Establishment of Irreversible Growth Arrest in Myogenic Differentiation Requires the RB LXCXE-Binding Function

The crystal structure of the A-B domain of RB has defined the binding pocket for the LXCXE peptide motif. Using the crystal structure as a guide, we have inactivated the LXCXE-binding pocket by replacing N757 with Phe [to obtain RB(N757F)]. RB(N757F) does not bind to viral oncoproteins but retains the ability to bind and inhibit E2F. RB(N757F) is less effective than the wild-type RB [RB(WT)] in repressing E2F-regulated transcription, and its repression activity is not affected by trichostatin A, an inhibitor of histone deacetylases. However, RB(N757F) is as effective as RB(WT) in suppressing cell growth. Interestingly, RB(N757F) cannot establish an irreversible growth arrest in differentiated myocytes. Differentiated myocytes with RB(WT) become refractory to serum. By contrast, differentiated myocytes with RB(N757F) undergo DNA synthesis and phosphorylate RB(N757F) in response to serum, despite a high level of p21Cip1 expression. Mutation of the phosphorylation sites in RB(N757F) rescued its defect and allowed myocytes to permanently withdraw from the cell cycle. These results demonstrate that it is possible to inactivate the LXCXE-binding pocket without compromising the overall integrity of RB. Moreover, the LXCXE-binding pocket is dispensable for the intrinsic growth suppression function of RB. However, the LXCXE-binding function is essential for RB to establish the serum-refractory state in differentiated myocytes.

视网膜母细胞瘤蛋白（Retinoblastoma protein, RB）的A-B结构域的晶体结构已明确了LXCXE肽基序（LXCXE peptide motif）的结合口袋。以该晶体结构为研究指导，我们通过将N757替换为苯丙氨酸（Phenylalanine, Phe），成功失活了该LXCXE结合口袋，获得突变体RB(N757F)。RB(N757F)无法结合病毒癌蛋白，但仍保留结合并抑制E2F转录因子（E2F）的能力。相较于野生型RB[RB(WT)]，RB(N757F)在抑制E2F调控的转录过程中效果更弱，且其转录抑制活性不受组蛋白去乙酰化酶抑制剂曲古抑菌素A（trichostatin A）的影响。然而，RB(N757F)在抑制细胞增殖方面的效果与RB(WT)相当。

值得注意的是，RB(N757F)无法在分化的肌细胞中建立不可逆的生长阻滞。表达野生型RB的分化肌细胞会进入血清不应答状态；与之形成鲜明对比的是，表达RB(N757F)的分化肌细胞在血清刺激下会重新进入DNA合成期，并对RB(N757F)进行磷酸化修饰，即便此时细胞内p21Cip1的表达水平仍处于较高水平。若对RB(N757F)的磷酸化位点进行突变，则可修复其功能缺陷，使肌细胞能够永久退出细胞周期。

上述实验结果证实，在不破坏RB整体结构完整性的前提下失活LXCXE结合口袋是可行的。此外，LXCXE结合口袋对于RB内在的生长抑制功能并非必需。但LXCXE结合功能对于RB在分化肌细胞中建立血清不应答状态是必不可少的。