Regulation of BAD Protein by PKA, PKCδ and Phosphatases in Adult Rat Cardiac Myocytes Subjected to Oxidative Stress

  • Cieslak, Danuta (Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki) ;
  • Lazou, Antigone (Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki)
  • Received : 2007.02.20
  • Accepted : 2007.06.08
  • Published : 2007.10.31

Abstract

$H_2O_2$, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM $H_2O_2$, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with $H_2O_2$. On the contrary, inhibition of PKA or specifically $PKC{\delta}$ resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in $H_2O_2$ treated cells after inhibition of PKA or $PKC{\delta}$ whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, $PKC{\delta}$ and phosphatases.

Keywords

Apoptosis;BAD;Cardiac Myocyte;$H_2O_2$;Phosphatase;Protein Kinase

References

  1. Condorelli, F., Salomoni, P., Cotteret, S., Cesi, V., Srinivasula, S. M., et al. (2001) Caspase cleavage enhances the apoptosisinducing effects of BAD. Mol. Cell Biol. 21, 3025−3036
  2. Maruyama, R., Takemura, G., Aoyama, T., Hayakawa, K., Koda, M., et al. (2001) Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videomicroscopy and electron microscopy. Am. J. Pathol. 159, 683−691
  3. Shizukuda, Y. and Buttrick, P. M. (2002) Subtype specific roles of ${\beta}-adrenergic$ receptors in apoptosis of adult rat ventricular myocytes. J. Mol. Cell. Cardiol. 34, 823−831
  4. Suzuki, K., Kostin, S., Person, V., Elsässer, A., and Schaper, J. (2002) Time course of the apoptotic cascade and effects of caspase inhibitors in adult rat ventricular cardiomyocytes. J. Mol. Cell Cardiol. 33, 983−994
  5. Tapia, J. A., Jensen, R. T., and Garcia-Marin, L. J. (2006) Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non- PKCdelta -dependent mechanism. Biochim. Biophys. Acta 1763, 25−38
  6. Yang, L., Omori, K., Suzukawa, J., and Inagaki, C. (2004) Calcineurin- mediated BAD Ser155 dephosphorylation in ammmonia- induced apoptosis of cultured rat hippocampal neurons. Neurosci. Lett. 357, 73−75
  7. Zheng, M., Zhang, S.-J., Zhu, W.-Z., Ziman, B., Kobilka, B. K., et al. (2000) beta 2-adrenergic receptor-induced p38 MAPK activation is mediated by protein kinase A rather than by Gi or gbeta gamma in adult mouse cardiomyocytes. J. Biol. Chem. 275, 40635−40640
  8. Haunstetter, A. and Izumo, S. (1998) Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ. Res. 82, 1111−1129
  9. Melling, C. W., Krause, M. P., and Noble, E. G. (2006) PKAmediated ERK1/2 inactivation and hsp70 gene expression following exercise. J. Mol. Cell. Cardiol. 41, 816−22
  10. Srivastava, J., Goris, J., Dilworth, S. M., and Parker P. J. (2002) Dephosphorylation of PKC delta by protein phosphatase 2Ac and its inhibition by nucleotides. FEBS Lett. 516, 265−269
  11. Li, D., Ueta, E., Kimura, T., Yamamoto, T., and Osaki, T. (2004) Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination. Cancer Sci. 95, 644−650
  12. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer S. J. (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL. Cell 87, 619−628
  13. Bishopric, N. H., Andreka, P., Slepak, T., and Webster, K. A. (2001) Molecular mechanisms of apoptosis in the cardiac myocyte. Curr. Opin. Pharmacol. 2, 141−150
  14. Hare, J. M. (2001) Oxidative stress and apoptosis in heart failure progression. Circ. Res. 89, 198−200
