Monitoring phosphorylation of the pyruvate dehydrogenase complex

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 El alpha subunit of pyruvate dehydrogenase (PDHE1 alpha). 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 PDHE1 alpha and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli. (C) 2009 Elsevier Inc. All rights reserved.