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Monitoring phosphorylation of the pyruvate dehydrogenase complex |
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| Authors: | Matthew J Rardin Sandra E Wiley Anne N Murphy |
| Institution: | a Department of Pharmacology, Department of Cellular and Molecular Medicine, and Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA b Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA c Mitochondrial and Metabolic Disease Center, University of California, San Diego, San Diego, CA 92103, USA d Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA |
| Abstract: | The pyruvate dehydrogenase multienzyme complex (PDC) is a key regulatory point in cellular metabolism linking glycolysis to the citric acid cycle and lipogenesis. Reversible phosphorylation of the pyruvate dehydrogenase enzyme is a critical regulatory mechanism and an important point for monitoring metabolic activity. To directly determine the regulation of the PDC by phosphorylation, we developed a complete set of phospho-antibodies against the three known phosphorylation sites on the E1 alpha subunit of pyruvate dehydrogenase (PDHE1α). We demonstrate phospho-site specificity of each antibody in a variety of cultured cells and tissue extracts. In addition, we show sensitivity of these antibodies to PDH activity using the pyruvate dehydrogenase kinase-specific inhibitor dichloroacetate. We go on to use these antibodies to assess PDH phosphorylation in a patient suffering from Leigh’s syndrome. Finally, we observe changes in individual phosphorylation states following a small molecule screen, demonstrating that these reagents should be useful for monitoring phosphorylation of PDHE1α and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli. |
| Keywords: | Pyruvate dehydrogenase Mitochondria Dichloroacetate Dichloroacetic acid Leigh&rsquo s syndrome Reversible phosphorylation Phospho-peptide Phospho-antibody |
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