  15. Remondino, A., Kwon, S. H., Communal, C., Pimentel, D. R., Sawyer, D. B., et al. (2003) ${\beta}-Adrenergic$ Receptor-stimulated apoptosis in cardiac myocytes is mediated by reactive oxygen species/c-Jun NH terminal kinase–dependent activation of the mitochondrial pathway. Circ. Res. 92,136−138
  16. Becker, L. B., Vandem Hoek, T. L., Shao, S. H., Li, C. Q., and Schuacker, P. T. (1999) Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am. J. Physiol. 277, H2240−H2246
  17. Muslin, A. J. and Xing, H. (2000) 14-3-3 proteins: regulation of subcellular localization by molecular interference. Cell Signal. 12, 703−709
  18. Seo, S. Y., Chen, Y., Ivanovska, I., Ranger, A. M., Hong, S. J., et al. (2004) BAD is a pro-survival factor prior to activation of its pro-apoptotic function. J. Biol. Chem. 279, 42240−42249
  19. Aoki, H., Kang, P. M., Hampe, J., Yoshimura, K., Noma T., et al. (2002) Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J. Biol. Chem. 277, 10244−10250
  20. Chio, C. C., Chang, Y. H., Hsu, Y. W., Chi, K. H., and Lin, W. W. (2004) PKA-dependent activation of PKC, p38 MAPK and IKK in macrophage: implication in the induction of inducible nitric oxide synthase and interleukin-6 by dibutyryl cAMP. Cell Signal. 16, 565−575
  21. Tokudome, T., Horio, T., Yoshihara, F., Suga, S., Kawano, Y., et al. (2002) Adrenomedullin inhibits doxorubicin-induced cultured rat cardiac myocyte apoptosis via a cAMP-dependent mechanism. Endocrinology 43, 3515−3521
  22. Valks, D. M., Cook, S. A., Pham, F. H., Morrison, P. R., Clerk, A. et al. (2002) Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signalregulated kinases 1/2 and protein kinase A. J. Mol. Cell Cardiol. 34, 749−763
  23. Yang, Y. and Yu, X. (2003) Regulation of apoptosis: the ubiquitous way. FASEB J. 17, 790−799
  24. Iwai-Kanai, E., Hasegawa, K., Araki, M., Kakita, T., Morimoto, T., et al. (1999) ${\alpha}$- and ${\beta}-Adrenergic$ pathways differentially regulate cell type–specific apoptosis in rat cardiac myocytes. Circulation 100, 305−311
  25. Yin, X. M., Oltyai, Z. N., and Korsmeyer, S. J. (1994) BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 369, 321−323
  26. Cheng, A. C., Huang, T. C., Lai, C. S., and Pan, M. H. (2005) Induction of apoptosis by luteolin through cleavage of Bcl-2 family in human leukemia HL-60 cells. Eur. J. Pharmacol. 509, 1−10
  27. Clerk, A., Cole, S. M., Cullingford, T. E., Harrison, J. G., Jormakka, M., et al. (2003) Regulation of cardiac myocyte cell death. Pharmacol. Ther. 97, 223−261
  28. Kim, B. C., Mamura, M., Choi K. S., Calabretta, B., and Lim, S. J. (2002) Transforming growth factor ${beta}1$ induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells. Mol. Cell. Biol. 22, 1369−1378
  29. Lopez-Neblina, F. and Toledo-Pereyra, L. H. (2005) Molecular biology of apoptosis in ischemia and reperfusion. J. Invest. Surg. 18, 335−350
  30. Kwon, S. H., Pimentel, D. R., Remondino, A., Sawyer, D. B., and Colucci, W. S. (2003) $H_2O_2$ regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways. J. Mol. Cell. Cardiol. 35, 615−621
  31. Alvarado-Kristensson, M. and Andersson T. (2005) Protein phosphatase 2A regulates apoptosis in neutrophils by dephosphorylating both p38 MAPK and its substrate caspase 3. J. Biol. Chem. 280, 6238−6244
  32. Murriel, C. L., Churchill, E., Inagaki, K., Szweda, L. I., and Mochly-Rosen, D. (2004) Protein kinase $C{\delta}$ activation induces apoptosis in response to cardiac ischemia and reperfusion damage. J. Biol. Chem. 279, 47985−47991
