Bartsch O, Horstmann S, Toprak K, Klempnauer K H, Ferrari S
Institute for Experimental Cancer Research, Tumor Biology Center, Freiburg, Germany.
Eur J Biochem. 1999 Mar;260(2):384-91. doi: 10.1046/j.1432-1327.1999.00191.x.
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.
B-myb是myb原癌基因家族中一个高度保守的成员，它编码一种在全身广泛表达的110-kDa序列特异性DNA结合蛋白。位于该蛋白C末端的一个调节结构域可抑制B-Myb对Myb诱导型启动子的反式激活作用。细胞周期蛋白A/Cdk2介导的磷酸化作用显然消除了这种负向限制，并触发了B-Myb的反式激活潜能。二维胰蛋白酶磷酸肽分析表明，体内大多数磷酸化位点在体外可被细胞周期蛋白A/Cdk2磷酸化。B-Myb中的六个位点符合被Cdk2识别的要求。通过将磷酸化位点突变为不可磷酸化的氨基酸，我们发现其中五个位点在体内是Cdk2的作用靶点。将其中一个残基（T524）突变为丙氨酸会削弱B-Myb促进报告基因转录的能力，这表明B-Myb在该位点的磷酸化对于细胞周期蛋白A/Cdk2对其活性的调节很重要。
