B-Myb function can be markedly enhanced by cyclin A-dependent kinase and protein truncation.

Abstract

Transcription of the B-Myb gene is cell cycle regulated by an E2F-dependent mechanism and its product, B-Myb, is itself a transcription factor required for S-phase entry. Previously, we have shown that B-Myb is specifically phosphorylated during S-phase and that similar modification to a less electrophoretically mobile form could be induced by baculovirus-expressed cyclin A/Cdk2 kinase. We report here that cyclin A-mediated phosphorylation of B-Myb is associated with a marked increase in transactivation function in U-2 OS cells. In contrast to previous studies, transactivation of the luciferase reporter was dependent upon Myb binding sites located upstream of the promoter. Enhancement of B-Myb activation function was also obtained by truncation of the C-terminus just downstream of a domain conserved in evolution. Potentiation of B-Myb activity by phosphorylation was not simply a consequence of overcoming the negative effect of the C-terminus, however, as the truncated protein was to a lesser extent also activated by cyclin A/Cdk2. Whereas wild-type B-Myb transactivation activity could not be potentiated by cyclin A/Cdk2 in NIH3T3 cells, the truncated protein was hyperactive. Finally, we showed that B-Myb synergises with cyclin A to promote U-2 OS cells into S-phase.