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.

Fig. 1

Schematic of phospho-peptides used to generate antisera. (A) Phospho-peptides corresponding to each of these sites were synthesized and used to immunize rabbits for the production of phospho-specific antibodies (numbers correspond to start methionine). (B) Amino acid comparison of PDHE1α orthologs across several species, with identical amino acids highlighted in gray. Colored bars and residues correspond to phospho-sites and peptides utilized to generate anti-sera (site 1-pSer²⁹³ in yellow, site 2-pSer³⁰⁰ in orange, and site 3-pSer²³² in green). Accession numbers follow the common name of each species.

Fig. 2

Phospho-PDHE1α antibody specificity. (A) 100 μg of crude rat kidney mitochondria were incubated with or without calf intestinal alkaline phosphatase (APase) for 30 min at 37°C. 5 μg of protein was separated by SDS-PAGE, and immunoblotted with the indicated antibodies. (B) Phospho-antibodies were incubated with a 20-fold excess of each of the phosphopeptides used for immunization. Crude mouse heart mitochondria were isolated; 5 μg of protein was separated by SDS-PAGE and immunoblotted with indicated phospho-antibodies (blots shown were exposed equally). (C) Immunofluorescence analysis of phosphorylation of endogenous PDHE1α in COS7 cells (green). In addition, cells were stained with the mitochondrial marker, MitoTracker Red, and the nuclear marker, DAPI (blue). Co-localization is represented in the merged images in yellow.

Fig. 3

Detecting changes in PDC activity. (A) Mouse liver extracts were treated with 5mM DCA for 4 hours and then analyzed by Western blot with the indicated phospho-antibodies and compared to total PDHE1α. (B) Phosphorylation status of PDHE1α in HEK293A cells over time treated with 5mM DCA. (C) Analysis of PDH phosphorylation by immunofluorescence in COS-7 cells treated with 5 mM DCA for 2 hours. Images were taken following equal exposure to those taken in Fig. 2C.

Fig. 4

Use of phospho-antibodies to detect changes in phosphorylation in a patient diagnosed with Leigh's Syndrome. (A) Sequence analysis of PDHE1α cDNA isolated from cultured HPFF from the LS patient and a control revealed a missense mutation (C412T) resulting in an amino acid substitution of L138F. Fluorescent images of cultured human primary fibroblasts from the LS patient versus the control (WT) probed with antibodies against pSer²⁹³ (B), total PDHE1α (C), and the PDC E2/E3 binding protein (D); equal exposure between samples. (E) 20 μg of protein was isolated from cultured fibroblasts, separated by SDS-PAGE and analyzed by Western blot for levels of pSer²⁹³, pSer³⁰⁰, pSer²³², total PDHE1α, and PDC E2/E3 binding protein. α-Tubulin and GAPDH were used as cytosolic loading controls, while NDUFB6 was used as an indicator of mitochondrial content.

Fig. 5

Pharmacological manipulation of the phospho-status of PDHE1α. Human primary fibroblasts were either left untreated or treated for 1 hour with various known inhibitors of oxidative phosphorylation or signaling pathways: oligomycin (2 μg/ml), rotenone (10 μM), FCCP (1 μM), doxorubicin (8 μM), BAY 11-7082 (20 μM), and UV light (4,000 μjoules). Whole cell lysates were prepared and 20 μg of protein were separated by SDS-PAGE. Western blot analysis was performed with indicated antibodies.