“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

Abstract

The mitochondrial pyruvate dehydrogenase complex (mtPDC) is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client (KiC) assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform “scanning mutagenesis” of the residues flanking the site of phosphorylation. Consistent with the results from “phylogenetic analysis” of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mtPDC by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

A Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology

“…We have previously established the utility of the KiC assay for analysis of defined PK/client pairs, as well as for PK-client discovery. ,, Although in this experiment, we limited ourselves to synthetic peptides corresponding to known in vivo phosphorylation sites of the proteins of Arabidopsis and B. napus developing seeds, a library including all of the potential Ser, Thr, and Tyr-containing tryptic peptides and maximum number of recombinant PK could scale up to encompass the majority of the potential clients for each PK. However, this would be impractical in terms of expense.…”

“…Instrument and search parameters settings have been previously described 15, 20 . For recombinant kinase-client assays, a composite target decoy database was constructed based upon all full length kinases and client protein sequences.…”

“…Analysis of the synthetic peptide screen was performed using a stand-alone LTQ-XL. Instrument settings were described previously , except the gradient was increased to 1–40% acetonitrile. Analysis of the recombinant protein kinase client assays was performed using a LTQ Orbitrap XL ETD.…”

“…The raw MS files were searched against a decoy database consisting of the random complement of the sequences comprising the peptide library, using SEQUEST algorithms (Proteome Discoverer 1.0, Thermo Fisher). Instrument and search parameters settings have been previously described. , For recombinant kinase-client assays, a composite target decoy database was constructed based upon all full-length kinases and client protein sequences. The search parameters were mass type, average precursor plus fragment; dynamic modifications, phosphorylation of Ser/Thr/Tyr (+79.9799 Da) and oxidation of Met (+15.9994 Da), and the static modification, of Cys-carboxyamidomethylation.…”

The role of mitochondria in physical activity and its adaptation on aging

“…E1 catalyzes the oxidative decarboxylation of pyruvate using thiamine pyrophosphate as a cofactor, followed by the reduced acetylation of the lipoil portion covalently bound to E2, which transfers the CoASH group releasing acetyl-CoA as a product. E3 contains FAD as a cofactor, and completes the cycle reaction by reoxidation of the lipoil groups of E2, using NAD as electron receptor (AHSAN, SWATEK, ZHANG et al, 2012).…”