Fig. 4.

Characterization of Tax amino acids important for Ran interaction. (A) A schematic overview of the different Tax mutants fused to GFP. CBP, CREB-binding protein; MOD, modulator domain; PCAF, p300–CREB-binding protein-associated factor. (B) Delineation of HTLV-1 Tax residues important for Tax–Ran interaction and centrosome localization. MEFs were cotransfected with Ran–HA and Tax deletion mutants (lanes 4–9). (Top) Ran–HA was immunoprecipitated with anti-HA. GFP fusion protein was detected by anti-GFP. (Middle and Bottom) Amount of Ran–HA in the immunoprecipitate (IP) and GFP–Tax in cell extracts were checked with anti-HA and anti-GFP. (C) Anti-pericentrin and anti-GFP were used to reveal the centrosomal localization of Tax in MEFs transfected with GFP–Tax deletion mutants. In these enlarged images, TaxTD1, TD254, and TD319 but not TD55, TD99, or TD150 localized to centrosomes. (D) Tax localization to the centrosome is insufficient to induce aberrant amplification. MEFs were cotransfected with GFP–Tax and Ran–HA. Anti-HA, anti-pericentrin, and anti-GFP were used to reveal the subcellular localizations. DNA was stained with Hoechst 33342. Whereas wild-type Tax (NF-κB⁺/CREB⁺) induced centrosome amplification, Tax S258A (NF-κB^–/CREB⁺), a mutant unable to activate NF-κB, localized to the centrosome (arrowheads) but did not induce centrosome amplification. In contrast, Tax S 274A (NF-κB^–/CREB^–), a CREB-defective mutant, can bind Ran but did not localize to the centrosome (arrowheads). Tax L320G (NF-κB⁺/CREB^–) behaved like GFP–Tax.