Identification of cyclin A/Cdk2 phosphorylation sites in B-Myb

B-myb is a highly conserved member of the myb proto-oncogene family that en codes a ubiquitously expressed 110-kDa sequence-specific DNA-binding protei n. Transactivation of Myb-inducible promoters by B-Myb is repressed by a re gulatory domain located at the C-terminus of the protein. Cyclin A/Cdk2-med iated phosphorylation apparently releases the negative constraint and trigg ers B-Myb transactivation potential. Two-dimensional tryptic phosphopeptide analysis indicated that the majority of the sites phosphorylated bl vivo a re targeted irt vitro by cyclin A/Cdk2. Six sites in B-Myb fulfil the requi rements for recognition by Cdk2. Using point mutation of the phosphorylatio n sites to nonphosphorylatable amino acids, we show that five of these site s are targets for Cdk2 in vivo. Mutation of one of these residues (T-524) t o alanine diminished the ability of B-Myb to promote transcription of a rep orter gene, suggesting that phosphorylation of B-Myb at this site is import ant for the regulation of its activity by cyclin A/Cdk2.