STRUCTURE OF AS160, AKT SUBSTRATE REGULATING GLUCOSE TRANSPORTER TRANSLOCATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Upon sensing high glucose levels in blood, pancreatic beta-cells secret insulin to lower the glucose level necessary for homeostasis. Insulin binds to insulin receptor and triggers multiple kinase cascades, which in the end lead to the translocation of glucose transporters from the intracellular storage vesicles to the cell membrane. Membrane bound glucose transporters allow the influx of the outside glucose into the cell to ultimately lower the blood glucose. High blood glucose levels caused by a decrease in this insulin action characterize the condition of the disease diabetes. The farthermost downstream target involving this glucose transporter translocation is the phosphorylation of a recently discovered protein AS160 (Akt substrate of 160kDa) by an insulin-activated kinase, Akt. Recent studies suggest that AS160 phosphorylation upon insulin action deactivates RabGTPases, which is a crucial event in the internalization of glucose transporters. Our structural target is this AS160 protein acting as a switch to turn on/off the insulin signal. We are especially interested in a 300-residue RabGAP domain at the C-terminus of AS160, which is responsible for activating the RabGTPase. We have obtained crystals of human AS160 RabGAP domain, which diffracts to 6¿¿¿ resolution at our home rotating anode X-ray source. We believe that a success in our project depends heavily on the access to a synchrotron facility. Since we were successful at incorporating heavy metals and stabilizing the crystals we intend to perform MAD experiments at a tunable beamline, which would be important for the phase determination. We believe that RabGAP domain by itself will be informative since structure can lead us in designing a drug that inactivates AS160 to simulate the action of insulin. This novel approach targeting the utmost downstream pathway in lowering blood glucose may be a promising treatment to test in diabetics to whom their response to insulin is failing.