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Journal of Bioenergetics and Biomembranes

, Volume 46, Issue 2, pp 135–145 | Cite as

Identification of phosphorylated form of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium

  • Tamara AzarashviliEmail author
  • Olga Krestinina
  • Anastasia Galvita
  • Dmitry Grachev
  • Yulia Baburina
  • Rolf Stricker
  • Georg Reiser
Article

Abstract

In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca2+ as a second messenger. One of the proteins, the 46 kDa phosphoprotein was found to be highly phosphorylated when Ca2+-induced permeability transition pore (mPTP) was opened in rat brain mitochondria (RBM). In the present study the 46 kDa phosphoprotein was identified as 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) after purification by 2D diagonal electrophoresis following mass spectrometric analysis and Western blot probed with anti-CNP antibody. CNPase was discovered in immunoprecipitates of mitochondria, phosphorylated under both conditions (control and with opened mPTP). Status phosphorylation of CNPase was found to be higher in the inmmunoprecipiates of calcium-overloaded RBM. The phospohoserine and phosphotyrosine residues were detected in phosphorylated 46 kDa band (CNPase) as well as in CNPase immunoprecipitates indicating possible participation of tyrosine and serine protein kinases in phosphorylation of CNPase in mitochondria. The levels of phospo-Ser and phospho-Tyr were increased in RBM with mPTP opened. It was found that CNPase substrate, 2′,3′-cAMP (5 μM) and, a non-competitive CNPase inhibitor, atractyloside (5 μM), were able to increase the level of CNPase phosphorylation in calcium-overloaded mitochondria, while CsA (mPTP blocker) was able to strong suppress the phosphorylation of the enzyme. Collectively, our results provide evidence that Ca2+-stimulated and mPTP-associated CNPase phosphorylation might be an important stage of mPTP regulation in mitochondria, revealing a new function of CNPase outside of myelin structure.

Keywords

Brain mitochondria CNPase Permeability transition pore Calcium transport Phosphoprotein 

