Identification of cyclin A/Cdk2 phosphorylation sites in B-Myb.
B-myb is a highly conserved member of the myb proto-oncogene family that encodes a ubiquitously expressed 110-kDa sequence-specific DNA-binding protein. Transactivation of Myb-inducible promoters by B-Myb is repressed by a regulatory domain located at the C-terminus of the protein. Cyclin A/Cdk2-mediated phosphorylation apparently releases the negative constraint and triggers B-Myb transactivation potential. Two-dimensional tryptic phosphopeptide analysis indicated that the majority of the sites phosphorylated in vivo are targeted in vitro by cyclin A/Cdk2. Six sites in B-Myb fulfil the requirements for recognition by Cdk2. Using point mutation of the phosphorylation sites to nonphosphorylatable amino acids, we show that five of these sites are targets for Cdk2 in vivo. Mutation of one of these residues (T524) to alanine diminished the ability of B-Myb to promote transcription of a reporter gene, suggesting that phosphorylation of B-Myb at this site is important for the regulation of its activity by cyclin A/Cdk2.
Bartsch
O
O
Institute for Experimental Cancer Research, Tumor Biology Center, Freiburg, Germany.
Horstmann
S
S
Toprak
K
K
Klempnauer
K H
KH
Ferrari
S
S
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D019925:Cyclin A / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011784:Quail; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; C000715470:MAS1 protein, human; C064352:MYBL2 protein, human; D000090063:Proto-Oncogene Mas; D015534:Trans-Activators; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases