Allosteric Regulation of Switch-II Domain Controls KRAS Oncogenicity.
Allosteric Regulation of Switch-II Domain Controls KRAS Oncogenicity.
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Switch-II 结构域的变构调节控制 KRAS 致癌性。
DOI：
10.1158/0008-5472.can-22-3210
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发表时间：
2023
期刊：
影响因子：
11.2
通讯作者：
Haigis,KevinM
中科院分区：
文献类型：
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作者：
Yang,MoonHee;Tran,TimothyH;Hunt,Bethany;Agnor,Rebecca;Johnson,ChristianW;Shui,Bing;Waybright,TimothyJ;Nowak,JonathanA;Stephen,AndrewG;Simanshu,DhirendraK;Haigis,KevinM
RAS蛋白是调节广泛的细胞过程的GTP酶。RAS活性依赖于其核苷酸结合状态，而核苷酸结合状态受鸟嘌呤核苷酸交换因子（GEF）和GTP酶激活蛋白（GAP）的调节。KRAS可以在赖氨酸104（K104）处被乙酰化，并且K104至谷氨酰胺（K104 Q）的乙酰化模拟突变通过中断GEF诱导的核苷酸交换来减弱致癌KRAS的体外转化能力。为了评估这种突变在体内的影响，我们使用CRISPR-Cas9来生成在野生型和条件性KrasLSL-G12 D等位基因中携带K104 Q点突变的小鼠模型。K104 Q的纯合子动物是可行的，可生育的，并出现在预期的孟德尔频率，表明K104 Q不是一个完全丧失功能的突变。然而，与我们先前的体外研究结果一致，KRASG 12 D的致癌活性因K104突变而显著减弱。生化和结构分析表明G12 D和K104 Q突变协同抑制GEF介导的核苷酸交换，解释了K104 Q对致癌KRAS的优先作用。此外，K104与开关II区的α2螺旋上的M72、R73和G75在变构网络中起作用。有趣的是，甘氨酸75到丙氨酸（G75 A）的点突变也显示出对KRASG 12 D的强负调节作用。这些数据表明，104位的赖氨酸对于突变型KRAS的全部致癌活性是至关重要的，并且表明调节其变构网络中的位点可以在表达突变型KRAS的癌症中提供独特的治疗方法。其可用于开发活化癌蛋白的抑制剂。
RAS proteins are GTPases that regulate a wide range of cellular processes. RAS activity is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP). KRAS can be acetylated at lysine 104 (K104), and an acetylation-mimetic mutation of K104 to glutamine (K104Q) attenuates thein vitro–transforming capacity of oncogenic KRAS by interrupting GEF-induced nucleotide exchange. To assess the effect of this mutationin vivo, we used CRISPR-Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional KrasLSL-G12Dalleles. Homozygous animals for K104Q were viable, fertile, and arose at the expected Mendelian frequency, indicating that K104Q is not a complete loss-of-function mutation. Consistent with our previous findings fromin vitrostudies, however, the oncogenic activity of KRASG12Dwas significantly attenuated by mutation at K104. Biochemical and structural analysis indicated that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic KRAS. Furthermore, K104 functioned in an allosteric network with M72, R73, and G75 on the α2 helix of the switch-II region. Intriguingly, point mutation of glycine 75 to alanine (G75A) also showed a strong negative regulatory effect on KRASG12D. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant KRAS and suggest that modulating the sites in its allosteric network may provide a unique therapeutic approach in cancers expressing mutant KRAS.SignificanceAn allosteric network formed by interaction between lysine 104 and residues in the switch-II domain is required for KRAS oncogenicity, which could be exploited for developing inhibitors of the activated oncoprotein.