A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation [Protein Structure and Folding]

The KRAS GTPase plays a critical role in control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also by posttranslational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, as mutations at this position impair GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate in its vitro and cell-based properties. Herein, we find that KRAS K104Q exhibits defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, likely due to offsetting defects in recognition of factors that upregulate (GEF) and downregulate (GAP) RAS activity.

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