Journal of Biological Chemistry
Volume 274, Issue 51, 17 December 1999, Pages 36741-36749
Journal home page for Journal of Biological Chemistry

CELL BIOLOGY AND METABOLISM
Phosphorylation of B-Myb Regulates Its Transactivation Potential and DNA Binding*

https://doi.org/10.1074/jbc.274.51.36741Get rights and content
Under a Creative Commons license
open access

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.

Cited by (0)

*

This work was supported in part by a pilot project from the University of Nebraska Medical Center/Eppley Cancer Center, Grant BE-260 from the American Cancer Society, and NCI Grant P30 CA36727 from the National Institutes of Health (to the Eppley Cancer Institute).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by NCI Health Training Grant CA09476 from the National Institutes of Health.

Present address: Dept. of Biochemistry and Molecular Genetics, University of Colorado Health Science Center, Denver, CO 80262.