Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

Abstract

The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3>or=PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4>or=PDK3>PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

Defective Branched-Chain Amino Acid Catabolism Disrupts Glucose Metabolism and Sensitizes the Heart to Ischemia-Reperfusion Injury

“…It has been well documented that the PDH activity is rapidly regulated by reversible phosphorylation of its E1α component (Holness and Sugden, 2003; Kaplon et al, 2013; Kolobova et al, 2001). However, there were no differences in total PDH protein expression and E1α phosphorylation between WT and KO, under either fed or fasting condition although fasting induced a significant increase of phosphorylation as expected (Figure S3D).…”

Abnormalities in the Tricarboxylic Acid Cycle in Huntington Disease and in a Huntington Disease Mouse Model

“…PDHC is continuously regulated by phosphorylation and dephosphorylation (25). The phosphatase coded for by PDP1 regulates dephosphorylation, or activation, of PDHC (26). mRNA levels do not predict protein levels, but the fact that it was 1 of only 4 mRNAs that were altered is indicative of the sensitivity of pyruvate metabolism to HD pathology.…”

The role of the p53 tumor suppressor in metabolism and diabetes

“…Analysis of the pentose phosphate pathway (PPP), an alternative mechanism to consume glucose for energy production, showed that it is blocked by p53, in part through direct interaction between p53 and the rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD) (Jiang, et al 2011). p53 also negatively regulates pyruvate dehydrogenase kinase-2 (PDK2) through both transcriptional and post-translational pathways to activate the PDH complex, which converts pyruvate to acetyl-CoA to tip the balance from glycolysis to mitochondrial respiration (Contractor and Harris 2012; Kolobova, et al 2001). Finally, p53 transactivates Parkin (PARK2), a gene associated with Parkinsons disease, in order to inhibit glycolysis (Zhang, et al 2011).…”