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Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.

Department of Pathology, University of California, San Diego, La Jolla 92093.

ABSTRACT

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.

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