A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5 Journal Article

Authors: Washington, C.; Chernet, R.; Gokhale, R. H.; Martino-Cortez, Y.; Liu, H. Y.; Rosenberg, A. M.; Shahar, S.; Pfleger, C. M.
Article Title: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5
Abstract: 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-A nd 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 RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,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 RasG12V 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. © 2020 Washington et al.
Keywords: signal transduction; controlled study; protein phosphorylation; unclassified drug; oncoprotein; janus kinase 2; nonhuman; protein function; animal cell; phenotype; animal tissue; amino acid substitution; protein metabolism; epidermal growth factor receptor; animal experiment; drosophila; in vivo study; in vitro study; protein tyrosine kinase; tyrosine; wild type; molecular mechanics; ubiquitination; amino terminal sequence; molecular recognition; recombinant protein; amino acid; ras protein; ubiquitin protein ligase e3; glutamic acid; protein variant; valine; polyclonal antibody; gain of function mutation; male; female; article; protein rabex 5; protein rabgef1
Journal Title: PLoS Genetics
Volume: 16
Issue: 6
ISSN: 1553-7390
Publisher: Public Library of Science  
Date Published: 2020-06-19
Start Page: e1008715
Language: English
DOI: 10.1371/journal.pgen.1008715
PUBMED: 32559233
PROVIDER: scopus
PMCID: PMC7329146
Notes: Article -- Export Date: 3 August 2020 -- Source: Scopus
Citation Impact
MSK Authors
  1. Hsiu Yu Liu
    3 Liu