p66Shc, a splice variant of the ShcA adaptor protein family, has been reported as an important regulator of mitochondrial reactive oxygen species (ROS) generation in the cells subjected to oxidant injury. This enzyme has also been proposed as the downstream of other pathways involved in ROS generation. A fraction of the cytosolic p66Shc translocates to mitochondrial intermembrane space upon various cellular stress signals, where it is dephosphorylated and in turn it oxidizes cytochrome c and reduces O2 into H2O2 by diverting electrons from complex IV of the electron transport chain, resulting in increased mitochondrial ROS generation. But whether and how this enzyme is activated in the heart subjected to ischemia/reperfusion (I/R) injury remains unclear! In this study, we determined the phosphorylation of p66Shc at Ser36 in whole tissue lysate and total p66Shc in cytosol and mitochondria fractions by immunoprecipitation and immunoblotting. We found that p66Shc phosphorylation was significantly increased in the whole lysate from the hearts subjected to 30-minute ischemia followed by 30- or 60-minute reperfusion (30I/30R, 30I/60R), but not from 10- or 20-minute ischemia followed by 30- or 60-minute reperfusion (10I/30R, 10I/60R, 20I/30R and 20I/60R) compared to that from sham control. 30I/60R noticeably exacerbated the p66Shc phosphorylation compared to 30I/30R. The level of total p66Shc substantially decreased in the cytosolic fraction, but remarkably increased in the mitochondria fraction isolated from hearts subjected to both 30I/30R and 30I/60R compared to sham control. The level of total p66Shc in the mitochondria fraction was negatively related to that of total p66Shc in the cytosol fraction, but positively related to the level of phosphorylated p66Shc. We also measured the phosphorylation of protein kinase C beta II (PKCβII) at Thr641 and activity of protein phosphatase 2A (PP2A) in whole tissue lysate using immunoblotting and the Ser/Thr Phosphatase Assay System, respectively. We found that both PKCβII phosphorylation and PP2A activity significantly increased in hearts subjected to 20I/30R and 20I/60R, 30I/30R and 30I/60R injury, but not in those subjected to 10I/30R or 10I/60R injury. Our data support that ischemia-reperfusion induces Ser36 phosphorylation of p66Shc very likely via activation of PKCβII and PP2A, and p66Shc phosphorylation facilitates its translocation from cytosol into mitochondria in myocardial I/R injury.

Activation of p66shc in the heart subjected to ischemia/reperfusion injury

2019 Fall National Meeting

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