Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

The enzymic activity of the mammalian pyruvate dehydrogenase complex is reg ulated by the phosphorylation of three serine residues (sites 1, 2 and 3) l ocated on the El component of the complex. Here we report that the four iso enzymes of protein kinase responsible for the phosphorylation and inactivat ion of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their a bilities to phosphorylate the enzyme. PDK1 can phosphorylate all three site s, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. A lthough PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporate d by each isocnzynic decreases in the order PDK1 > PDK3 greater than or equ al to PDK4 > PDK2. Significantly, binding of the coenzyme thiamin pyrophosp hate to pyruvate dehydrogenase alters the rates and stoichiometries of phos phorylation of the individual sites. First, the rate of phosphorylation of site I by all isoenzymes of kinasc is decreased. Secondly, thiamin pyrophos phate markedly decreases the amount of phosphate that PDK I incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenz yme does not significantly affect the total amount of phosphate incorporate d in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphoryla tion of this site. Furthermore, pyruvate dehydrogenase complex phosphorylat ed by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP 1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyr uvate dehydrogenase is phosphorylated by other isoenzymes, the rates of rea ctivation decrease in the order PDK4 >, PDK3 > PDK1. Taken together, result s reported here strongly suggest that the major determinants of the activit y state of pyruvate dehydrogenase in mammalian tissues include the phosphor ylation site specificity of isoenzymes of kinase in addition to the absolut e amounts of kinase and phosphatase protein expressed in mitochondria.