The diverse biochemical effects of different neurotransmitters or hormones that stimulate cAMP production have been proposed to occur through the site-specific localization of particular PKA subtypes. Localization of the type II PKA is maintained through protein-protein interactions between the regulatory subunit (RII) and specific A-kinase anchoring proteins (AKAPs). During the past funding period we have demonstrated that PKA anchoring proceeds through interaction of the RII dimer with an amphipathic helix on the AKAP, elucidated the roles of selected AKAPs in targeting PKA to specific subcellular compartments, and demonstrated that disruption of PKA anchoring uncouples certain cAMP-responsive events inside cells. Plans for the next funding period focus on extending these lines of inquiry.
Aim 1 will structurally elucidate the AKAP-binding site on RII by measuring changes in AKAP-binding affinity by plasmon resonance and equilibrium dialysis in selected RII mutants and by completing the structure of the RII fragment when complexed to an AKAP peptide by multidimensional NMR.
Aim 2 will define the peroxisomal targeting sequence on AKAP220, a novel AKAP which associates with peroxisomes.
Aim 3 will establish if manipulation of PKA localization alters cAMP-responsive events by determining whether correct PKA anchoring facilitates the phosphorylation of two physiological targets: the transcription factor CREB which is involved in the regulation of gene expression, and enzymes of the MAP kinase cascade which modulate cell growth.
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