Insights into K-Ras 4B regulation by post-translational lysine acetylation
Insights into K-Ras 4B regulation by post-translational lysine acetylation
复制标题
DOI：
10.1515/hsz-2016-0118
复制
发表时间：
2016-10-01
影响因子：
3.7
通讯作者：
Lammers, Michael
中科院分区：
文献类型：
--
作者：
Knyphausen, Philipp;Lang, Franziska;Lammers, Michael
RAS是一种分子开关，在GTP结合的活跃状态和GDP结合的非活跃状态之间循环。RAS的突变，主要影响开关，在许多人类肿瘤中发现。最近，有研究表明K-RAS 4B被赖氨酸乙酰化作用靶向于K104。根据对乙酰化模拟RAS突变体(K104Q)的研究结果，推测K104-乙酰化可能通过抑制SOS催化的鸟核苷酸交换来干扰其致癌活性。我们利用遗传密码扩展的概念制备了K104-乙酰化的K-RAS 4B和相应的致癌突变蛋白G12V。我们发现SOS催化的核苷酸交换，也是变构激活的SOS，既不受野生型K-RAS 4B中K104乙酰化的影响，也不受G12V突变体中K104乙酰化的影响，这表明谷氨酰胺在该位点不是乙酰化的模拟物。在体外，赖氨酸-乙酰转移酶CBP和p300均能乙酰化野生型和G12VK-RAS4B。除了K104外，我们还在K-RAS 4B中发现了更多的乙酰化位点，包括重要的G5/SAK基序中的K147。K-RAS 4B的K147-乙酰化不影响K-RAS 4B的固有和SOS催化的核苷酸交换。最后，我们证明了SIRT2和HDAC6在体外既不会去乙酰化K-RAS 4B，也不会去乙酰化K-RAS 4B。
Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.