Macrophage WEE1 Directly Binds to and Phosphorylates NF‐κB p65 Subunit to Induce Inflammatory Response and Drive Atherosclerosis

Abstract Atherosclerosis has an urgent need for new therapeutic targets. Protein kinases orchestrate multiple cellular events in atherosclerosis and may provide new therapeutic targets for atherosclerosis. Here, a protein kinase, WEE1 G2 checkpoint kinase (WEE1), promoting inflammation in atherosclerosis is identified. Kinase enrichment analysis and experimental evidences reveal macrophage WEE1 phosphorylation at S642 in human and mouse atherosclerotic tissues. RNA‐seq analysis, combined with experiment studies using mutant WEE1 plasmids, shows that WEE1 phosphorylation, rather than WEE1 expression, mediated oxLDL‐induced inflammation in macrophages. Macrophage‐specific deletion of WEE1 or pharmacological inhibition of WEE1 kinase activity attenuates atherosclerosis by reducing inflammation in mice. Mechanistically, RNA‐seq and co‐immunoprecipitation followed by proteomics analysis are used to explore the mechanism and substrate of WEE1. p‐WEE1 promoted inflammatory response through activating NF‐κB shown and further revealed that WEE1 can directly bind to the p65 subunit. It is confirmed that p‐WEE1 directly interacts with the RHD domain of p65 and phosphorylates p65 at S536, thereby facilitating subsequent NF‐κB activation and inflammatory response in macrophages. The findings demonstrate that macrophage WEE1 drives NF‐κB activation and atherosclerosis by directly phosphorylating p65 at S536. This study identifies WEE1 as a new upstream kinase of p65 and a potential therapeutic target for atherosclerosis.