Role of individual phosphorylation sites in inactivation of pyruvate dehydrogenase complex in rat heart mitochondria

Abstract

1. A method is described using trypsin/formic acid cleavage for unambiguously measuring occupancies of phosphorylation sites in rat heart pyruvate dehydrogenase [I Piphosphate complexes. 2. In mitochondria oxidizing 2-oxoglutarate + L-malate relative initial rates of phosphorylation were site 1 > site 2 > site 3. 3. Dephosphorylation and reactivation of fully phosphorylated complex was initiated in mitochondria by inhibiting the kinase reaction. Using dichloroacetate relative rates of dephosphorylation were site 2 > (1 = 3). Using sodium dithionite or sodium pyruvate or uncouplers + sodium arsenite or steady state turnover (31P replacing 32p in inactive complex) relative rates were site 2 > site 1 > site 3. With dithionite reactivation was faster than site 3 dephosphorylation, i.e. site 3 is apparently not inactivating. 4. The steady state proportion of inactive complex was varied (92-48%) in mitochondria oxidizing 2-oxoglutarate/L-malate by increasing extramitochondrial Ca2+ (0-2.6gM). This action of Ca2+ induced dephosphorylation (site 3 > site 2> site 1). These experiments enable prediction of site occupancies in vivo for given steady state proportions of inactive complexes. 5. The proportion of inactive complex was related linearly to occupancy of site 1. 6. Sodium dithionite (10mM) and Ca2+ (0.5,uM) together resulted in faster dephosphorylations of each site than either agent alone; relative rates were site 2 > (1 = 3). 7. Dephosphorylation and possibly phosphorylation of sites 1 and 2 was not purely sequential as shown by detection of complexes phosphorylated in site 2 but not in site 1. Estimates of the contribution of site 2 phosphorylation to inactivation ranged from 0.7 to 6.4%. 8. It is concluded that the primary function of site 1 phosphorylation is inactivation, phosphorylation of site 2 is not primarily concerned with inactivation and that phosphorylation of site 3 is non-inactivating.

Tissue-specific kinase expression and activity regulate flux through the pyruvate dehydrogenase complex

“…These outcomes were virtually identical regardless of whether the samples were made from diaphragm or skeletal muscle, where PDK1 is a fairly minor isozyme, or from heart muscle, where PDK1 accounts for a significant proportion of the total kinase mass. A number of studies originating from the Randle laboratory and later confirmed by others demonstrated that the overall inactivation rate of PDC in a kinase reaction correlates with the phosphorylation rate of site 1 located on the ␣ chain of the E1 component (35,36). Thus, the lack of the effect of PDK1 ablation on the rate of PDC inactivation strongly suggests that the native PDK1, at least in tissues of muscle origin, does not appreciably contribute to the overall inactivation rate of PDC in kinase reaction, presumably due to its inability to compete with other isozyme(s) of PDK for phosphorylation site 1.…”

Reversible phosphorylation of pyruvate dehydrogenase in rat skeletal-muscle mitochondria. Effects of starvation and diabetes

“…Mitochondria were then incubated in KCI medium (containing 0.2mM-[32P]P,; sp. radioactivity 1500-8500d.p.m./pmol) and samples taken at prescribed times into Triton medium at 0°C for assay of 32p in PDH complex and in tryptic and tryptic/formic acid [32P]phosphopeptides as described by Sale & Randle (1982a). Analytical methods PDH complex (active form) was assayed spectrophotometrically by coupling to arylamine acetyltransferase (Coore et al, 1971); citrate synthase was assayed by the method of Srere et al (1963), as modified by Coore et al (1971).…”

“…Protein-bound 32p was assayed as described by Sale & Randle (1982a,b). Tryptic and tryptic/formic acid phosphopeptides were prepared with Tos-Phe-CH2Cltreated trypsin, separated by high-voltage paper electrophoresis and assayed for 32P as described by Sale & Randle (1981, 1982a. ATPase activity was assayed by the method of Cooper et al (1974).…”

Recognition of the Inner Lipoyl-Bearing Domain of Dihydrolipoyl Transacetylase and of the Blood Glucose-Lowering Compound AZD7545 by Pyruvate Dehydrogenase Kinase 2

“…Inactivation of E1 occurs as a result of phosphorylation of three serine residues (S264 or site 1, S271 or site 2, and S203 or site 3) located on the α chain of E1 (9). Although all three phosphorylation sites were shown to be inactivating (10,11), the regulation of PDC in vivo largely correlates with phosphorylation of site 1 (12), which is the site most rapidly modified by PDHK (10,11). Under starvation and in diabetes, all three sites of E1 become heavily phosphorylated, reflecting an increase in intramitochondrial concentrations of NADH and acetyl-CoA, which promote PDHK activity, as well as an increase in the intramitochondrial concentration of the PDHK protein, which comes about as a result of de novo synthesis (1,2).…”