Lysines K117 and K147 play conserved roles in Ras activation from Drosophila to mammals

Ras signaling plays an important role in growth, proliferation, and developmental patterning. Maintaining appropriate levels of Ras signaling is important to establish patterning in development and to prevent diseases such as cancer in mature organisms. The Ras protein is represented by Ras85D in Drosophila and by HRas, NRas, and KRas in mammals. In the past dozen years, multiple reports have characterized both inhibitory and activating ubiquitination events regulating Ras proteins. Inhibitory Ras ubiquitination mediated by Rabex-5 or Lztr1 is highly conserved between flies and mammals. Activating ubiquitination events at K117 and K147 have been reported in mammalian HRas, NRas, and KRas, but it is unclear if these activating roles of K117 and K147 are conserved in flies. Addressing a potential conserved role for these lysines in Drosophila Ras activation requires phenotypes strong enough to assess suppression. Therefore, we utilized oncogenic Ras, Ras^G12V, which biases Ras to the GTP-loaded active conformation. We created double mutants Ras^G12V,K117R and Ras^G12V,K147R and triple mutant Ras^{G12V,K117R,K147R} to prevent lysine-specific post-translational modification of K117, K147, or both, respectively. We compared their phenotypes to Ras^G12V in the wing to reveal the roles of these lysines. Although Ras^G12V,K147R did not show compelling or quantifiable differences from Ras^G12V, Ras^G12V,K117R showed visible and quantifiable suppression compared to Ras^G12V, and triple mutant Ras^{G12V,K117R,K147R} showed dramatic suppression compared to Ras^G12V and increased suppression compared to Ras^G12V,K117R. These data are consistent with highly conserved roles for K117 and K147 in Ras activation from flies to mammals.