AKAP220, a novel targeting and anchoring protein for protein kinase A I
MetadataShow full item record
Many hormones elicit loading of GTP onto the alpha-subunit of the GTP binding protein Gs. Gsalpha-GTP dissociates from betagamma-subunits and stimulates, via direct binding, adenlylate cyclase (AC) at the plasma membrane. Despite (1) the low abundance and modest specific activity of AC and (2) robust degradation of cAMP by cyclic nucleotide phosphodiesterases, signals carried by cAMP are disseminated to microdomains in the cytoplasm, the surfaces of organelles and cytoskeleton. Four protein kinase A (PKA) isoforms are the principal intracellular targets for cAMP. Regulatory (RII) subunits of PKAIIalpha and PKAIIbeta bind with high-affinity tethering sites on A&barbelow; K&barbelow;inase A&barbelow;nchor P&barbelow;roteins (AKAPs). Discrete and unique domains in AKAPs anchor tethered PKAII isoforms at various intracellular locations. AKAP-mediated clustering of PKAs enables co-localization of kinase with substrate/effector proteins and increases sensitivity to low levels of CAMP by elevating local PKA concentration.;Most classic AKAPs either fail to bind or only weakly ligate PKAI. However, a growing body of evidence suggests that ubiquitous PKAIalpha is partially cytoplasmic and partially anchored. The main goals of this thesis were to (1) characterize a PKAIalpha-selective AKAP; (2) determine similarities and differences between RI and RII tethering sites; (3) establish the subcellular location(s) of an RIalpha (PKAIalpha)-selective AKAP; and (4) link an RI-selective AKAP to a functional role of PKAI.;I discovered that AKAP220 selectively complexes RIalpha (and PKAIalpha) in vitro and in intact cells, irrespective of the concentration of PKAII (or RII). Two factors confer unique properties on AKAK220. First, systematic mutagenesis/expression analysis revealed that the anchor protein contains 9 independent, clustered RIalpha binding sites. Second, the classical RII-binding, amphipathic helix ((LIV)X3(AV)X2(LIV)(LIV)X2(ALIV)(I)) is modified in AKAP220. The amino acid located at position 9 in the tethering alpha-helix has a small side chain (Ala, Ser, Thr) and serves as a "gate keeper" that permits entry of RIalpha to the binding site, while excluding RII isoforms. RIalpha binding specificity is also determined and sustained by novel (relative to RII binding sites), collective interactions of small (Ala/Gly) or aromatic amino acids at positions 4, 5 and 16 in the tethering domain. Monospecific, high-affinity IgGs (directed against, central, C- and N-terminal segments of AKAP220) were developed to elucidate the intracellular distribution of the anchor protein by immunofluorescence microcopy. Unlike most other anchor proteins, perinuclear portion of cytoskeleton that contains vimentin, an intermediate filament (IF) protein. When the PKAIalpha level increases, AKAP220 efficiently recruits the kinase to the perinuclear IF-based cytoskeleton. Moreover, recruitment of PKAIalpha promotes phosphorylation of vimentin (at Ser39) and decompacting of IFs, suggesting that AKAP220-PKAIalpha complexes regulate interactions between IFs and organelles or other parts of the cytoskeleton.