Investigation of Kinase/s to Determine the Catalytic Activity of SGKl at a Site Regulated by Drugs of Abuse
Moore, D. Blaine, 1972-
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Drug addiction is a serious health issue with limited and inadequate treatment options. Although dysregulation of the brain reward circuitry has been implicated in addiction, our understanding of signaling changes within these pathways is not well understood. Previous work from our lab demonstrated that chronic administration of cocaine and morphine increases phosphorylation and activity of serum and glucocorticoid-inducible kinase 1 (SGKl) in the ventral tegmental area of mice. However, the kinase/s responsible for phosphorylation of S78 in SGKl are currently unknown. Previously an in silico approach was used, identifying four candidate kinases potentially capable of phosphorylating SGKl S78: glycogen synthase kinase 3 beta (GSK3p), extracellular-signal regulated kinase 5 (ERK5), ERK 1/2,and cyclin dependent kinase 5 (CDK5). To determine whether these candidate kinases are capable of phosphorylating SGKl S78, a series of pharmacological inhibitors were tested in Neuro2A cells, a mouse Neuroblastoma cell line. Following serum starvation, the cells were incubated with insulin and specific kinase inhibitors, then lysed and the protein was isolated for Western blot analyses. Insulin was used in place of drugs of abuse as they had previously been determined to elicit similar increases in SGKl catalytic activity. Importantly, multiple concentrations of inhibitors were used in order to generate an inhibition curve to identify the inhibitor with the greatest potency for decreasing SGKl S78 phosphorylation. While inhibitor trials were inconclusive, they revealed that increased doses of inhibitors may be required to decrease SGKl catalytic activity. Future studies should further investigate the role of SGKl S78 phosphorylation, in combination with additional phosphorylation sites to determine a potential therapeutic target avenue for drug addiction.