FIGURE 1:
The binding of mRNA export complex proteins, as measured by FRAP, is regulated by PI3 kinase, AKT, and mTOR. HeLa cells were transfected with EGFP-fusion proteins for UAP56, ALY/REF, and NXF1. After 48 h, cells were treated with 20 μM of the PI3 kinase inhibitor LY294002 (green circles) for 2 h, 5 μM of AKT inhibitor VIII (blue triangles) for 3.5 h, and 100 nM of the mTOR inhibitor rapamycin (red squares) for 5 h. Multiple cells were examined by FRAP over a 2-h interval in all treatment groups. The times between drug addition and photobleaching are reported as a mean. Black squares, untreated cells. Normalized fluorescence recovery curves are presented for EGFP-UAP56, EGFP-TAP/NXF1, and EGFP-ALY/REF. Also shown is EGFP-UAP56 K95N, a point mutant that cannot bind ATP. Each colored arrow marks the t1/2 of recovery for the curve of the same color. Statistical analysis of the differences between averaged FRAP curves was by paired one-tailed t test: untreated EGFP-UAP56 vs. LY294002 (p = 0.0083), AKT (p < 0.0001), rapamycin (p = 0.2799); untreated EGFP-ALY/REF vs. LY294002 (p < 0.0001), AKT (p < 0.0001), rapamycin (p < 0.0001); untreated EGFP-NXF1 vs. LY294002 (p = 0.0002), AKT (p = 0.0004), rapamycin (p = 0.4102). Means are plotted with error bars for standard errors. n, number of cells in each treatment group.