Antelman D, Perry S, Hollingsworth R, Gregory R J, Driscoll B, Fung Y K, Bookstein R
Canji Inc., San Diego, California 92121, USA.
Oncogene. 1997 Dec 4;15(23):2855-66. doi: 10.1038/sj.onc.1201465.
We have constructed a panel of substitution mutants which affect one or more of the putative cdk target sites of the RB protein. We have examined the activity of these mutants relative to wild-type RB by both a transcriptional repression assay and by measuring growth suppression in vitro. We find that some phosphorylation site mutants of pRB can repress E2 transcription more strongly than wild-type RB. These mutants are partially resistant to phosphorylation by cdks and can arrest tumor cells in G1 in vitro. Our results indicate a functional correlation between the ability to repress E2F-dependent transcription and the ability to suppress tumor cell growth in vitro. In addition, we describe two classes of RB mutants: N-terminal truncated p56RB and a novel mutant of RB containing multiple substitutions near its nuclear localization signal. Both classes of RB mutants have greater activity than the wild-type protein. Because RB is a key regulator of cell cycle progression, expression of a more potent, phosphorylation resistant RB may have utility in both RB(-/-) and RB(+/+) tumors as well as in hyperproliferative disorders.
我们构建了一组替代突变体,这些突变体影响RB蛋白的一个或多个假定的细胞周期蛋白依赖性激酶(cdk)靶位点。我们通过转录抑制试验以及测量体外生长抑制作用,研究了这些突变体相对于野生型RB的活性。我们发现,pRB的一些磷酸化位点突变体比野生型RB能更强烈地抑制E2转录。这些突变体对cdks介导的磷酸化具有部分抗性,并且在体外能使肿瘤细胞停滞于G1期。我们的结果表明,抑制E2F依赖性转录的能力与体外抑制肿瘤细胞生长的能力之间存在功能相关性。此外,我们描述了两类RB突变体:N端截短的p56RB和一种在其核定位信号附近含有多个替代的新型RB突变体。这两类RB突变体都比野生型蛋白具有更强的活性。由于RB是细胞周期进程的关键调节因子,表达一种更强效、抗磷酸化的RB可能在RB(-/-)和RB(+/+)肿瘤以及增殖性疾病中都具有应用价值。