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.

Carbenoxolone induces permeability transition pore opening in rat mitochondria via the translocator protein TSPO and connexin43

“…6 shows a semi-quantitative analysis of the phosphoprotein bands from SDS–PAGE after separation of Ca 2+ -loaded RBM in the presence or absence of Cbx and anti-TSPO antibody. We investigated the concentration-dependent effect of Cbx on protein phosphorylation and found that Cbx at 1 µM and 10 µM significantly stimulated the incorporation of [γ- 32 P]ATP into 43–46 kDa proteins (where Cx43 and CNP were found) [6,27], 17 kDa proteins (myelin basic protein isoforms) [28], as well as of 3.5 kDa (subunit c of ATP synthase) (Fig. 6A).…”

“…The phosphorylation status of these proteins and peptide was shown to change depending on the opened/closed state of the pore [26]. These phosphoproteins were identified: 46 kDa phosphoprotein is 2′,3′-cyclic nucleotide-3′-phosphodiestearase [27], 21 kDa and 17 kDa phosphoproteins are isoforms of myelin basic protein (MBP) [28], and the 3.5 kDa phosphopeptide is subunit c of ATP synthase [29]. Incubation of the rat brain mitochondria (RBM) with anti-TSPO antibodies specifically prevented these phosphorylations, suggesting that TSPO participates in the modulation of mPTP opening.…”

Astaxanthin Prevents Mitochondrial Impairment Induced by Isoproterenol in Isolated Rat Heart Mitochondria

“…Beutner and coauthor suggested that CyP-D regulated the activity of oxidative phosphorylation, presumably altering the activity of the respiratory chain and respirasome assembly and inhibiting the activity of ATP synthase and synthasome assembly of ETC complexes [34]. Earlier, we showed that CyP-D was co-localized with 2 ,3 -cyclic nucleotide-3 -phosphodiasterase (CNPase) in rat brain and liver mitochondria (RBM, RLM) [35], which was identified in our laboratory [36]. We observed that CNPase was associated with each complex of ETC, was colocalized with the adenine nucleotide translocator (ANT), voltage-dependent anion channel, CyP-D, and α-tubulin [35], and performed the protective function in RBM and RLM [37,38].…”

Acetyl‐CoA production from pyruvate is not necessary for preservation of myelin

“…This could be a consequence of haploinsufficiency for Cnp in animals expressing Cnp Cre/+ , since the Cre recombinase is knocked‐in in one of the Cnp alleles (Lappe‐Siefke et al, ). The molecular function of CNP in myelinating glia is still unknown, but CNP was identified in the mitochondrial fraction, might play a role in mitochondrial permeability (Azarashvili et al, ; McFerran & Burgoyne, ), and can have a neuroprotective function in OLs (Verrier et al, ). Interestingly, mice deficient or haplodeficient for Cnp developed axonal swelling and neurodegeneration in the nervous system at 3.5 months and 19 months, respectively (Hagemeyer et al, ; Lappe‐Siefke et al, ), two features also observed in Pdha1 f/Y ; Cnp Cre/+ axonopathy.…”

“…This could be a consequence of haploinsufficiency for Cnp in animals expressing Cnp Cre/1 , since the Cre recombinase is knocked-in in one of the Cnp alleles(Lappe-Siefke et al, 2003). The molecular function of CNP in myelinating glia is still unknown, but CNP was identified in the mitochondrial fraction, might play a role in mitochondrial permeability(Azarashvili et al, 2014;…”