In vivo and in vitro phosphorylation of the adenovirus type 5 single-strand-specific DNA-binding protein

Abstract

The type 5 adenovirus single-strand-specific DNA-binding protein can be labeled with 32PO 4 added as inorganic phosphate to the medium of infected monkey or human cells. The DNA binding protein in the infected cell can be isolated as a phosphoprotein at early and late times after infection. Most or all of the [ 32P]phosphate can be removed from the DNA-binding protein by alkaline phosphatase or hydrolysis at alkaline pH. The dephosphorylated protein retains the ability to bind specifically to single-stranded DNA. H5 ts125 is a mutation in the structural gene of the 72,000 MW single-strand-specific DNA-binding protein. When cells infected with this viral mutant at the permissive temperature are shifted to the nonpermissive temperature, the phosphorylation of the 72,000 MW protein decreased rapidly (within 30 min) even though approximately normal (wild type) levels of this protein (labeled with 35S]methionine for 30 min) were detected in H5 ts125-infected cells shifted to the nonpermissive temperature. An in vitro protein kinase assay utilizing nuclei from infected or uninfected cells has been developed. This in vitro assay can detect the phosphorylation of at least three adenovirus-induced phosphoproteins (100,000 MW; 72,000 MW; 36,000 MW) that are also observed when adenovirus-infected cells are labeled with inorganic [ 32P] phosphate in vivo. The protein kinase activity detected in this in vitro assay is not dependent upon or stimulated by the addition of exogenous cyclic AMP or cyclic GMP. The 72,000 M W protein phosphorylated in vitro by this phosphokinase activity has been shown to be the adenovirus single-strand-specific DNA-binding protein by a specific immunoprecipitation test.