Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains.
The simian virus 40 large T antigen is phosphorylated at eight or more sites that are clustered in an amino-terminal region and a carboxy-terminal region of the protein. Mutants carrying exchanges at these phosphorylation sites have been generated in vitro by bisulfite or oligonucleotide-directed mutagenesis and analyzed for their phosphorylation patterns. Two-dimensional phosphopeptide analyses of the mutant large T antigens confirmed most of the previously identified phosphorylation sites, namely, serine residues 106, 112, 123, 639, 677, and 679 and threonine residues 124 and 701. In addition, serine residue 120 was identified as a new site, whereas serines residues 111 and 676 were excluded. Interestingly, several of the mutants exhibited secondary effects in that a mutation in the amino-terminal region affected phosphorylation at distant and even carboxy-terminal sites and vice versa. Thus, the amino- and carboxy-terminal domains appear to be in close proximity in the three-dimensional structure of large T antigen. The possible consequences of the above findings and the role of phosphorylation are discussed.
Scheidtmann
K H
KH
Abteilung Molekulargenetik, Universität Bonn, Germany.
Buck
M
M
Schneider
J
J
Kalderon
D
D
Fanning
E
E
Smith
A E
AE
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D051381:Rats; D012694:Serine; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D010748:Phosphopeptides; D012694:Serine