Covalent Modification of CDK2 by 4-Hydroxynonenal as a Mechanism of Inhibition of Cell Cycle Progression

Oxidative stress is a contributing factor in a number of chronic diseases; including cancer, atherosclerosis, and neurodegenerative diseases. Lipid peroxidation-that occurs during periods of oxidative stress results in the formation of lipid electrophiles, which can modify a multitude of proteins in the cell. 4-Hydroxy-2-nonenal (HNE') is one of the most well-studied lipid electrophiles and has previously been shown, to, arrest :cells at the Gl/S transition. Recently, proteomic data have shown, that HNE is capable of covalently modifying CDK2, the kinase responsible for the Gl/S transition. Here, we identify the sites adducted: by HNE using recombinant CDK2 and show that HNE treatment suppresses the kinase activity of the enzyme. We further identify sites of adduction in HNE-treated intact human colorectal carcinoma cells.(RKO), and show that HNE-dependent modification in cells is long-lived, disrupts CDK2 function, and correlates with a delay of progression of the cells into S-phase. We propose that adduction of CDK2 by HNE directly alters its activity, contributing to the cell cycle delay.