Further Characterization of the Phosphate Moiety of the Adenovirus Type 2 DNA‐Binding Protein

Abstract

The adenovirus type 2 DNA‐binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in molecular weight from 72000 to 70000. The dephosphorylated protein binds to single‐stranded and double‐stranded DNA as well as the phosphorylated protein does. Controlled chymotrypsin treatment cleaves the DNA‐binding protein into two subspecies of Mr about 45000 and 25000. The 45000‐Mr polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25000‐Mr polypeptide contains all the phosphate groups and shows no binding to DNA. Isoelectric focusing gels show heterogeneity of the DNA‐binding protein and 15 subspecies with different charges can be observed after partial dephosphorylation by alkaline phosphatase. After extensive dephosphorylation two or three basic species with a molecular weight around 70000 are observed. Quantitative immunoprecipitation from cells labeled to equilibrium with inorganic 32PO4 gives a molar ratio of phosphate to protein of 4–7 and direct chemical determination of the phosphate residues yields 4 mol Pi/mol protein. These results suggest that there exist subspecies of the protein moiety of the adenovirus DNA‐binding protein. The DNA‐binding protein isolated from infected cells after a short ‘pulse’ of [35S]methionine has a molecular weight which corresponds to that of the dephosphorylated protein. After a ‘chase’ period the molecular weight increases to 72000, but alkaline phosphatase treatment converts it to a species with the same molecular weight as the newly synthesized DNA‐binding protein, indicating that the modification of the protein is due to phosphorylation.

Protein Kinases Associated with the Adenovirus Single‐Stranded DNA‐Binding Protein

“…It has been shown that the Ad 72000-M, DNA-binding protein is phosphorylated in viva [22] and in vitro [21,23,25]. In an attempt to characterize the kinase(s) involved, an Ad5-infected cell extract was prepared and the material was purified by affinity chromatography on DNA-cellulose and gel filtration through Ultrogel.…”

“…As isolated from infected cells, it is a phosphoprotein [21,22], but the influence of phosphorylation on its activity remains unclear.…”

Phosphorylation of Adenovirus DNA-binding Protein

“…Mutations which affect the ability of the virus to grow in monkey cells appear to be confined to the N-terminal region (Anderson et al, 1983;Brough et al, 1985) whereas the functions relating to DNA replication and to DNA-and RNA-binding are located in the C-terminal domain (Ariga et al, 1980;Cleghon & Klessig, 1986;Seiberg et al, 1989). It has been suggested (Linne & Philipson, 1980;Klein et al, 1979) that these two domains can be separated by chymotrypsin treatment of the DBP; indeed some evidence of specific breakdown can be seen in extracts of infected cells. However the exact border of the domains is unknown with reported C-terminal fragments ranging from 34K to 45K.…”

“…In adenovirus type 2 (Ad2) and 5 this polypeptide, designated the DNA-binding protein (DBP), has an apparent Mr of 72K from analysis by SDS-PAGE, a value significantly higher than the value of 59K calculated from sequence data. Isoelectric focusing has resolved purified DBP into as many as 15 subspecies, of which at least some result from differing degrees of phosphorylation (Klein etal., 1979;Linne & Philipson, 1980). However Linne & Philipson (1980) showed that even after extensive treatment with bacterial alkaline phosphatase its apparent Mr was reduced by only 2K.…”

“…Isoelectric focusing has resolved purified DBP into as many as 15 subspecies, of which at least some result from differing degrees of phosphorylation (Klein etal., 1979;Linne & Philipson, 1980). However Linne & Philipson (1980) showed that even after extensive treatment with bacterial alkaline phosphatase its apparent Mr was reduced by only 2K. Therefore the mobility upon electrophoresis has been ascribed mainly to its relatively high proline content and to its highly asymmetrical configuration (van der Vliet et al, 1978).…”