The Ras Mutant D119N Is Both Dominant Negative and Activated

Abstract

The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras-a strong decrease in nucleotide affinity-enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.

Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3‐effector‐GAP complex

“…This supports observations by us and others that the mutation of the P-loop Ser/Thr in the GxxxxGKS/T motif does not necessarily, as generally assumed, reflect an exclusive binding of GDP [24,27]. In general it should be kept in mind that the corresponding mutation, T30N in Arl2, destabilizes G proteins and also strongly affects the binding to effectors, as shown for Ras, where the S17N mutation no longer bind effector molecules [28,29].…”

Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway

“…Thus, the inhibition of membrane traffic by dominantnegative RAB-D1 in tobacco leaf epidermis or Arabidopsis seedlings could be substantially restored by coexpressing wild-type c and e). Note that the RAB-D2a [N121] mutant is not significantly overexpressed under these conditions and that it accumulates less efficiently than the wild type, consistent with previous observations that small GTPases are unstable in the nucleotide-free state when not bound to the exchange factor (Okkonen and Stenmark, 1997;Cool et al, 1999).…”

“…Dominant-negative Rab GTPase mutants inhibit membrane trafficking by titration of an essential interacting partner such as a nucleotide exchange factor (Jones et al, 1995;Olkkonnen and Stenmark, 1997;Walch-Solimena et al, 1997;Cool et al, 1999;Goh et al, 2007). If this interactor has additional activities that are independent of its interaction with the mutated Rab GTPase, the effects of the dominant-negative mutant might not reflect the true activity of the Rab GTPase.…”

An unusual H-Ras mutant isolated from a human multiple myeloma line leads to transformation and factor-independent cell growth

“…Single amino-acid substitutions in these positions (116-119) generated by site-directed mutagenesis result in a reduction in binding affinity for GTP (Clanton et al, 1986;Walter et al, 1986). It has been suggested that Ras (D119N) behaves as an activated mutant because of the strongly reduced nucleotide affinity enabling the mutant to bind GTP in a GEF-independent manner (Cool et al, 1999). Since the physiological concentrations of GTP and GDP are thought to be in the ratio of 25 : 1 (Walter et al, 1986), this increased exchange rate is proposed to favor the formation of the active GTP-bound form of Ras protein.…”