Abstract 18590: Molecular Mechanisms Underlying Protein Kinase C Targeting To Mitochondria
Protein Kinase C (PKC) isoform PKCε has been shown to translocate to subcellular organelles including mitochondria upon activation. However, the molecular machineries responsible for translocation of PKC to mitochondria are largely unknown. The present study was designed to identify the mechanism that regulates the mitochondrial translocation of PKCε. Isolated mitochondria from adult rat cardiac myocytes and H9c2 were used to examine the effect of adenosine on mitochondrial PKCε and the role of heat shock protein 90 (HSP90). Immunofluorescence imaging of isolated mitochondria from cardiac myocytes showed that PKCε (but not PKCδ) was localized in mitochondria and this mitochondrial localization of PKCε was significantly increased by adenosine treatment for 5 minutes (PKCε-positive mitochondria normalized to the total mitochondria, adenosine: 82.8 ± 7.0%, control: 21.1 ± 4.5%, n=3, p<0.01). The adenosine-induced increase in PKCε-positive mitochondria was significantly blocked not only by PKC inhibitor chelerythrine (10 μM, 51.3 ± 6.2%, n=3, p<0.05 vs. adenosine) but also by inhibiting HSP90 function with geldanamycin (1 μM, 54.1 ± 3.3%, n=3, p<0.05 vs. adenosine). Similar results were obtained in H9C2 cells where suppressing HSP90 expression with siRNA targeting HSP90 significantly inhibited adenosine-induced increase in PKCε-positive mitochondria (control: 14.5 ± 2.1%, adenosine: 53.6 ± 4.7%, HSP90 siRNA: 32.5 ± 6.2%, n=3, p<0.05 vs. adenosine). Furthermore, immunoblot analysis from percoll-purified mitochondrial fraction from cardiac myocytes revealed that adenosine induced a significant increase in mitochondrial PKCε (not PKCδ), which was blocked by inhibiting PKC activity with chelerythrine (normalized to control, adenosine: 274.4 ± 92.1%, n=3, p<0.05 vs. control; chelerythrine: 166.7 ± 16.5%, p=NS vs. control). In addition, co-immunoprecipitation data indicated that PKCε co-precipitated with HSP90 was enhanced by adenosine (normalized to control, 196.9 ± 23.9%, n=3, p<0.05 vs. control). In conclusion, we demonstrate that adenosine induces mitochondrial translocation of PKCε, possibly through association of PKCε with HSP90. These results point out a novel mechanism in regulating PKC in mitochondria.
- © 2010 by American Heart Association, Inc.