Table II. The tyrosine phosphorylation of Ad2 E4orf4 regulates its cytoplasmic membrane activity but not its nuclear activity.
| Constructs | C-33A | 293T | 293 | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NL | Apoptosis | NL | Apoptosis | NL | Apoptosis | |||||||||||
| % cells
|
||||||||||||||||
| 24 h | 48 h | 48 h | 72 h | 24 h | ||||||||||||
| GFP | – | 10 | 8 | – | 4 | – | – | 9 | ||||||||
| GFP-NLS | 69 | 5 | 4 | 76 | 3 | 4 | – | 11 | ||||||||
| GFP-CAAX | <1 | 11 | 13 | 1 | 7 | – | – | 7 | ||||||||
| orf4-GFP | 29 | 37 | 56 | 35 | 56 | – | 42 | 37 | ||||||||
| 3YF-GFP | 64 | 21 | 41 | 77 | 13 | 26 | – | 20 | ||||||||
| orf4-GFP-NLS | 65 | 14 | 34 | 94 | – | 33 | 92 | 21 | ||||||||
| 3YF-GFP-NLS | 92 | 17 | 34 | 91 | – | 30 | – | – | ||||||||
| orf4-GFPCAAX | 2 | 39 | 51 | 2 | 42 | – | – | 48 | ||||||||
| 3YF-GFPCAAX | 9 | 13 | 16 | 19 | 14 | – | – | 10 | ||||||||
| n > 600 | n > 900 | n > 600 | n > 900 | n = 500 | n = 1,000 | |||||||||||
293T, 293, and C-33A cells were transfected with the indicated GFP constructs; (3Y-F), nonphosphorylated mutant E4orf4. At the indicated times after transfection, cells were fixed and DNA was labeled with DAPI. Protein targeting efficiencies and apoptotic activities (nuclear condensation and fragmentation) were evaluated by fluorescence microscopy and are expressed as the percentage of nuclear localization (NL), or the percentage of apoptosis, relative to the total number of GFP-positive cells. Data are the means ± SE of four independent experiments.