Characterization of the 72 kDa inositol polyphosphate 5-phosphatase

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Cells respond to external signals and the enviroment to undergo cell growth, secretion and or other specialized functions including control of cell death and or cell size. We have identified a new enzyme (72 kDa 5-phosphatase) which resides inside the cell, which we have evidence plays a role in regulating both the movement of intracellular vesicles and also lipid signals stimulated by insulin. We have characterised the phospholipids that the enzyme cleaves and demonstrated the generation of new cell signals at specific subcellular localizations on intracellular membranes. We predict the generation of these specific lipid signals may play a significant role in controlling the transport of intracellular cargo to specific sites in the cell. In this grant proposal we aim to examine the regulation of specialised cargo called the glucose transporter, which is found in fat and muscle cells, and also the mannose 6-phosphate receptor, which regulates the trafficking of specific enzymes which mediate digestion of proteins. These studies include the clarification of which phospholipid signals the enzyme terminates and where in the cell this occurs. Secondly, we will examine the movement of the glucose transporter GLUT-4 in unstimulated cells and in response to insulin and furthermore how expression of the novel enzyme regulates its movement. We will also examine the movement of the mannose 6-phosphate receptor and the specific phospholipid signals which control the route the receptor traffics, using inhibitors of lipid signals and expression of lipid phosphatases and kinases. We will also examine how our novel enzyme forms complexes with other molecules in the cell and characterise these novel molecules using basic biochemical assessment of enzyme activity and function. Finally we will examine the regulation of intracellular messages by our novel enzyme following insulin stimulation, which facilitates glucose uptake into the cell.