RAS蛋白是GTP酶，可以调节多种细胞过程。RAS活性取决于其核苷酸结合状态，其由鸟嘌呤核苷酸交换因子（GEFs）和GTP酶活化蛋白（GAPs）调节。KRAS可以在赖氨酸104（K104）位点乙酰化，将K104乙酰模拟突变为谷氨酰胺（K104Q）则会通过干扰GEF诱导的核酸交换来减弱 KRAS的体内转化能力。为了评估这种突变对体内的影响，我们使用CRISPR-Cas9技术在野生型和条件性KrasLSL-G12D等位基因中引入了K104Q点突变，以产生小鼠模型。K104Q的纯合动物不但能生存繁殖，而且其出现频率符合预期的孟德尔定律，表明K104Q不是完全丧失功能的突变。然而，与我们之前的体外研究结果一致，K104的突变显著减弱了KRASG12D的致癌活性。生化和结构分析表明，G12D和K104Q突变具有协同抑制GEF介导的核苷酸交换的作用，从而解释了K104Q对致癌性KRAS的优先影响。此外，K104与α2螺旋的开关-II区域上的M72、R73和G75构成一个异构网络。有趣的是，谷氨酸75点突变为丙氨酸（G75A）对KRASG12D表现出强烈的负调控作用。这些数据表明，位于104位置的赖氨酸对突变型KRAS的完整致癌活性至关重要，并且调节其异构网络中的位点可能为表达突变型KRAS的肿瘤提供了独特的治疗方法。

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 (GEFs) and GTPase-activating proteins (GAPs). KRAS can be acetylated at lysine 104 (K104), and an acetylation-mimetic mutation of K104 to glutamine (K104Q) attenuates the in vitro transforming capacity of oncogenic KRAS by interrupting GEF induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR-Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional KrasLSL-G12D alleles. 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 from in vitro studies, however, the oncogenic activity of KRASG12D was 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 glutamine 75 to alanine (G75A) 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.