Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector.
The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.
Höss
A
A
Institute for Biochemistry, Munich, Federal Republic of Germany.
Moarefi
I
I
Scheidtmann
K H
KH
Cisek
L J
LJ
Corden
J L
JL
Dornreiter
I
I
Arthur
A K
AK
Fanning
E
E
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000302:isolation & purification; D016203:CDC2 Protein Kinase; D002460:Cell Line; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D010750:Phosphoproteins; D011494:Protein Kinases; D016203:CDC2 Protein Kinase