My research focuses on how a family of enzymes collectively referred to as protein kinase C (PKC) plays key roles in cardiac protection and damage. There are 10 known isozymes of PKC and often different PKC isozymes have opposing roles in biochemical processes. We study the catalytic and cellular targeting mechanisms of PKC isozymes in the context of cardiac ischemic preconditioning and ischemia / reperfusion injury. Our current research is focusing on how individual PKC isozymes modulate the activities of electron transport chain complexes and other mitochondrial enzymes to contribute cardiac protection or damage. We are using cell-based and in vivo models combined with biochemical, proteomic and other approaches to better understand the roles of PKC isozymes in these processes.
Cytochrome c oxidase (also referred to as complex IV) is the last enzymatic step in the mammalian electron transport chain. It transfers electrons from cytochrome c to molecular oxygen and acts as 1 of 3 proton pumps in the ETC. These activities are linked to maintenance of the mitochondrial proton gradient and ATP production. In addition inhibition of complex IV leads to the "back up" of electrons at complexes I and III of the ETC which allows electron leak from these complexes and the consequent production of damaging superoxide. Our recent studies have demonstrated a key interaction between the ePKC isozyme and the number IV subunit of cytochrome c oxidase in cardiac preconditioning. This interaction correlates with a 2-fold enhancement of cytochrome oxidase activity which we believe augments cardiac ATP production and reduces damaging electron leakage and superoxide production from the ETC. There are 13 subunits in the cytochrome c oxidase enzyme complex and we are currently studying the interactions between individual PKC isozymes and each cytochrome c oxidase subunit. Our approaches include density gradient purification of mitochondria, assays of ETC complex activities, 2-dimensional electrophoresis, mass spectrometric and phosphor-peptide analyses, co-immunoprecipitation, Western blot and other analyses. By identifying the protein-protein interaction sites between PKC isozymes and cytochrome c oxidase we hope to develop peptidomimetic compounds that will augment or interfere with these interactions to protect heart cells from ischemia / reperfusion injury. We are currently expanding our studies to include other enzymes in oxidative phosphorylation and oxidative stress.
Johnson, J. A., Goka, T. G., and Clark, R. B. (1986) Phorbol ester induced augmentation and inhibition of epinephrine-stimulated adenylate cyclase in S49 lymphoma cells. J. Cyclic Nucleotide and Protein Phosphorylation Research 11: 199-215.
Clark, R. B., Friedman, J.,
Johnson, J. A., Kunkel, M. (1987) Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg++. FASEB J. 1(4) : 289-297.
Clark, R. B., Kunkel, M., Friedman, J., Goka, T. and
Johnson, J. A. (1988) Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase in S49 wild-type lymphoma cells. Proc. Natl. Acad, Sci. U.S.A 85: 1442-14446.
Johnson, J. A. and Clark, R. B. (1990) Multiple nonspecific effects of sphingosine on adenylate cyclase and cyclic AMP accumulation in S49 lymphoma cells preclude its use as a specific inhibitor of protein kinase C. Biochem. J. 268: 507-511.
Johnson, J. A., Clark, R. B., Friedman, J., Dixon, R. A. F., and Strader, C. D. (1990) Identification of a specific domain in the beta-adrenergic receptor required for phorbol ester-induced inhibition of catecholamine-stimulated adenylyl cyclase. Mol. Pharmacol. 38: 289-293.
Johnson, J. A., Clark, R. B., Friedman, J., Dixon, R. A. F., and Strader, C. D. (1990) Identification of a specific domain in the beta-adrenergic receptor required for phorbol ester-induced inhibition of catecholamine-stimulated adenylyl cyclase. Mol. Pharmacol. 38: 289-293.
Johnson, J. A., Friedman, J., Halligan, R.D., Birnbaumer, M., and Clark, R. B. (1991) Sensitization of adenylyl cyclase by P2 purinergic and M5 muscarinic receptor agonists in L cells. Mol. Pharmacol. 39: 539-546.
