Mechanisms regulating PKA phosphorylation of the key cardiac proteins Troponin I and Hsp20

[摘要] Reversible modification of protein function by PKA phosphorylation is critical to a variety of processes in the heart, including the modulation of cardiac output in response to stress, and the transduction of cardioprotective signals. As a result, PKA phosphorylation events must be tightly regulated. This regulation is achieved by compartmentalisation of cAMP/PKA signal transduction cascades, which is mediated by phosphodiesterase enzymes (PDEs) and A-kinase anchoring proteins (AKAPs). PDEs provide the only means of hydroysing cAMP in cells, and in recent years, the use of genetically encoded FRET-based cAMP sensors has allowed direct visualisation of discrete cAMP microdomains created by subcellular tethering of PDEs in cardiac cells. The physical compartmentalisation of PKA by AKAPs ensures that only a small subpopulation of PKA is activated by these cAMP microdomains. Macromolecular complexes nucleated by AKAPs bring together cAMP effectors, signal terminating enzymes and other scaffold proteins, to facilitate PKA signaling events.The aim of this thesis was to investigate the signaling elements which participate in these complexes to modulate PKA phosphorylation of the myofilament protein troponin I and the small heat shock protein Hsp20 in the heart. Both of these proteins are phosphorylated by PKA at their N termini in response to β-adrenergic stimulation, resulting in increased cardiac output and enhanced cardioprotection. PKA phosphorylation of TnI mediates the fight or flight response, by reducing the Ca2+ sensitivity of the myofilament and altering the strength of contraction and rate of relaxation of the heart to increase cardiac output. In the first part of this thesis, I investigated the specific PDE isoforms which regulate cAMP/PKA signaling at the myofilament. A novel FRET-based cAMP sensor which is targeted to troponin I at the myofilament demonstrated that PDE4 is the main phosphodiesterase family which modulates cAMP levels in the subcellular compartment around troponin I. Biochemical studies identified a specific association between troponin I and the long phosphodiesterase isoform PDE4D9. The binding site for PDE4D9 on troponin I was mapped using peptide array technology to a region on the flexible C terminus of the myofilament protein, and the sequence information used to design a cell permeable peptide disruptor of this interaction. Disruption of the troponin I-PDE4D9 complex in cells using this peptide was sufficient to promote PKA phosphorylation of troponin I in the absence of other stimuli. This approach may be of benefit in the treatment of diseases such as heart failure, where PKA phosphorylation of myofilament proteins is known to be reduced. Control of post-translational modifications at the level of the myofilament by specific PDE isoform inhibitors represents a promising therapeutic avenue which has not yet been explored.In the second part of the thesis I investigated the signaling components which regulate PKA phosphorylation of Hsp20. Hsp20 is known to protect against ischaemic injury and cardiac hypertrophy, and its cardioprotective actions are enhanced by PKA phosphorylation on Ser16. Biochemical studies identified an interaction between Hsp20 and AKAP-Lbc in cardiac cells, and selective knockdown of AKAP-Lbc confirmed that this interaction is required for β-adrenergic-induced PKA phosphorylation of Hsp20 and the anti-apoptoticeffects of Hsp20 in cardiac myocytes. FRET-based studies using a cAMP sensor targeted to Hsp20 demonstrated the role of PDE4 isoforms in modulating cAMP levels around Hsp20, and biochemical techniques identified PDE4D isoforms in the macromolecular complex formed by Hsp20 and AKAP-Lbc. The AKAP-Lbc/PKA/Hsp20/PDE4D complex appears to play a key role in cardioprotection via the modulation of Hsp20 Ser16 phosphorylation, and selective targeting of signaling elements that enhance this modification represents a new therapeutic avenue for the prevention and treatment of pathological cardiac remodelling and ischaemic injury.

[发布机构] University:University of Glasgow;Department:Institute of Molecular Cell and Systems Biology

[关键词] cAMP, PKA phosphorylation, phosphodiesterase, PDE4, AKAP, myofilament, troponin I, AKAP-Lbc, Hsp20, cardioprotection