• Identification and characterization of an A-kinase anchoring domain in Chromodomain Helicase Binding Protein 8.

      Shanks, Maureen O'Donnell; Bond, Meredith, Ph.D. (2012)
      A-kinase anchoring proteins (AKAPs) localize protein kinase A (PKA) to discrete microdomains in the cell, and act as scaffolds for the phosphorylation targets and regulatory proteins of the PKA signaling pathway. Reduced phosphorylation of PKA substrates and reduced anchoring of PKA by AKAPs has been observed in heart failure. The upregulation of developmental genes is also characteristic of failing hearts. Chromodomain helicase binding protein 8 (Chd8) is a chromatin remodeling protein that is expressed at high levels in embryogenesis, and regulates transcription of apoptotic genes in the context of large protein complexes. A screen for novel AKAPs in the human heart identified Chd8 as a PKA-binding protein. Here we show that Chd8 contains an AKAP domain in its amino terminus, and that phosphorylation of the PKA regulatory subunit modulates anchoring of PKA by Chd8. We have also identified Chd8 in nuclear and perinuclear domains in three cell types. Chd8 was found to be expressed in high levels in embryonic and post-natal heart, and bound to the p53-responsive P2 promoter of the MDM2 gene. These results demonstrate a novel function for Chd8 as an AKAP and characterize Chd8 in the context of cardiac development. We therefore conclude that Chd8 is a novel AKAP and propose that Chd8 plays a role in the regulation of MDM2, a key regulator of p53.
    • PKA as the Effector of Beta-2-Adrenoreceptor Signaling Regulating Airway Smooth Muscle Relaxation

      Morgan, Sarah; Penn, Raymond B. (2013)
      Inhaled beta-agonists are effective at reversing bronchoconstriction in asthma but exhibit significant side effects including tachyphylaxis and possibly increased asthma-related mortality. The mechanisms by which beta-agonists exert both their beneficial and detrimental effects are controversial and poorly understood. The cAMP-dependent protein kinase (PKA) is the historically accepted effector of beta-agonist-mediated bronchorelaxation, though this assumption is based on associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac)--and not PKA--mediates the relaxation of ASM observed with beta-agonist treatment. This study aims to clarify the role of PKA in the pro-relaxant effects of beta-agonists on airway smooth muscle as well as the ability of the kinase to exert negative feedback on this signaling. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased beta-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, beta-agonist-mediated relaxation of contracted smooth muscle. These findings demonstrate that PKA is the predominant and physiologically relevant effector through which beta-agonists exert their bronchorelaxant effects. To investigate compartmentalized cAMP and PKA signaling and regulation related to beta-agonists, analyses of mRNA and protein expression identified 11 A-kinase anchoring proteins (AKAPs) in human ASM. Disruption of AKAP-PKA interactions via peptides AKAP-IS or Ht31 has minimal effects on whole cell cAMP signaling stimulated by beta-agonist, but significantly increases the duration of plasma membrane-delineated cAMP. Thus PKA, in addition to its effector function, significantly contributes to homologous desensitization of the beta-2-AR in ASM through mechanisms dependent on its localization near the plasma membrane. While the role of specific AKAPs in ASM remains undefined, these findings clearly demonstrate the importance of targeted PKA activity in beta-agonist-mediated signaling. Specific manipulation of PKA activity or AKAP complexes may provide a means to improve upon current bronchodilator therapies for the treatment of asthma.