RAS ubiquitylation modulates effector interactions.

RAS proteins function as molecular switches that regulate cellular growth by cycling between active GTP- and inactive GDP bound states. While RAS activity is modulated by factors (guanine nucleotide exchange and GTPase activating proteins) that control levels of active Ras-GTP, RAS proteins also undergo a number of post-translational modifications that regulate their function. One such modification is ubiquitylation. Monoubiquitylation of KRAS at lysine 147 (mUbRAS) enhances Ras activation and promotes signaling through the RAF and Phosphoinositide 3-Kinase (PI3K) signaling pathways. We have previously shown that mUbRAS leads to activation of RAS through a defect in GTPase activating protein (GAP) mediated downregulation, similar to the action of most oncogenic mutations. Consistent with these findings, we now show that mUbRASimpairsRAS binding to the p120 GAP catalytic domain. Mutations in activated G12V RAS that prevent ubiquitylaton at 147 show a decrease in tumorigenesis, suggesting that in addition to activating KRAS, monoubiquitylation at this site may promote downstream signaling and transformation. To investigate whether mUbRAS alters RAS effector interactions, we chemically ubiquitylated KRAS at residue 147 and characterized binding of mUbRAS to RAS binding domains (RBDs) from three distinct downstream effectors that play key roles in RAS-mediated transformation. Results from these studies show a decrease in binding of mUbRAS (7-10-fold) relative to the CRAF RAS Binding Domain (RBD), the catalytic subunit of Phosphoinositide 3-Kinase catalytic gamma (PI3Kcγ) and RALGDS RBD. Intriguingly, we find that mUbRAS shows greatly enhanced (> 40-fold) binding to the CRAF RBD when bound to GDP. These findings, taken together, suggest that mUbRASmay promoteactivation of RAS through a GAP defect, and facilitate RAF association and MAPK signaling in a nucleotide independent manner.