Fig. 3. Nmyc1T50A and Nmyc1S54A show transactivation capacity equivalent to wild-type Nmyc1 but have enhanced stability. (A) Nmyc1, Nmyc1T50A and Nmyc1S54A were compared for their ability to activate target gene expression as determined by a luceriferase reporter assay. The graph depicts relative luciferase activity in HeLa cells co-transfected with Nmyc1 or Nmyc1 phosphorylation mutant retroviral constructs, and a reporter plasmid containing luciferase driven by either the human Cdk4 promoter (dark bars) or the same promotor lacking E boxes, Myc proteins' cognate DNA recognition sequence (light bars). Results shown are the average of three independent experiments±s.e.m. No significant differences in transactivation capacity between wild-type Nmyc1 or the phosphomutants were detected. (B) Nmyc1T50 and Nmyc1S54 are more stable than wild-type Nmyc1. Primary CGNP cultures were infected with retroviruses expressing wild-type Nmyc1, Nmyc1T50A or Nmyc1S54A. After 24 hours, the cells were pulsed with cycloheximide for 30-90 minutes (three trials per time point) to prevent the synthesis of new protein and to allow an assessment of remaining previously synthesized protein. Protein lysates were prepared and blotted for Nmyc1. A representative western blot is shown from a 90 minute cycloheximide pulse. By 90 minutes, levels of retrovirally expressed wild-type Nmyc1 had substantially declined. Levels of Nmyc1T50A and Nmyc1S54A proteins were not strongly affected by cycloheximide treatment. Relative to retrovirally expressed wild type Nmyc1, increased levels of Nmyc1T50A and Nmyc1S54A protein levels can be seen (left), despite similar levels of retroviral transcript expression, as determined by northern blotting for Nmyc1 (right), consistent with an inability to be phosphorylated leading to protein stabilization and accumulation. (C) Nmyc1T50A and Nmyc1S54A enhance proliferation in human neuroblastoma cells, in comparison with ectopically expressed wild-type Nmyc1. SK-N-SH neuroblastoma cells were infected as indicated and proliferation was assessed after 48 hours using flow cytometry. The graph shows levels of cells in S phase, relative to GFP-infected cells. (D) Mutation of T50 or S54 to aspartic acid has different effects on Nmyc1 turnover. Cycloheximide chase assay was performed as described, and a representative western blot from a 90-minute cycloheximide chase is shown. Nmyc1S54E shows similar turnover characteristics to wild-type Nmyc1, whereas Nmyc1T50E is more stable than wild-type Nmyc1. These results indicate that the effect of phosphorylation at that site requires the presence of the phosphate group and does not depend merely upon the negative charge transferred by phosphorylation.