ZSTK474 is a new PI3K inhibitor with strong antitumor activity against human cancer xenografts without toxic effects in critical organs. ZSTK474 merits further investigation as an anticancer drug.

Analyses of mice lacking glycosyltransferase have suggested that their pathological phenotypes are not attributable to the overall change of the sugar modification, but instead the result of changes of the glycan structures on a specific 'target' glycoprotein. Therefore, detecting or monitoring the glycosylation status of a specific protein in living cells is important, but no such methods are currently available. Here we demonstrate the detection of glycoforms of a specific glycoprotein using the fluorescence resonance energy transfer technique. using model proteins, we detect characteristic fluorescence resonance energy transfer signals from the specific glycoform-bearing target glycoprotein. We also show that, upon insulin removal, sialylated glycoforms of green fluorescent protein-tagged GLuT4 seem to be internalized more slowly than non-sialylated GLuT4. This novel analytical imaging tool allows studying the roles of specific glycan modifications of a protein of interest.

The facilitative glucose transporter GLUT4 plays a key role in regulating whole body glucose homeostasis. GLUT4 dramatically changes its distribution upon insulin stimulation, and insulin-resistant diabetes is often linked with compromised translocation of GLUT4 under insulin stimulation. To elucidate the functional significance of the sole N-glycan chain on GLUT4, wild-type GLUT4 and a GLUT4 glycosylation mutant conjugated with enhanced GFP were stably expressed in HeLa cells. The N-glycan contributed to the overall stability of newly synthesized GLUT4. Moreover, cell surface expression of wildtype GLUT4 in HeLa cells was elevated upon insulin treatment, whereas the glycosylation mutant lost the ability to respond to insulin. Subcellular distribution of the mutant was distinct from that of wild-type GLUT4, implying that the subcellular localization required for insulin-mediated translocation was impaired in the mutant protein. Interestingly, kifunensine-treated cells also lost sensitivity to insulin, suggesting the functional importance of the N-glycan structure for GLUT4 trafficking. The K m or turnover rates of wild-type and mutant GLUT4, however, were similar, suggesting that the N-glycan had little effect on transporter activity. These findings underscore the critical roles of the N-glycan chain in quality control as well as intracellular trafficking of GLUT4.

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