Adenylate kinase(AK)-catalyzed energetic and AMP metabolic signaling (AK->AMP->AMP-sensors) is increasingly recognized among major stress-response elements in cardiac cells, critical in regulating diverse cellular processes. At present, however, molecular and cellular mechanisms regulating AK-catalyzed phosphotransfer, the main AMP signal generator, AK interactions with AMP-metabolic sensors and intracellular AMP signal dynamics, are largely unknown. We have obtained evidence that AK phosphotransfer communicates energetic signals from mitochondria to myofibrils, the nucleus and the plasma membrane, securing efficient energy supply and metabolic sensing. The central hypothesis of this proposal is that dynamics of AK phosphotransfer and AMP signal generation, regulated by functional, hormonal and metabolic state, promotes efficient cellular energetics and, by association with AMP-sensing modules, integrates AMP signal transduction into adaptive response to metabolic stress. This hypothesis is supported by preliminary data that indicate: 1) AK phosphotransfer flux and AMP metabolic pool size are regulated by the cell's functional and hormonal states and respond rapidly to metabolic stress; 2) AK1 associates/co-localizes with metabolic sensor AMP-activated protein kinase (AMPK); 3) AK1 deficiency is associated with defective AMP signaling and stress response. Based on this in Aim #1 we propose to define the cellular regulatory mechanisms coupling AK-catalyzed phosphotransfer, AMP signal dynamics and metabolic sensors response;
In Aim #2, we will determine mechanisms and the significance of AK co- localization and molecular/functional interactions with AMPK in metabolic signaling. Finally, in Aim #3, we will determine the significance of the AK->AMP->AMP-sensors system in transducting metabolic signals triggering cardioprotective response. The proposed aims will be addressed using wilde type and transgenic AK1-deficient animals. AK phosphotransfer flux and AMP turnover will be quantified using 180-assisted 31P NMR and mass spectrometric techniques. Molecular, proteomic and imaging techniques will be employed to define AK intracellular interactions and new AMP signaling targets. As a long-term objective, this proposal will establish cellular and molecular mechanisms that regulate AK phosphotransfer and associated AMP signaling circuits sustaining efficient and stress tolerant myocardial energetics. ? ? ?
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