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

English version

Autoři: Chalita Washington ^aff001; Rachel Chernet ^aff001; Rewatee H. Gokhale ^aff001; Yesenia Martino-Cortez ^aff001; Hsiu-Yu Liu ^aff006; Ashley M. Rosenberg ^aff001; Sivan Shahar ^aff001; Cathie M. Pfleger ^aff001
Působiště autorů: Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York, United States of America ^aff001; University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America ^aff002; The Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York, United States of America ^aff003; The Tisch Cancer Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, United States of America ^aff004; Tufts University School of Medicine, Boston, Massachusetts, United States of America ^aff005; Memorial Sloan Kettering Cancer Center, New York, New York, United States of America ^aff006; Columbia University, New York, New York, United States of America ^aff007; New York Medical College, Valhalla, New York, United States of America ^aff008
Vyšlo v časopise: A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5. PLoS Genet 16(6): e32767. doi:10.1371/journal.pgen.1008715
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008715

Souhrn

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.

Klíčová slova:

Carcinogenesis – Drosophila melanogaster – Eyes – Phenotypes – Phosphorylation – Ras signaling – RNA interference – Ubiquitination

Zdroje

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