Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins.
Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.
Culleré
X
X
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Rose
P
P
Thathamangalam
U
U
Chatterjee
A
A
Mullane
K P
KP
Pallas
D C
DC
Benjamin
T L
TL
Roberts
T M
TM
Schaffhausen
B S
BS
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D017931:DNA Primers / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011120:Polyomavirus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D027601:Polyomavirus Infections / Q000209:etiology; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D018411:Spodoptera; D014162:Transfection; D014412:Tumor Virus Infections / Q000209:etiology; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D011506:Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D011505:Protein-Tyrosine Kinases