|Rosca MG, Hoppel CL (2010) Activation of distinct mitochondrial cAMP-PKA compartments induces different effects on mitochondrial function.
Link: Abstracts Session 1
Reversible phosphorylation of proteins is a main cellular regulatory mechanism. Despite the protection of mitochondrial proteins against signalling cascades initiated by cytosolic kinases, the list of mitochondrial phosphoproteins is expanding. There is also accumulating evidence that reversible phosphorylation at specific serine and threonine amino acid residues induced by cAMP-activated protein kinase A (PKA) changes the function of mitochondrial proteins (1-3).
• O2k-Network Lab: US_OH Cleveland_Hoppel CL
Organism: Rat Tissue;cell: Heart Preparation: Isolated mitochondria Enzyme: Adenine nucleotide translocase, Complex IV;cytochrome c oxidase, Complex V;ATP synthase
- Reversible phosphorylation of proteins is a main cellular regulatory mechanism. Despite the protection of mitochondrial proteins against signalling cascades initiated by cytosolic kinases, the list of mitochondrial phosphoproteins is expanding. There is also accumulating evidence that reversible phosphorylation at specific serine and threonine amino acid residues induced by cAMP-activated protein kinase A (PKA) changes the function of mitochondrial proteins (1-3).
- In cardiomyocytes, this signalling pathway is initiated by the β1 sympathetic receptor coupled with the stimulatory G protein-bound adenylyl cyclase (AC) which synthesizes cAMP. Binding of cAMP to regulatory subunits of the nearby cytosolic PKA induces the dissociation of the holoenzyme and phosphorylation of substrates responsible for the positive chronotropic and inotropic effects. The new finding of a functional mitochondrial AC activated by the tricarboxylic cycle-generated bicarbonate which stimulates mitochondrial Complex IV (CIV) (4,5) via phosphorylation of CIV subunits suggests the presence of an independent mitochondrial cAMP-PKA pathway that regulates mitochondrial function.
- Protein kinase A was identified on mitochondrial inner and outer membranes, and in the matrix. Since cAMP is poorly diffusible, we hypothesized that the activation of PKA localized in different mitochondrial compartments by cAMP of either extramitochondrial origin or synthesized within the mitochondria will cause specific phosphorylation events on mitochondrial proteins that will drive distinct effects on mitochondrial function. Oxidative phosphorylation was assessed as the integrative function of mitochondria in both individual saponin-permeabilized rat heart muscle fibres and isolated subsarcolemmal (SSM) and interfibrillar (IFM) cardiac mitochondria.
- High-resolution respirometry of permeabilized rat heart fibres in the presence of CI substrates (electron transport through CI/CIII/CIV) shows that State 3 respiratory rates of cAMP (membrane permeable, dibutyryl cAMP)-incubated fibres were 70% of the control. The addition of succinate, which provides electron input to CII, resulted in a much greater increase in oxygen flux in control (80%) compared with cAMP-incubated fibres (27%); therefore, the oxygen flux of cAMP-incubated fibres was 50% of control. The decrease in oxygen flux of cAMP-incubated fibres was not relieved upon collapsing mitochondrial potential with the uncoupler Dinitrophenole, showing that the defect induced by cAMP resides in the electron transport system (ETS) and not in the phosphorylation apparatus. Oxygen flux through CII/CIII/CIV measured upon addition of rotenone (inhibits CI) also was decreased in cAMP-incubated fibres. TMPD+ascorbate donates the reducing equivalents to cytochrome c, which is then oxidized by cytochrome c oxidase with final tetravalent reduction of oxygen to water. The incubation with cAMP resulted in a decrease in TMPD+ascorbate supported respiration. Therefore, cAMP leads to a decrease in oxidative phosphorylation with CI, CII, and CIV substrates, suggesting defects in the ETS localized at least at the level of cytochrome c oxidase.
- In contrast, the incubation of intact freshly-isolated cardiac IFM with 100 µm Sp-cAMP induces a 30% increase in State 3 respiratory rates with CI substrates which was not changed by the uncoupler. The data point to enzymes of the ETS as targets for the effect of exogenous permeable cAMP.
- Activation of mitochondrial AC with bicarbonate leads to a dramatic decrease in the ADP-dependent respiratory rates in both cardiac SSM and IFM. The improvement of respiratory rates by collapsing the mitochondrial membrane potential with an uncoupler shows that the intramitochondrial-generated cAMP causes defects in the phosphorylation apparatus rather than in the ETS responsible for the limitation of oxidative phosphorylation. In conclusion, the activation of distinct mitochondrial cAMP-PKA compartments induces different effects on the function of cardiac mitochondria.
- Lee I, Salomon AR, Ficarro S, Mathes I, Lottspeich F, Grossman LI (2005) cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity. J Biol Chem 280:6094-100.
- Lee I, Salomon AR, Yu K, Doan JW, Grossman LI, Huttemann M (2006)New prospects for an old enzyme: mammalian cytochrome c is tyrosine-phosphorylated in vivo. Biochemistry 45:9121-8.
- Helling S, Vogt S, Rhiel A, Ramzan R, Wen L, Marcus K (2008) Phosphorylation and kinetics of mammalian cytochrome c oxidase. MolCell Proteomics.
- Acin-Perez R, Salazar E, Brosel S, Yang H, Schon EA, Manfredi G (2009) Modulation of mitochondrial protein phosphorylation by soluble adenylyl cyclase ameliorates cytochrome oxidase defects. EMBO Molecular Medicine 1:392-406.
- Acin-Perez R, Salazar E, Kamenetsky M, Buck J, Levin LR, Manfredi G (2009) Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation. Cell Metabolism 9:265-76.