title: Identification of phosphorylated form of 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium.
abstract: In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca(2+) as a second messenger. One of the proteins, the 46 kDa phosphoprotein was found to be highly phosphorylated when Ca(2+)-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 Ca(2+)-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.
authors: Institute of Theoretical and Experimental Biophysics Russian Academy of Science, Institutskaya St, 3, RU-142290, Pushchino, Moscow region, Russia, azartam@yandex.ru.
authors: Tamara
authors: T
authors: Azarashvili
authors: Olga
authors: O
authors: Krestinina
authors: Anastasia
authors: A
authors: Galvita
authors: Dmitry
authors: D
authors: Grachev
authors: Yulia
authors: Y
authors: Baburina
authors: Rolf
authors: R
authors: Stricker
authors: Georg
authors: G
authors: Reiser
mesh_terms: D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D051381:Rats
chemical_list: D010750:Phosphoproteins; D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; D002118:Calcium