Project Summary/Abstract At the cellular level, impaired signaling cascades both contribute to and are characteristic of metabolic syndrome, a constellation of interrelated disorders including hyperlipidemia and hyperglycemia. However, the mechanisms of pathogenesis are complex, multifactorial, and incompletely described. Recent studies suggest a role for protein S-palmitoylation, a dynamic lipid post-translational modification (PTM) that affects protein function and activity, in the regulation of metabolic signaling events. As the addition of the palmitate lipid to cysteine is dependent on lipid levels – which are in turn altered in metabolic syndrome – protein lipidation represents a potential mechanistic link between aberrant nutrient factors and changes in signaling protein activity. We have observed that the extracellular signal-regulated kinase (ERK), whose signaling-responsive activity regulates cellular metabolism/energy homeostasis and is altered in metabolic syndrome, is subject to a rapid, signaling- induced increase in palmitoylation. Moreover, the basal level of ERK palmitoylation is increased in a mouse model of obesity, confirming that its lipidation status is sensitive to diet. In this project, with ERK as the focal point, we aim to map the connections between regulatory S-palmitoylation, signal transduction, and the sequelae of metabolic syndrome. In Aim 1, we will use a combination of molecular biological, biochemical, and chemical approaches to determine the molecular mechanisms of ERK palmitoylation and to elucidate the impact of dynamic S-palmitoylation on ERK-mediated signal transduction. In order to assess the contribution of increased ERK palmitoylation to metabolic syndrome, in Aim 2 we will develop a chemical inhibitor of ERK S-palmitoylation. We will then employ it in a mouse model of metabolic syndrome, evaluating changes in ERK activity and the mitigation of markers of metabolic syndrome, including insulin resistance and circulating fatty acid levels. This work will establish a novel regulatory framework for ERK, as well as new chemical tools for probing the mechanisms and consequences of its activity. It will also provide a model for how dynamic protein lipidation can function in cell signaling events and contribute to cellular pathophysiologies. !