title: Monitoring phosphorylation of the pyruvate dehydrogenase complex.
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 (PDHE1alpha). 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 PDHE1alpha and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Rardin
authors.forename: Sandra E
authors.initials: SE
authors.lastname: Wiley
authors.forename: Robert K
authors.initials: RK
authors.lastname: Naviaux
authors.forename: Anne N
authors.initials: AN
authors.lastname: Murphy
authors.forename: Jack E
authors.initials: JE
authors.lastname: Dixon