Dysregulation of the Ras oncogene in development causes developmental disorders, “Rasopathies,” whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and Ras^G12V proteins when incubated with JAK2 or SRC kinases but not of Ras^Y4F or Ras^Y4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of Ras^G12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.

发育过程中 Ras 癌基因的失调会导致发育障碍（“Rasopathies”），而分化组织中 Ras 的突变激活或扩增会导致癌症。Rabex-5（也称为 RabGEF1）通过促进 Ras 单泛素化和二泛素化来抑制 Ras。我们在这里报告 Rabex-5 介导的 Ras 泛素化需要 Ras 酪氨酸 4 (Y4)，这是一个已知的磷酸化位点。对 Y4 磷酸化不敏感的 Ras 取代突变体不会在细胞中经历 Rabex-5 介导的泛素化，并在体内表现出 Ras 功能获得表型。Ras Y4 磷酸化取代增加了细胞中 Rabex-5 介导的泛素化。致癌 Ras 中的 Y4 拟磷化取代根据 Rabex-5 的存在阻断体内与致癌 Ras 相关的形态表型。我们开发了针对 Y4 磷酸化 N 端 Ras 肽的多克隆抗体。当与 JAK2 或 SRC 激酶一起孵育时，这些抗磷酸 Y4 抗体表现出对重组野生型 Ras 和 Ras ^G12V蛋白的显着识别，但不能识别 Ras ^Y4F或 Ras ^Y4F、G12V重组蛋白，表明 JAK2 和 SRC 可以促进 Ras 蛋白的磷酸化Y4体外。当与 EGFR 一起孵育时，抗磷酸 Y4 抗体也显示出对 Ras ^G12V蛋白的识别，但不能识别野生型 Ras。JAK2、SRC 和 EGFR（通过 Ras 激活信号传导的众所周知的激酶）促进 Ras Y4 磷酸化的作用可能代表限制 Ras 激活的反馈机制，从而建立 Ras 稳态。值得注意的是，在小脑胶质母细胞瘤中发现了 Y4 位点 Ras 的罕见变异。因此，我们的工作确定了生理相关的 Ras 泛素化信号，并强调了 Rabex-5 抑制 Ras 所需的 Y4，以维持 Ras 途径稳态并防止组织转化。