Petrovas Constantinos, Jeay Sébastien, Lewis Robert E, Sonenshein Gail E
Department of Biochemistry, Boston University Medical School, MA 02118-2394, USA.
Oncogene. 2003 Apr 3;22(13):2011-20. doi: 10.1038/sj.onc.1206231.
B-Myb is a widely expressed member of the myb oncogene family that has been shown to act as either an activator or repressor of gene transcription in a cell-type-specific fashion. For example, in aortic smooth muscle cells B-Myb represses transcription of the alpha2(V) collagen gene. Recently, phosphorylation of B-Myb by cyclin A was shown to enhance greatly its ability to transactivate. Here, we have tested the effects of cyclin A on the ability of B-Myb to repress. We report that coexpression of cyclin A abolished repression of the alpha2(V) collagen promoter, whereas a dominant-negative cyclin-dependent kinase 2 (cdk2) enhanced repression by ectopic and endogenous B-Myb protein. Mutation of 10 of 22 putative cyclin A sites, which greatly reduces the effects of cyclin A on transactivation by B-Myb, had no effect on the ability of cyclin A to alleviate B-Myb-mediated repression of alpha2(V) collagen promoter activity. Furthermore, the stability of the mutant B-Myb protein was largely unaffected by cyclin A, although ectopic expression of cyclin A enhanced the rate of decay of wild-type B-Myb protein. Thus, the mechanisms of repression and activation appear distinct, for example, mediated by different critical phosphorylation sites or protein-protein interactions. B-Myb mutants with either deletion of aa 374-581 (B-Myb-Mut3) or C-terminal truncation beyond aa 491 (B-Myb-491) positively regulated alpha2(V) collagen promoter activity, and were not affected by cyclin A. Thus, our findings indicate that the ability of B-Myb to function as a repressor of matrix promoter activity is abolished by cyclin A, and maps the sites mediating negative regulation by B-Myb to the region between aa 491 and 582.
B-Myb是myb癌基因家族中广泛表达的成员，已证明它在细胞类型特异性方式中可作为基因转录的激活剂或抑制剂。例如，在主动脉平滑肌细胞中，B-Myb抑制α2(V)胶原蛋白基因的转录。最近，细胞周期蛋白A对B-Myb的磷酸化被证明极大地增强了其反式激活能力。在此，我们测试了细胞周期蛋白A对B-Myb抑制能力的影响。我们报告称，细胞周期蛋白A的共表达消除了对α2(V)胶原蛋白启动子的抑制作用，而显性负性细胞周期蛋白依赖性激酶2（cdk2）通过异位和内源性B-Myb蛋白增强了抑制作用。22个假定的细胞周期蛋白A位点中的10个发生突变，这大大降低了细胞周期蛋白A对B-Myb反式激活的影响，但对细胞周期蛋白A减轻B-Myb介导的α2(V)胶原蛋白启动子活性抑制的能力没有影响。此外，突变型B-Myb蛋白的稳定性在很大程度上不受细胞周期蛋白A的影响，尽管细胞周期蛋白A的异位表达增强了野生型B-Myb蛋白的降解速率。因此，抑制和激活机制似乎是不同定的，例如，由不同的关键磷酸化位点或蛋白质-蛋白质相互作用介导。缺失aa 374-581（B-Myb-Mut3）或C末端截短超过aa 491（B-Myb-491）的B-Myb突变体正向调节α2(V)胶原蛋白启动子活性，且不受细胞周期蛋白A影响。因此，我们的研究结果表明，细胞周期蛋白A消除了B-Myb作为基质启动子活性抑制剂的功能，并将介导B-Myb负调节的位点定位到aa 491和582之间的区域。
