The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

Computational Structural Biology Section Frederick National Laboratory for Cancer Research in the Laboratory of Cancer ImmunoMetabolism National Cancer Institute Frederick MD 21702 USA

Department of Hematooncology University Hospital Ostrava Ostrava Czech Republic

Department of Human Molecular Genetics and Biochemistry Sackler School of Medicine Tel Aviv University Tel Aviv 69978 Israel

Department of Oncology KU Leuven 3000 Leuven Belgium

School of Neurobiology Biochemistry and Biophysics The George S Wise Faculty of Life Sciences Tel Aviv University Ramat Aviv 69978 Tel Aviv Israel

VIB KU Leuven Center for Cancer Biology VIB 3000 Leuven Belgium

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