FIGURE 6.

DDN interacts and activates the insulin receptor. A, DDN does not complete with insulin for IR binding site. FITC-insulin was incubated with CHO-IR cells in the presence of various concentrations of DDN (top panel) or unlabeled insulin (middle panel). Parental CHO cells were used as a negative control (bottom panel). The fluorescence-labeled cells were analyzed on flow cytometry. B, DDN binds to IRTK. UV-visible spectra were scanned from 200 to 800 nm for IRTK fragment in the presence of DDN or its analog. When DDN binds to IRTK, it elicits the absorption alteration at 560 and 602 nm. C, DDN protects IRTK from proteolysis. GST-IRTK was subjected to limited trypsin digestion in the presence of DDN (50 μm), ATPγS (5 mm), or both. The reaction mixture was resolved by SDS-PAGE, followed by silver staining. D, DDN increases IRTK activity in vitro. Recombinant GST-IRTK was incubated with different concentrations of DDN or its inactive analog 2NH₂-NQ, and the kinase activity of IRTK was examined by monitoring the phosphorylation of histone H2B using autoradiography.