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References

  1. Agrawal HC, Sprinkle TJ, Agrawal D (1990) 2′,3′cyclic nucleotide-3′-phosphodiesterase in peripheral nerve myelin is phosphorylated by a phorbol ester-sensitive protein kinase. Biochem Biophys Res Commun 170:817–823CrossRefGoogle Scholar
  2. Augereau O, Claverol S, Boudes N, Basurko MJ, Bonneu M, Rossignol R, Mazat JP, Letellier T, Chary-Prigent J (2005) Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery. Cell Mol Life Sci 62:1478–1488CrossRefGoogle Scholar
  3. Azarashvili TS, Odinokova IV, Evtodienko YV (1999) Phosphorylation of a low-molecular-weight polypeptide in rat liver mitochondria and dependence of its phosphorylation on mitochondrial functional state. Biochemistry (Mosc) 64:556–560Google Scholar
  4. Azarashvili TS, Tyynela J, Odinokova IV, Grigorjev PA, Baumann M, Evtodienko YV, Saris NE (2002) Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria. J Bioenerg Biomembr 34:279–284CrossRefGoogle Scholar
  5. Azarashvili T, Krestinina O, Odinokova I, Evtodienko Y, Reiser G (2003) Physiological Ca2+ level and Ca2+ -induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria. Cell Calcium 34:253–259CrossRefGoogle Scholar
  6. Azarashvili T, Krestinina O, Yurkov I, Evtodienko Y, Reiser G (2005) High-affinity peripheral benzodiazepine receptor ligand, PK11195, regulates protein phosphorylation in rat brain mitochondria under control of Ca(2+). J Neurochem 94:1054–1062CrossRefGoogle Scholar
  7. Azarashvili T, Krestinina O, Galvita A, Grachev D, Baburina Y, Stricker R, Evtodienko Y, Reiser G (2009) Ca2+ -dependent permeability transition regulation in rat brain mitochondria by 2′,3′-cyclic nucleotides and 2′,3′-cyclic nucleotide 3′-phosphodiesterase. Am J Physiol Cell Physiol 296:C1428–C1439CrossRefGoogle Scholar
  8. Azarashvily TS, Tyynela J, Baumann M, Evtodienko YV, Saris NE (2000) Ca(2+)-modulated phosphorylation of a low-molecular-mass polypeptide in rat liver mitochondria: evidence that it is identical with subunit c of F(0)F(1)-ATPase. Biochem Biophys Res Commun 270:741–744CrossRefGoogle Scholar
  9. Bradbury JM, Thompson RJ (1984) Photoaffinity labelling of central-nervous-system myelin. Evidence for an endogenous type I cyclic AMP-dependent kinase phosphorylating the larger subunit of 2′,3′-cyclic nucleotide 3′-phosphodiesterase. Biochem J 221:361–368Google Scholar
  10. Brustovetsky N, Klingenberg M (1996) Mitochondrial ADP/ATP carrier can be reversibly converted into a large channel by Ca2+. Biochemistry 35:8483–8488CrossRefGoogle Scholar
  11. Cho SJ, Jung JS, Jin I, Moon IS (2003) 2′, 3′-cyclic nucleotide 3′-phosphodiesterase is expressed in dissociated rat cerebellar cells and included in the postsynaptic density fraction. Mol Cells 16:128–135Google Scholar
  12. Davis FF, Allen FW (1956) A specific phosphodiesterase from beef pancreas. Biochim Biophys Acta 21:14–21CrossRefGoogle Scholar
  13. Douglas AJ, Thompson RJ (1993) Structure of the myelin membrane enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase: evidence for two human mRNAs. Biochem Soc Trans 21:295–297Google Scholar
  14. Dreiling CE, Schilling RJ, Reitz RC (1981) 2′,3′-cyclic nucleotide 3′-phosphohydrolase in rat liver mitochondrial membranes. Biochim Biophys Acta 640:114–120CrossRefGoogle Scholar
  15. Drummond G, Iyer N, Keith J (1962) Hydrolysis of Ribonucleoside 2′,3′-Cyclic Phosphates by a Diesterase from Brain. J Biol Chem 237:3535–3539Google Scholar
  16. Galvita A, Grachev D, Azarashvili T, Baburina Y, Krestinina O, Stricker R, Reiser G (2009) The brain-specific protein, p42(IP4) (ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+. J Neurochem 109:1701–1713CrossRefGoogle Scholar
  17. Giulian D, Moore S (1980) Identification of 2′:3′-cyclic nucleotide 3′-phosphodiesterase in the vertebrate retina. J Biol Chem 255:5993–5995Google Scholar
  18. Gravel M, DeAngelis D, Braun PE (1994) Molecular cloning and characterization of rat brain 2′,3′-cyclic nucleotide 3′-phosphodiesterase isoform 2. J Neurosci Res 38:243–247CrossRefGoogle Scholar
  19. Kim T, Pfeiffer SE (1999) Myelin glycosphingolipid/cholesterol-enriched microdomains selectively sequester the non-compact myelin proteins CNP and MOG. J Neurocytol 28:281–293CrossRefGoogle Scholar
  20. Koonin EV (1993) A superfamily of ATPases with diverse functions containing either classical or deviant ATP-binding motif. J Mol Biol 229:1165–1174CrossRefGoogle Scholar
