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Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Sanderson TH, Mahapatra G, Pecina P, Ji Q, Yu K, Sinkler C, Varughese A, Kumar R, Bukowski MJ, Tousignant RN, Salomon AR, Lee I, Hüttemann M - PLoS ONE (2013)

Bottom Line: Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration.This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion.These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

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In vivo induction of cytochrome c Tyr97 phosphorylation in rat brain by insulin treatment.(A) Cytc was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cytc under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cytc is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cytc species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.
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pone-0078627-g005: In vivo induction of cytochrome c Tyr97 phosphorylation in rat brain by insulin treatment.(A) Cytc was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cytc under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cytc is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cytc species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.

Mentions: In the previous section we showed that insulin applied to ischemic pig brain tissue in vitro leads to Tyr97 phosphorylation. To be fully consistent with the data of the release of Cytc from the mitochondria, which is suppressed by insulin treatment (Fig. 1), we purified Cytc from rat brains after the animals were subjected to the same treatment regimen. To do so we modified our standard purification protocol used for large tissue samples by increasing the volume of extraction buffer and by bypassing the second round of anion and cation exchange chromatography to further increase yield (see materials and methods section). The resultant Cytc was of high purity (Fig. 5A, bottom). Consistent with the in vitro data (Figs. 3 and 4), rat brain Cytc is not tyrosine phosphorylated under control conditions or after global brain ischemia (Fig. 5A, lanes 1 and 2). Importantly, in vivo insulin treatment leads to strong induction of tyrosine phosphorylation (Fig. 5A, lane 3), and this effect is persistent after global brain ischemia (Fig. 5A, lane 4). Analysis by mass spectrometry of both phosphorylated species after insulin treatment +/− global insulin treatment unambiguously revealed that the same residue, Tyr97, was phosphorylated in an insulin dependent manner (Fig. 5B and C). These in vivo data are consistent with the in vitro pig brain results showing phosphorylation of the same residue after insulin treatment (Fig. 4).


Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Sanderson TH, Mahapatra G, Pecina P, Ji Q, Yu K, Sinkler C, Varughese A, Kumar R, Bukowski MJ, Tousignant RN, Salomon AR, Lee I, Hüttemann M - PLoS ONE (2013)

In vivo induction of cytochrome c Tyr97 phosphorylation in rat brain by insulin treatment.(A) Cytc was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cytc under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cytc is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cytc species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.
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pone-0078627-g005: In vivo induction of cytochrome c Tyr97 phosphorylation in rat brain by insulin treatment.(A) Cytc was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cytc under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cytc is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cytc species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cytc after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.
Mentions: In the previous section we showed that insulin applied to ischemic pig brain tissue in vitro leads to Tyr97 phosphorylation. To be fully consistent with the data of the release of Cytc from the mitochondria, which is suppressed by insulin treatment (Fig. 1), we purified Cytc from rat brains after the animals were subjected to the same treatment regimen. To do so we modified our standard purification protocol used for large tissue samples by increasing the volume of extraction buffer and by bypassing the second round of anion and cation exchange chromatography to further increase yield (see materials and methods section). The resultant Cytc was of high purity (Fig. 5A, bottom). Consistent with the in vitro data (Figs. 3 and 4), rat brain Cytc is not tyrosine phosphorylated under control conditions or after global brain ischemia (Fig. 5A, lanes 1 and 2). Importantly, in vivo insulin treatment leads to strong induction of tyrosine phosphorylation (Fig. 5A, lane 3), and this effect is persistent after global brain ischemia (Fig. 5A, lane 4). Analysis by mass spectrometry of both phosphorylated species after insulin treatment +/− global insulin treatment unambiguously revealed that the same residue, Tyr97, was phosphorylated in an insulin dependent manner (Fig. 5B and C). These in vivo data are consistent with the in vitro pig brain results showing phosphorylation of the same residue after insulin treatment (Fig. 4).

Bottom Line: Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration.This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion.These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Show MeSH
Related in: MedlinePlus