Johnson, J. A. and Mochly-Rosen, D. (1995) Inhibition of the spont-aneous rate of contraction of neonatal cardiac myocytes by protein kinase C isozymes. A putative role for the e isozyme. Circ. Res. 76: 654-663
Johnson, J.A., Adak, S., and Mochly-Rosen, D. (1995) Prolonged phorbol ester treatment down-regulates protein kinase C isozymes and increases contraction rate in neonatal cardiac myocytes. Life Sciences 57: 1027-1038.
Johnson, J.A., Gray, M.O., Chen, C.-H., and Mochly-Rosen, D. (1996) A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J. Biol. Chem. 271: 24962-24966.
Johnson, J. A., Gray, M., Karliner, J., Chen,C., and Mochly-Rosen, D. (1996) An improved permeabilization protocol for the introduction of peptides into cardiac myocytes: application to protein kinase C research. Circ. Res. 79:1086-1099.
Yedovitzki, M., Mochly-Rosen, D.,
Johnson, J. A., Gray, M., Ron, D., Abramovitch, E., Cerasi, E. and Nesher, R. (1997) Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic b-cells. J. Biol. Chem. 272:1417-1420.
Zhang, Z.,
Johnson, J.A., Nabil E., Mochly-Rosen, D., and Boutjdir, M. (1997) C2-Region derived peptides of b-protein kinase C regulate cardiac calcium channels Circ. Res. 80:720-729.
Johnson, J.A. and Waller, J. (2002) Transforming growth factor beta-1 attenuates endothelin-1-induced functions in neonatal cardiac myocytes. Life Sciences 71:99-113.
Johnson, J.A. (2002) An ePKC-selective inhibitor attenuates back phosphorylation of a low molecular weight protein in cardiac myocytes. Cellular Signaling 15: 123-130.
Pipkin, W.L.,
Johnson, J.A., Creazzo, T., Burch, B.S. and Brophy, C. (2003) Phosphorylation of the small heat shock protein Hsp20 modulates neonatal cardiac myocyte function. Circulation 107: 469-476.
Wingard, C.J.,
Johnson, J.A., Holmes, A., and Prikosh, A. (2003) Improved erectile function following Rho-kinase inhibition in a rat castrate model of erectlie dysfunction. Am. J. Physiol. 284: R1572-R1579.
Abou-Mohamed, G.,
Johnson, J.A., Jin, L., El-Remessy, A.B., Do, K., Kaesemeyer, W.H., Caldwell, R.B., and Caldwell, R.W. (2004) Roles of superoxide, peroxynitrite and protein kinase C in the development of tolerance to nitroglycerin. J. Pharmacol. Exp. Therapeutics 308: 289-299.
Johnson, J.A. (2004) Differential inhibition by a and ePKC pseudosubstrate sequences:a putative mechanism for preferential ePKC activation in neonatal cardiac myocytes. Life Sciences 74: 3153-3172.
Johnson, J.A., and Barman, S.A. (2004) Protein kinase C modulation of cyclic GMP in rat neonatal pulmonary vascular smooth muscle. Lung 182:79-89.
Ogbi, M., Chew, C.S., Pohl, J., Stuchlik, O., Ogbi, S. and
Johnson, J.A. (2004) Cytochrome c oxidase subunit IV as a marker of ePKC function in neonatal cardiac myocytes: Implications for cytochrome c oxidase activity. Biochem. J. 382 (3): 923-932.
Ogbi, M., Wingard, C.J., Ogbi, S. and
Johnson, J.A. (2004) Epsilon protein kinase C lengthens the quiescent period between spontaneous contractions in rat ventricular cardiac myocytes and trabecula. Naunyn-Schmiedeberg's Archives of Pharmacology 370:251-261.
Ogbi, M. and
Johnson, J.A. (2006) Protein kinase Ce interacts with cytochrome c oxidase subunit IV and enhances cytochrome c oxidase activity in neonatal cardiac myocyte preconditioning. 393:191-199
Ma, G., Al-Shabrawey, M.,
Johnson, J., Datar R., Caldwell, R.B., Caldwell, R.W. Protection against myocardial ischemia/reperfusion injury by short term diabetes: role of VEGF-induced angiogenesis and activation of cell survival signaling. Naunyn-Schmiedeberg's Archives of Pharmacology. In Press
