Cytochrome c oxidase subunit IV as a marker of protein kinase Cε function in neonatal cardiac myocytes: Implications for cytochrome c oxidase activity

Mourad Ogbi, Catherine S. Chew, Jan Pohl, Olga Stuchlik, Safia Ogbi, John A. Johnson

Research output: Contribution to journalArticle

56 Scopus citations

Abstract

We have previously demonstrated that low concentrations of phorbol esters stimulate the selective translocation of protein kinase C (PKC) α and ε from the cell soluble to the particulate fraction in NCMs (neonatal rat cardiac myocytes). We therefore determined if the in vitro phosphorylation of substrates in these fractions could be used as assays of PKCα or ε activation. Intact cell phorbol ester treatment caused a decline in the in vitro 32P-incorporation into several proteins in the cell-soluble fraction. These declines occurred in the presence or absence of in vitro Ca 2+ and probably reflected the exit of PKC isoenzymes from the soluble fraction. In contrast, an approx. 18 kDa protein incorporated 32p in particulate fractions isolated from 4β-PMA-treated cells in a Ca 2+-independent manner. Proteomic and immunoprecipitation analyses indicated that the protein is subunit IV of the cytochrome c oxidase complex (COIV). In vitro phosphorylation of COIV was attenuated by PKC pseudosubstrate peptides. Introduction of an PKCε-selective translocation inhibitor [Johnson, Gray, Chen and Mochly-Rosen (1996) J. Biol. Chem. 271, 24962-24966] into NCMs before 4β-PMA treatments also attenuated the in vitro phosphorylation of COIV. In mitochondrial extracts from 4β-PMA-treated NCMs, the PKCε isoenzyme coimmunoprecipitated with COIV, and cytochrome c oxidase activity was enhanced 2-fold. The in vitro phosphorylation of COIV reflects a novel approach for monitoring PKCε function in NCMs. Furthermore, PKCε probably interacts with COIV in NCM mitochondria to enhance electron-transport chain complex IV activity.

Original languageEnglish (US)
Pages (from-to)923-932
Number of pages10
JournalBiochemical Journal
Volume382
Issue number3
DOIs
StatePublished - Sep 15 2004

Keywords

  • Cardiac myocyte
  • Cytochrome c oxidase
  • Immunoprecipitation
  • Mitochondria
  • Phosphorylation
  • Protein kinase Cε (PKCε)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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