Functional Alterations in CDK2 by Lipid Electrophile Modification

Vanderbilt University, Nashville TN

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Abstract

? DESCRIPTION (provided by applicant): Chromic inflammation, as a result of continuous oxidative stress, has been shown to be a contributing factor to the development of cancer by both experimental and epidemiological studies. Inflammation can contribute to tumorigenesis by a number of different pathways, including promotion of genomic instability, alterations of epigenetic markers, increases in proliferation, and resistance to apoptosis. While reactive oxygen species (ROS) alone are capable of inducing these different pathways to promote transformation, the high reactivity of ROS results in reactions near the site of production. Polyunsaturated lipids within membrane bilayers, most notably mitochondrial membranes, are key targets of ROS. Reactions between these lipids and ROS result in lipid peroxidation and, in some cases, can generate small, reactive lipid electrophiles. 4-Hydroxynonenal (HNE) is the most commonly studied lipid electrophile. Due to its lower reactivity relative to ROS, HNE can diffuse throughout the cell and covalently modify both DNA and proteins; modification of cellular proteins can result in altered function. Previous proteomic data have shown a number of proteins susceptible to modification by HNE, one of which is CDK2. Cyclin-dependent kinase 2 (CDK2) is a cell cycle protein responsible for the G1/S transition and for maintaining progression through S-phase. Interestingly, microarray studies have shown down-regulation of genes associated with S-phase following HNE treatment; many of these genes are controlled upstream by CDK2 kinase activity. Together, these data suggest that HNE-modification of CDK2 could alter its function, resulting in protein inactivation and cell cycle arrest. Our preliminary data hve shown that CDK2 is modified by HNE on a number of histidine and lysine residues. Treatment of synchronized cells with HNE causes a delay in progression into S-phase. Most notably, treatment of cells treated with HNE results in a decrease in CDK2 kinase activity. Given these data, we hypothesize that HNE covalently modifies CDK2 and inhibits its kinase activity, thereby resulting in G1-phase arrest until HNE-modified CDK2 is degraded. I propose two aims to test this hypothesis: 1) Assess the functional impact of HNE modification of CDK2 and 2) Quantify the extent of CDK2 modification by HNE in RKO cells. The research proposed in this application will assess the functional significance of HNE modification on a main cell cycle regulator and kinase, thereby increasing our understanding of altered protein function as a result of HNE modification. The aims proposed will provide a mechanism for how small changes in protein structure from the adduction by HNE can significantly disrupt interactions within the cell.