TY - JOUR
T1 - Phosphorylation of B-Myb regulates its transactivation potential and DNA binding
AU - Johnson, Teresa K.
AU - Schweppe, Rebecca E.
AU - Septer, John
AU - Lewis, Robert E.
PY - 1999/12/17
Y1 - 1999/12/17
N2 - The transcription factor B-Myb is a cell cycle-regulated phosphoprotein and a potent regulator of cell cycle progression. Previous studies demonstrated that B-Myb was phosphorylated at the onset of S phase, suggesting that it could be due to cyclin-dependent kinases. We identified 10 B-Myb phosphorylation sites by automated peptide radiosequencing of tryptic phosphopeptides derived from in vivo 32P-labeled B-Myb. Each B-Myb phosphorylation site contained a phosphoserine or phosphothreonine followed by a proline, suggesting that this phosphorylation is due to a proline- directed kinase. Cyclin A-Cdk2 and cyclin E-Cdk2 complexes each phosphorylated B-Myb in a cell-free system on the same sites as in intact cells. Furthermore, the ability of B-Myb to activate a reporter plasmid was enhanced by the cotransfection of cyclin A, whereas mutagenesis of the 10 identified phosphorylation sites from B-Myb blocked the effect of cyclin A coexpression. Additional analysis revealed that the effect of phosphorylation on B-Myb transactivation potential was enhanced by phosphorylation sites in its carboxyl-terminal half. One phosphorylation site (Ser581) appeared to negatively regulate DNA binding, as mutation of this site enhanced the ability of B-Myb to bind a Myb-binding sequence. These data suggest that B- Myb is a target for phosphorylation by cyclin-Cdk2 and that phosphorylation of B-Myb regulates its transcriptional activity.
AB - The transcription factor B-Myb is a cell cycle-regulated phosphoprotein and a potent regulator of cell cycle progression. Previous studies demonstrated that B-Myb was phosphorylated at the onset of S phase, suggesting that it could be due to cyclin-dependent kinases. We identified 10 B-Myb phosphorylation sites by automated peptide radiosequencing of tryptic phosphopeptides derived from in vivo 32P-labeled B-Myb. Each B-Myb phosphorylation site contained a phosphoserine or phosphothreonine followed by a proline, suggesting that this phosphorylation is due to a proline- directed kinase. Cyclin A-Cdk2 and cyclin E-Cdk2 complexes each phosphorylated B-Myb in a cell-free system on the same sites as in intact cells. Furthermore, the ability of B-Myb to activate a reporter plasmid was enhanced by the cotransfection of cyclin A, whereas mutagenesis of the 10 identified phosphorylation sites from B-Myb blocked the effect of cyclin A coexpression. Additional analysis revealed that the effect of phosphorylation on B-Myb transactivation potential was enhanced by phosphorylation sites in its carboxyl-terminal half. One phosphorylation site (Ser581) appeared to negatively regulate DNA binding, as mutation of this site enhanced the ability of B-Myb to bind a Myb-binding sequence. These data suggest that B- Myb is a target for phosphorylation by cyclin-Cdk2 and that phosphorylation of B-Myb regulates its transcriptional activity.
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U2 - 10.1074/jbc.274.51.36741
DO - 10.1074/jbc.274.51.36741
M3 - Article
C2 - 10593981
AN - SCOPUS:0033579527
SN - 0021-9258
VL - 274
SP - 36741
EP - 36749
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 51
ER -