TORC-dependent CREB activity is regulated by glucose in beta cells
Creators: name: Jansson, Deidre
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2009
Subjects: subject: Biology, Molecular.

description: Our bodies go through cycles of feeding and fasting, and in order to control blood glucose levels, will respond by releasing hormones such as glucagon and insulin. After feeding, insulin is released from the pancreatic beta cells to reduce blood glucose levels. When our bodies are unable to do this the result is hyperglycemia, leading to diabetes, of which there are two types. Type I diabetes is an autoimmune disease where the body's T cells attack and destroy the insulin producing beta cells, rendering these patients insulin-dependent. Type 2 diabetes however, results from chronic high blood glucose levels. This will lead to insulin resistance and eventually beta cell apoptosis. Recently it was observed that the transcription factor cAMP Response Element Binding (CREB) protein is required for beta cell survival. Expression of a dominant-negative CREB under the control of the beta cell specific rat insulin promoter leads to progressive reduction of beta cell mass and diabetes in mice. Interestingly, the same signals that lead to insulin secretion, cAMP and Ca2+, are required for activation of CREB. Transducers of Regulated CREB activity (TORCs) were recently identified as CREB coactivators that are responsible for the synergistic activation of CREB target genes by cAMP and Ca2+. Under resting conditions phosphorylated TORCs are sequestered in the cytoplasm by 14-3-3 proteins. After stimulation with cAMP and Ca2+ TORCs become dephosphorylated and enter the nucleus, where they bind to the bZIP domain of CREB to activate transcription. In the absence of these stimuli, TORC2 is phosphorylated by the salt inducible kinase 2 (SIK2) at S171. Treatment of 293 cells with cAMP leads to the phosphorylation of SIK2 and therefore the dissociation of this kinase from TORC2. Additionally, the calcium responsive phosphatase calcineurin (Cn) in response to calcium stimuli dephosphorylates TORC2 leading to dissociation of 14-3-3 proteins and hence TORC2 nuclear entry. Interestingly while 293 cells require no more than a cAMP stimulus for TORC2 nuclear entry, beta cells remain in the cytoplasm following the same treatment. Additionally, only after treatment with cAMP and calcium does TORC2 lose 14-3-3 binding and enter the nucleus of beta cells to activate CREB. My hypothesis was that there remained additional regulatory phosphorylation site(s) that governed TORC2: 14-3-3 binding and therefore nuclear entry of TORC2 in beta cells. Work in this thesis reveals that s275 is the remaining phosphorylation site that governs 14-3-3 binding and is regulated by glucose/Ca2+ stimuli in beta cells. This evidence provides an explanation for the synergistic activation of TORC2 and therefore CREB in response to cAMP and Ca2+ . Additionally the potential kinase responsible for phosphorylation of this site was identified to be MARK2, a member of the AMPK family of kinases. I have identified an additional phosphorylation site in TORC2, S275 that is involved in 14-3-3 binding. This serine, in addition to S171 must be dephosphorylated to induce TORC2 nuclear entry and subsequent CREB activation. I was also able to show that the phosphorylation of this site is responsive to glucose in beta cells and in primary mouse islets. The goal of this thesis was to identify these remaining sites in order to uncover the regulatory mechanisms of TORC2-dependent CREB activation in response to cAMP and Ca2+ in the insulin producing beta cells.