CYCLIC AMP AND PROTEIN KINASES IN CELL REGULATION

Cyclic AMP-dependent protein kinase IIbeta (PKA IIbeta), the principal mediator of cAMP actions in brain, is anchored at specific sites in the cytoskeleton of neurons. In part, this is due to the avid binding of the regulatory subunit (RIIbeta) of the kinase by novel, A Kinase Anchor Proteins (AKAPs). AKAPs simultaneously bind with proteins in the cytoskeleton. This arrangement places PKA IIbeta near both substrate/effector proteins and adenylate cyclase in post-synaptic regions of neurons, thereby creating target sites for the reception and propagation of signals carried by cAMP. Structurally-divergent AKAPs adapt PKA IIalpha or PKA IIbeta for other functions by linking the kinases to other organelles in neurons and non-neuronal cells. The overall goals of this research program are to (a) determine the biochemical and molecular basis for the anchoring of PKA Il isoforms at effector sites and (b) elucidate physiological functions of AKAP-PKA Il complexes. Studies will focus on the neuronal anchor proteins AKAP75, AKAP150 and MAP2CV. AKAP75 contains two N terminal targeting/attachment domains (T1, T2). Structural features in T1 and T2 that are essential for targeting PKA Il isoforms to microtubules and/or the cortical actin cytoskeleton will be determined by mutagenesis and transfection/expression analysis. AKAP docking proteins will be purified and characterized; their cDNAs will be cloned, sequenced and subjected to mutational analyses. The physicochemical properties of highly-purified AKAP75 and AKAP150 will be delineated. Affinities of the AKAPs and MAP2CV for RIIbeta and RIIalpha will be accurately quantified. Structural features in RII subunits and RII binding sites (in AKAPs) that govern the affinity, isoform-selectivity and stoichiometry of AKAP-RII complex formation will be elucidated. The calmodulin binding domain of AKAP75 will also be characterized. A mutant anchor protein will be used to disrupt the normal localization of PKA Il isoforms in a model neuronal system PC12 cells. Potential roles for PKAII-AKAP complexes in enzyme activation, secretion, cytoskeletal organization and gene transcription will be studied. Finally, we will investigate the regulated expression, biochemical properties and physiological function of the S-AKAP84, a novel anchor protein that may play an important, stage-specific role in spermatocyte differentiation.