  33. Wang, H.-G., Pathan, N., Ethell, I. M., Krajewski, S., Yamaguchi, Y., et al. (1999) $Ca^2$-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339−343
  34. Rodriguez, M. and Schaper, J. (2005) Apoptosis: measurement and technical issues. J. Mol. Cell. Cardiol. 38, 15−20
  35. von Harsdorf, R., Li, P. F., and Dietz, R. (1999) Signalling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation 99, 2934−2941
  36. Wood, D. E. and Newcomb, E. W. (2000) Cleavage of Bax enhances its cell death function. Exp. Cell Res. 256, 375−382
  37. Ayllon, V., Cayla, X., García, A., Roncal, F., Fernandez, R. et al. (2001) Bcl-2 targets protein phosphatase $1{\alpha}$ to bad. J. Immunol. 166, 7345−7352
  38. Cook, S. A., Sugden, P. H., and Clerk, A. (1999) Regulation of Bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes. Circ. Res. 12, 940−949
  39. Gross, A., McDonnell, J. M., and Korshmeyer, S. J. (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 1899−911
  40. Ojeda, F., Folch, H., Guarda, M., Jastorff, B., and Diehl, H. A. (1995) Induction of apoptosis in thymocytes: new evidence for an interaction of PKC and PKA pathways. Biol. Chem. Hoppe. Seyler. 376, 389−393
  41. Markou, T., Hadzopoulou-Cladaras, M., and Lazou, A. (2004) Phenylephrine induces activation of CREB in adult rat cardiac myocytes through MSK1 and PKA signaling pathways. J. Mol. Cell. Cardiol. 37, 1001−1011
  42. Zaugg, M., Xu, W., Lucchinetti, E., Shafiq, S. A., Jamali, N. Z., et al. (2000) ${\beta}-Adrenergic$ receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation 102, 344−350
  43. Sawyer, D. B., Siwik, D. A., Xiao, L., Pimentel, D. R., Singh, K., et al. (2002) Role of oxidative stress in myocardial hypertrophy and failure. J. Mol. Cell. Cardiol. 34, 379−388
  44. Baines, C. P. and Molkentin, J. D. (2005) STRESS signalling pathways that modulate cardiac myocyte apoptosis. J. Mol. Cell Cardiol. 38, 47−62
  45. Cheng, E. H., Kirsch, D. G., Clem, R. J., Ravi, R., Kastan, M. B., et al. (1997) Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 278, 1966−1968
  46. Schonthal, A. H. (1998) Role of PP2A in intracellular signal transduction pathways. Front Biosci. 3, 1262−1273
  47. Steinberg, S. F. (2004) Distinctive activation mechanisms and functions for protein kinase $C{\delta}$. Biochem. J. 384, 449−459
  48. Voss, O., Kim, S., Wewers, M. D., and Doseff, A. (2005) Regulation of monocyte apoptosis by the protein kinase ${\delta};\(PKC{\delta})$-dependent phosphorylation of caspase-3. J. Biol. Chem. 280, 17371−17379
  49. Chiang, C. W., Kanies, C., Kim, K. W., Fang, W. B., Parkhurst, K., et al. (2003) Protein phosphatase 2A dephosphorylation of phosphoserine 112 plays the gatekeeper role for Badmediated apoptosis. Mol. Cell. Biol. 23, 6350−6362
  50. Klumpp, S., Mäurer, A., Zhu, Y., Aichele, D., Pinna, L. A., et al. (2004) Protein kinase CK2 phosphorylates BAD at threonine- 117. Neurochem. Int. 45, 747−752
  51. Willis, S. N. and Adams, J. M. (2005) Life and balance: how BH-3 only proteins induce apoptosis. Curr. Opin. Cell Biol. 17, 1−9
  52. Communal, C., Singh, K., Pimentel, D. R., and Colucci, W. S. (1998) Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the ${\beta}-adrenergic$ pathway. Circulation 98, 1329−1334
  53. Dhalla, N. S., Temsah, R. M., and Netticadan, T. (2000) Role of oxidative stress in cardiovascular diseases. J. Hypertens 18, 655−673