FIG. 8.

A Tax point mutant neither coimmunoprecipitates with cyclin D3 nor induces increased cyclin D3-cdk6 kinase activity. (A) Inducible expression of Tax in JPX9 and JPX9m cell lines. Expression of Tax wild-type and Tax mutant was assessed by Western blotting with a monoclonal antibody to Tax. +, Tax wild type and mutant induced in JPX9 and JPX9m strains with zinc; −, control mock-treated cells. JPX9m expresses a transcriptionally inactive Tax mutant that contains a single amino acid (arginine) inserted at position 62. (B) JPX9 or JPX9m cells were treated (lanes 2 and 4) or mock treated with zinc (lanes 1 and 3) for 16 h. After solubilization in lysis buffer, complexes were immunoprecipitated under native conditions with polyclonal antisera against cyclin D3. The presence of Tax in complexes was assessed by Western blotting with a monoclonal antibody to Tax. (C) Tax mutant protein in JPX9m does not compel G₁-to-S progression as measured by [³H]thymidine incorporation. JPX9 and JPX9m cells were synchronized by serum starvation and then treated with or without zinc for 16 h. Cells were then plated into a 96-well plate and labeled with [³H]thymidine. Cell cycle progression from G₁ to S phase was measured by [³H]thymidine incorporation. The relative values for JPX9 and JPX9m cells were normalized against parallel values obtained in similarly treated Jurkat cells, with thymidine incorporation for JPX9 set at 100%. (D) Comparison of kinase activity of cyclin D3-cdk6 in cells expressing Tax mutant or Tax wild type. In vitro kinase assays with GST-Rb as substrate were performed. JPX9 or JPX9m cells were synchronized by serum starvation for 20 h. Cells grown in the presence (+; lanes 6, 8, 10, and 12) or absence (−; lanes 5, 7, 9, and 11) of zinc for 8 h were harvested, and cell extracts were immunoprecipitated with either anti-cdk6 (lanes 5 to 8) or anti-cyclin D3 (lanes 9 to 10) antibody. The immunoprecipitates were tested for kinase activity with GST-Rb as the substrate.