UC San Diego

Mitochondria represent an underappreciated site of regulation by reversible phosphorylation. Our work has focused on the identification of proteins involved in regulating mitochondria by reversible phosphorylation as well as the development of tools to study phosphorylation in mitochondria. Here we show two poorly characterized dual specific phosphatases (DSP) 18 and 21 are resident members of mitochondria. DSP18 and DSP21 are catalytically active phosphatases, and while DSP18 is widely expressed in tissues, DSP21 is selectively expressed in the testes. We show that DSP18 and 21 are directed to mitochondria by cryptic internal localization signals. Subfractionation of rat kidney mitochondria demonstrated that DSP18 is peripherally associated with the mitochondrial inner membrane facing the intermembrane space compartment. In contrast, DSP21 is shown to be localized to the matrix compartment in rat testis mitochondria as a peripheral membrane protein of the inner membrane. Furthermore, we demonstrate that a previously reported substrate for DSP18, the stress activated protein kinase, does not localize to mitochondria in several tissues making it an unlikely substrate for DSP18. In addition we show that DSP18 is released from the intermembrane space following induction of apoptosis by staurosporine treatment. This work establishes the localization of two DSPs on opposing sides of the mitochondrial inner membrane. In addition we have developed phospho-antibodies against three sites on the mitochondrially localized pyruvate dehydrogenase E1[alpha] subunit. We demonstrate these antibodies are both phospho- sensitive as well as site specific. Furthermore, we show that treatment of cells with dichloroacetic acid, a specific inhibitor of pyruvate dehydrogenase kinases, abolishes the phospho-signal with no change in total protein. In addition we show that phosphorylation of these sites are differentially regulated. Interestingly, phosphorylation at Ser232 is more widespread in tissues then previously thought. These antibodies will provide useful tools in monitoring pyruvate dehydrogenase activity in both the disease state as well as in response to a myriad of physiological stimuli