that cAMP or cGMP binds to activate PKA. In turn, if point mutations are induced to the Ser 99 and replaced with Ala and Glu the phosphorylation site will not be phosphorylated. Our materials and method include inducing and growing the RIa protein in BL21 cells. We will purify and express the RIa protein, including those with point mutations and will use gel filtration chromatography to ensure that we localize and purify the RIa proteins. We will also chemically tag and label the point mutated RIa proteins so that we can purify the proteins that are unable to bind to cAMP or cGMP. Our goals in this paper are to further study the PKA/PKG phosphorylation site at Ser 99 and become effective in purifying the point mutated RIa proteins.