DAPK2 Regulates PKM2 Phosphorylation at Threonine 45 to Facilitate Disturbed Flow-Induced Atherosclerosis

Oscillatory shear stress (OSS), resulting from disturbed blood flow, is implicated in atherosclerotic plaque formation by incompletely understood mechanisms. This study aims to elucidate the involvement of death-associated protein kinase (DAPK) 2 in OSS-induced endothelial cell (EC) activation and atherosclerosis.

Publicly available resources, including genome-wide microarray, RNA sequencing, and single-cell RNA sequencing, were utilized to identify key OSS-sensitive regulatory factors. Techniques such as mass spectrometry, immunoprecipitation, proximity ligation assay, and RNA sequencing were employed to identify pyruvate kinase M2 (PKM2) as the binding protein of DAPK2 and determine the specific site of PKM2 phosphorylation by DAPK2. To assess the role of Dapk2 in vivo, EC-specific Dapk2-deficient mice on an Apoe^-/- (apolipoprotein E^-/-) background were utilized in carotid artery ligation and Western diet-induced atherosclerosis models. Mice with EC-specific overexpression of Pkm2 harboring either a phospho-refractory mutation (Pkm2^T45A) or a phospho-mimicking mutation (Pkm2^T45E) were subjected to carotid artery ligation to further elucidate the functional implications of Pkm2 phosphorylation.

DAPK2 expression was elevated in OSS-exposed regions of human and murine arteries. Mechanistically, Krüppel-like factor 2 (KLF2) suppressed DAPK2 transcription, whereas OSS-induced KLF2 downregulation led to DAPK2 upregulation. EC-specific Dapk2 deficiency or pharmacological inhibition suppressed EC activation and atherogenesis in Apoe^-/- mice, effects reversed by overexpression of wild-type Dapk2 but not by a dominant-negative mutant. Mass spectrometry identified that DAPK2 interacted with the key glycolytic enzyme PKM2 and directly phosphorylated it at threonine 45, leading to PKM2 dimerization and nuclear translocation. Nuclear PKM2 activated the expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 by directly interacting with and activating signal transducer and activator of transcription 1. Elevated PKM2^T45 and signal transducer and activator of transcription 1^Y701 phosphorylation were observed in atheroprone endothelium. Finally, EC-specific overexpression of Pkm2^T45A mitigated disturbed flow-induced atherogenesis, whereas Pkm2^T45E overexpression abrogated the protective effects of Dapk2 deficiency in Apoe^-/- mice.

DAPK2-driven phosphorylation of PKM2 at threonine 45 orchestrates endothelial inflammatory responses to disturbed flow, identifying a novel mechanistic axis and potential therapeutic target in atherosclerosis.