Establishment of irreversible growth arrest in myogenic differentiation requires the RB LXCXE-binding function
Establishment of irreversible growth arrest in myogenic differentiation requires the RB LXCXE-binding function
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DOI：
10.1128/mcb.20.15.5571-5580.2000
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发表时间：
2000-08-01
影响因子：
5.3
通讯作者：
Wang, JYJ
中科院分区：
文献类型：
--
作者：
Chen, TT;Wang, JYJ
RE的A-B结构域的晶体结构定义了LXCXE肽基序的结合口袋，以晶体结构为指导，我们通过用Phe取代N757来灭活LXCXE结合口袋[以获得RB（N757 F）]，RB（N757 F）不与病毒癌蛋白结合，但保留了结合和抑制E2 F的能力。RB（N757 F）在抑制E2 F调节的转录方面不如野生型RE [RB（WT）]有效，并且其抑制活性不受组蛋白去乙酰化酶抑制剂A的影响。然而，RB（N757 F）在抑制细胞生长方面与RB（WT）一样有效。有趣的是，RB（N757 F）不能在分化的肌细胞中建立不可逆的生长停滞。与RB（WT）分化的心肌细胞变得对血清不敏感。相反，RB（N757 F）分化的心肌细胞响应于血清进行DNA合成并磷酸化RB（N757 F），尽管高水平的p21 Cip 1表达。RB（N757 F）磷酸化位点的突变挽救了其缺陷，并允许肌细胞永久退出细胞周期。这些结果表明，可以在不损害RE的整体完整性的情况下使LXCXE结合口袋变小。此外，LXCXE结合口袋是由于RE的内在生长抑制功能。然而，LXCXE结合功能是RE在分化的肌细胞中建立血清不应状态所必需的。
The crystal structure of the A-B domain of RE 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 RE [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 RE. Moreover, the LXCXE-binding pocket is dispensable for the intrinsic growth suppression function of RE. However, the LXCXE-binding function is essential for RE to establish the serum-refractory state in differentiated myocytes.