Induction of p19INK4d and inhibition of cyclin D–dependent Rb kinase activity in zinc-induced 32D clones. (A) Immunoblot analysis of parental 32D cells (lane 1), a polyclonal population of control cells receiving the empty pMT-CB6+ vector (lanes 2 and 3), and six single cell-derived subclones electroporated with the pMT-CB6+p19 vector (lanes 4 to 15) before (even numbered lanes) or after (odd numbered lanes) treatment with 75 μmol/L zinc for 4 hours. An Sf9 cell lysate containing recombinant p19 was used as an internal control to mark the mobility of the protein in the gel (lane 16). (B) Time course after zinc-addition, and dose-dependence of p19 expression. A p19-inducible clone was grown in IL-3 and 75 μmol/L zinc for the indicated times in hours, or treated with the indicated concentrations (μmol/L) of zinc for 5 hours. Sf9 cell lysates containing p19 were used to mark the position of p19 in the gel (right lane, Sf9-p19). (C) Induced p19 inhibits cyclin D3–dependent kinase activity. Cell lysates were prepared from clone 2C at the indicated times in hours after zinc-addition and were precipitated with an irrelevant control monoclonal antibody (lane C) or with a monoclonal antibody to mouse cyclin D3 (D3-19D5-13). Washed immune complexes were assayed for kinase activity in vitro using bacterial GST-Rb fusion protein as substrate. Lysates from untreated cells (time = 0) were treated as indicated for 5 minutes at room temperature with 1 μg recombinant GST-p19 or GST alone to verify that p19 inhibits the Rb kinase activity precipitated from these cells. Recombinant cyclin D3-CDK4 complexes were prepared in Sf9 cells and used as a positive control for enzyme activity (right lane). (D) The same lysates as in (C) were immunoprecipitated with nonimmune rabbit serum (lane C) or with rabbit antiserum (RZ ) directed to the CDK4 C-terminus, and washed precipitates were assayed for Rb kinase activity as in (C).
