Phosphorylation of simian virus 40 large T antigen: cytoplasmic and nuclear phophorylation sites differ in their metabolic stability.

The turnover of phosphate residues in simian virus 40 (SV40) large T antigen (large T) was determined by pulse-chase labeling with 32Pi and subsequent two-dimensional peptide mapping. Three groups of phosphorylation sites with respect to metabolic stability were distinguished with half-lives of about 8, 3 to 5, or 2 hr, respectively. Sites phosphorylated in the cytoplasm were relatively stable, whereas most of the sites phosphorylated in the nucleus exhibited high turnover rates. We suggest that sites with low turnover represent permanent modifications whereas sites with high turnover might contribute to the dynamic properties of large T, e.g., its interaction with the viral genome. When the phosphate turnover in various subclasses of large T was compared the monomeric and oligomeric forms showed no significant differences. Likewise, the DNA-binding and nonbinding fractions exhibited similar phosphate turnover. However, in the DNA-binding fraction the 3H label decreased faster than the 32P label indicating that large T in this fraction had been replaced by newly synthesized molecules which were not labeled with 3H but nevertheless with 32P. this latter result suggests that a certain degree of phosphorylation might be required for DNA binding.