Shan B, Durfee T, Lee W H
Center for Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio 78245, USA.
Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):679-84. doi: 10.1073/pnas.93.2.679.
The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression.
视网膜母细胞瘤蛋白（RB）被认为通过与细胞蛋白如转录因子E2F形成复合物来发挥细胞增殖负调节因子的作用。为了研究RB/E2F-1相互作用的生物学后果，利用酵母双杂交系统分离出不能与RB结合的E2F-1点突变体。序列分析显示，在E2F-1与RB结合所需的最小18个氨基酸肽段内，五个残基，即酪氨酸（第411位）、谷氨酸（419）以及天冬氨酸-亮氨酸-苯丙氨酸（423 - 425）至关重要。这些氨基酸在已知的E2F家族成员中是保守的。虽然这五个氨基酸中的任何一个发生突变都会消除与RB的结合，但所有突变体都保留了其完全的反式激活潜能。与野生型相比，突变型E2F-1的表达显著加速了进入S期及随后的细胞凋亡。这些结果为RB/E2F相互作用的生物学意义提供了直接的遗传学证据，并强烈表明RB与E2F之间的相互作用对正常细胞周期进程至关重要。