  21. Kurihara T, Monoh K, Sakimura K, Takahashi Y (1990) Alternative splicing of mouse brain 2′,3′-cyclic-nucleotide 3′-phosphodiesterase mRNA. Biochem Biophys Res Commun 170:1074–1081CrossRefGoogle Scholar
  22. Lee J, Gravel M, Zhang R, Thibault P, Braun PE (2005) Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein. J Cell Biol 170:661–673CrossRefGoogle Scholar
  23. Lee J, O’Neill RC, Park MW, Gravel M, Braun PE (2006) Mitochondrial localization of CNP2 is regulated by phosphorylation of the N-terminal targeting signal by PKC: implications of a mitochondrial function for CNP2 in glial and non-glial cells. Mol Cell Neurosci 31:446–462CrossRefGoogle Scholar
  24. Lefkimmiatis K, Leronni D, Hofer AM (2013) The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics. J Cell Biol 202:453–462CrossRefGoogle Scholar
  25. Lewandrowski U, Sickmann A, Cesaro L, Brunati AM, Toninello A, Salvi M (2008) Identification of new tyrosine phosphorylated proteins in rat brain mitochondria. FEBS Lett 582:1104–1110CrossRefGoogle Scholar
  26. Moreau B, Nelson C, Parekh AB (2006) Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration. Curr Biol 16:1672–1677CrossRefGoogle Scholar
  27. Myllykoski M, Raasakka A, Lehtimaki M, Han H, Kursula I, Kursula P (2013) Crystallographic analysis of the reaction cycle of 2′,3′-cyclic nucleotide 3′-phosphodiesterase, a unique member of the 2H phosphoesterase family. J Mol Biol 25(22):4307–4322Google Scholar
  28. Pagliarini DJ, Dixon JE (2006) Mitochondrial modulation: reversible phosphorylation takes center stage? Trends Biochem Sci 31:26–34CrossRefGoogle Scholar
  29. Rais I, Karas M, Schagger H (2004) Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification. Proteomics 4:2567–2571CrossRefGoogle Scholar
  30. Reiser G, Kunzelmann U, Steinhilber G, Binmoller FJ (1994) Generation of a monoclonal antibody against the myelin protein CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase) suitable for biochemical and for immunohistochemical investigations of CNP. Neurochem Res 19:1479–1485CrossRefGoogle Scholar
  31. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858CrossRefGoogle Scholar
  32. Sprinkle TJ (1989) 2′,3′-cyclic nucleotide 3′-phosphodiesterase, an oligodendrocyte-Schwann cell and myelin-associated enzyme of the nervous system. Crit Rev Neurobiol 4:235–301Google Scholar
  33. Stingo S, Masullo M, Polverini E, Laezza C, Ruggiero I, Arcone R, Ruozi E, Dal PF, Malfitano AM, D’Ursi AM, Bifulco M (2007) The N-terminal domain of 2′,3′-cyclic nucleotide 3′-phosphodiesterase harbors a GTP/ATP binding site. Chem Biol Drug Des 70:502–510CrossRefGoogle Scholar
  34. Stricker R, Lottspeich F, Reiser G (1994) The myelin protein CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase): immunoaffinity purification of CNP from pig and rat brain using a monoclonal antibody and phosphorylation of CNP by cyclic nucleotide-dependent protein kinases. Biol Chem Hoppe Seyler 375:205–209Google Scholar
  35. Tsukada Y, Nagai K, Suda H (1980) A rapid micro method for 2′,3′-cyclic nucleotide 3′-phosphohydrolase assay using micro high performance liquid chromatography. J Neurochem 34:1019–1022CrossRefGoogle Scholar
  36. Valsecchi F, Ramos-Espiritu LS, Buck J, Levin LR, Manfredi G (2013) cAMP and mitochondria. Physiology (Bethesda) 28:199–209CrossRefGoogle Scholar
  37. Vogel US, Thompson RJ (1988) Molecular structure, localization, and possible functions of the myelin-associated enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase. J Neurochem 50:1667–1677CrossRefGoogle Scholar
  38. Weissbarth S, Maker HS, Raes I, Brannan TS, Lapin EP, Lehrer GM (1981) The activity of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in rat tissues. J Neurochem 37:677–680CrossRefGoogle Scholar
  39. Winkler HH (1969) Localization of the atractyloside-sensitive nucleotide binding sites in rat liver mitochondria. Biochim Biophys Acta 189:152–161CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Tamara Azarashvili
    • 1
    • 2
    Email author
  • Olga Krestinina
    • 1
    • 2
  • Anastasia Galvita
    • 1
  • Dmitry Grachev
    • 1
    • 2
  • Yulia Baburina
    • 2
  • Rolf Stricker
    • 1
  • Georg Reiser
    • 1
  1. 1.Institute of Theoretical and Experimental Biophysics Russian Academy of ScienceMoscow regionRussia
  2. 2.Institut für NeurobiochemieOtto-von-Guericke-Universität Magdeburg, Medizinische FakultätMagdeburgGermany

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