Phosphorylation at Ser - 181 of oncogenic KRAS is required for tumor growth 1

26 KRAS phosphorylation has been reported recently to modulate the activity of mutant 27 KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site 28 required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant 29 S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D 30 exhibited an enhanced tumor formation capacity, compared to the wild-type KRAS 31 protein. Reduced growth of tumors composed of cells expressing the non32 phosphorylatable KRAS S181A mutant was correlated with increased apoptosis. 33 Conversely, increased growth of tumors composed of cells expressing the 34 phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT 35 and ERK, two major downstream effectors of KRAS. Pharmacological treatment with 36 PKC inhibitors impaired tumor growth associated with reduced levels of 37 phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell 38 lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was 39 essential for survival and tumorigenic activity. Further, we identified phosphorylated 40 KRAS in a panel of primary human pancreatic tumors. Taken together, our findings 41 establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, 42 implying that its inhibition represents a relevant target to attack KRAS-driven tumors. 43