KRAS, HRAS and NRAS proto-oncogenes belong to a family of 40 highly homologous genes, which in turn are a subset of a superfamily of >160 genes encoding small GTPases. RAS proteins consist of a globular G-domain (aa1-166) and a 22-23 aa unstructured hypervariable region (HVR) that mediates membrane targeting. The evolutionary origins of the RAS isoforms, their HVRs and alternative splicing of the KRAS locus has not been explored. We found that KRAS is basal to the RAS proto-oncogene family and its duplication generated HRAS in the common ancestor of vertebrates. In a second round of duplication HRAS generated NRAS and KRAS generated an additional RAS gene we have designated KRASBL, absent in mammals and birds. KRAS4A arose through a duplication and insertion of the 4^th exon of NRAS into the 3^rd intron of KRAS. We found evolutionary conservation of a short polybasic region (PBR1) in HRAS, NRAS and KRAS4A, a second polybasic region (PBR2) in KRAS4A, two neutralized basic residues (NB) and a serine in KRAS4B and KRASBL, and a modification of the CaaX motif in vertebrates with farnesyl rather than geranylgeranyl polyisoprene lipids, suggesting that a less hydrophobic membrane anchor is critical to RAS protein function. The persistence of four RAS isoforms through >400 million years of evolution argues strongly for differential function.

KRAS、HRAS和NRAS原癌基因属于 40 个高度同源基因的家族，而这些基因又是由超过 160 个编码小 GTP 酶的基因组成的超家族的子集。RAS 蛋白由球状 G 结构域 (aa1-166) 和介导膜靶向的 22-23 aa 非结构化高变区 (HVR) 组成。RAS 亚型、其 HVR 和KRAS基因座的选择性剪接的进化起源尚未被探索。我们发现KRAS是RAS原癌基因家族的基础，其复制在脊椎动物的共同祖先中产生了HRAS 。在第二轮复制中，HRAS生成了NRAS，KRAS生成了一个额外的RAS基因，我们将其命名为KRASBL，在哺乳动物和鸟类中不存在。KRAS4A是通过将NRAS的第四^个外显子复制并插入到KRAS的第三^个内含子中而产生的。我们在 HRAS、NRAS 和 KRAS4A 中发现了一个短多碱基区域 (PBR1)，在 KRAS4A 中发现了第二个多碱基区域 (PBR2)，在 KRAS4B 和 KRASBL 中发现了两个中和的碱性残基 (NB) 和一个丝氨酸，以及 CaaX 的修饰脊椎动物中含有法尼基而不是香叶基香叶基聚异戊二烯脂质的基序，表明疏水性较低的膜锚对于 RAS 蛋白功能至关重要。四种 RAS 同工型在超过 4 亿年的进化过程中持续存在，有力地证明了其功能差异。