Type II cAMP-dependent protein kinase (PKA) isoforms are localize to microtubules, centrosomes and Golgi in cells through interactions of their regulatory subunits (RII) with structurally distinct proteins containing A-kinase anchoring (AKA)-motifs. The central hypothesis that guides the studie proposed in this application is that anchoring of PKA to A-kinase anchoring proteins (AKAPs) is a regulated process for localizing PKA to specific subcellular structures and through this anchoring a cAMP signal can be channeled to discrete subcellular locations. The objective of the research program is to test the hypothesis that RII-beta anchoring to AKAPs is regulate through the phosphorylation of RII-beta at a threonine and/or serine residue near the AKAP interaction domain.
The specific aims of this proposal are to: 1 Characterize the effects of phosphorylation of RII-beta by cyclin-dependent protein kinase on the binding parameters of RII-beta to MAP2, AKAP75/79, AKAP6 and TAKAP80. In vitro overlay binding studies using AKAP-fusion proteins and synthetic peptides will be used to characterize and quantify the binding parameters. 2. Identify the amino acid (threonine/serine) in the N-terminus of RII-beta that is phosphorylated by cyclin-dependent protein kinase. In vitro phosphorylation of RII-beta will be characterized by site directed mutagenesis peptide mapping and mass spectrometry to identify the amino acid(s) phosphorylated by p34cdc2 and cdk2. 3. Show that RII-beta is phosphorylated at threonine 69 and/or serine 71 in intact cells and attempt to show effects of phosphorylation on binding to endogenous AKAPs. The in vivo studies will be accomplished by comparing 32P-labeled tryptic peptide maps of RII-beta phosphorylated in vitro by p34cdc2/cdk2 with the 32P-peptides generated from RII-beta immunopreciptitated from MSN neuroblastoma cells incubated with 32P-orthophosphate. The long range goal will be to see if there is cell cycle regulation of RII-beta phosphorylation and anchoring of PKAII to AKAPs in the intact cell. 4. The 3-D structure of the RII-AKAP interaction domains will be resolved by using Nuclear Magnetic Resonance (NMR) to solve the solution structure of synthetic peptides corresponding to the known RII-tethering sites in four AKAPs (MAP2, AKAP75/79, AKAP64 and TAKA80) and of RII-alpha and RII-beta N-terminal 50 amino acid residue peptides. Achieving the goals of thi proposal will increase our understanding of cAMP-mediated signal transduction in normal and disease states.
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| Erlichman, J; Gutierrez-Juarez, R; Zucker, S et al. (1999) Developmental expression of the protein kinase C substrate/binding protein (clone 72/SSeCKS) in rat testis identification as a scaffolding protein containing an A-kinase-anchoring domain which is expressed during late-stage spermatogenesis. Eur J Biochem 263:797-805 |
