Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-Phosphorylation of Pyruvate Dehydrogenase

Abstract

Abstract:A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1α subunits. When synthetic peptides including site 1 were treated with H 2 O 2 , the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no longer phosphorylated by E1α-kinase. Isolated mitochondria were incubated under state III or IV conditions, lysed, the pyruvate dehydrogenase complex (PDC) immunoprecipitated, and tryptic peptides analyzed by MALDI-TOF mass spectrometry. In all instances both Met and MetSO site 1 tryptic-peptides were detected. Similar results were obtained when suspension-cultured cells were incubated with chemical agents known to stimulate production of reactive oxygen species within the mitochondria. Treatment with these agents had no effect upon the amount of total PDC, but decreased the proportion of P-PDC. We propose that the redox-state of the Met residue adjacent to phosphorylation site 1 of pyruvate dehydrogenase contributes to overall regulation of PDC activity in vivo.

Methionine residues around phosphorylation sites are preferentially oxidized in vivo under stress conditions

“…In addition to the direct effect on protein function, methionine sulfoxidation has been recently postulated as a mechanism to couple oxidative signals to changes in protein phosphorylation. This hypothesis derives from studies on two unrelated plant enzymes, mitochondrial pyruvate dehydrogenase20 and cytoplasmic nitrate reductase19, both of which have a methionine residue proximal to a regulatory phosphorylation site. In both cases, the authors demonstrated that the oxidation of these methionine residues resulted in the inhibition of phosphorylation, suggesting that the oxidation status of these methionines allows the enzymes to monitor oxidative stress and subsequently code this information in terms of phosphorylation patterns.…”

“…In this regard, the activities of calcineurin, a protein phosphatase, as well as Ca 2+ /calmodulin-dependent protein kinase II, have been previously shown to be modulated by specific methionine oxidation1718. Furthermore, it has been reported that the oxidation of methionine residues within the phosphorylation motif of nitrate reductase19 and pyruvate dehydrogenase20 inhibits the phosphorylation of nearby sites, providing a mechanism to couple oxidative signals to changes in protein phosphorylation. These isolated observations imply that reversible methionine oxidation might serve as a rheostat to control the phosphorylation of proximal phospho-acceptors21.…”

Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

“…Oxidation of the +4 Met residue, which serves as a hydrophobic recognition element for the kinase, markedly inhibited phosphorylation. In a related study, also using synthetic peptides and a recombinant kinase, Miernyk et al (2009) observed that oxidation of the +1 Met of pyruvate dehydrogenase (PDH) strongly inhibited phosphorylation of the adjacent Ser residue.…”

“…However, the common occurrence of Met adjacent to Ser/ Thr/Tyr residues (Rao et al 2013) and the frequent occurrence of MetSO (Ghesquière et al 2011;Liu et al 2013;Salvato et al 2014) near O-phosphorylation-sites (Song et al 2012) suggests that crosstalk between these two PTM's is widespread. (Hardin et al 2009;Miernyk et al 2009), oxidation of Met might directly inhibit O-phosphorylation of a nearby Ser/Thr/Tyr-residue by interfering with the kinase recognition/binding (Fig. 1).…”

“…Furthermore, msrA-null mutants showed lower degradation of α-synuclein protein. Two studies (Hardin et al 2009;Miernyk et al 2009) have specifically tested the effect of Met oxidation on the subsequent phosphorylation of nearby Ser residues. Using a multiple combinations of synthetic peptides and recombinant kinases, Hardin et al (2009) found that Met oxidation strongly inhibits phosphorylation in vitro.…”