Structural Basis for Phosphorylation and Lysine Acetylation Cross-talk in a Kinase Motif Associated with Myocardial Ischemia and Cardioprotection

Benjamin L Parker,Nicholas E Shepherd,Sophie Trefely,Nolan J Hoffman,Melanie Y White,Kasper Engholm-Keller,Brett D Hambly,Martin R Larsen,David E James,Stuart J Cordwell

doi:10.1074/jbc.m114.556035

Abstract

Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.

Highlights

Myocardial ischemia and cardioprotection induce signal networks mediated by post-translational modification
Protein abundance displayed very low variation, whereas larger variations were observed for phosphopeptides and lysineacetylated peptides, suggesting the myocardial response to acute ischemia and IPC is dominated by changes in Post-translational modification (PTM) rather than protein abundance (Fig. 1B)
Perfusion of rat hearts with 10 ␮M splitomicin, without subsequent ischemia or IPC, increased global acetyl-Lys by ϳ10% and phosphorylation by ϳ2.5%. 20 min of ischemia (20I) increased global phosphorylation and acetyl-Lys, and both were further increased by splitomicin

Summary

Background

Myocardial ischemia and cardioprotection induce signal networks mediated by post-translational modification. Myocardial ischemia and cardioprotection by ischemic preconditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase.

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION