Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

Abstract

Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.

Figure 1

Monoubiquitination of Ras. (a) Reaction of Ubiquitin^G76C with Ras or a Ras^K147C mutant, under non–reducing conditions. The product of the reaction contains mUbRas, Ras, Ubiquitin–Ubiquitin dimer (Ub–Ub), and free Ubiquitin (Ub). (b) HSQC spectra of ¹⁵N–Ras^K147C bound to Mg–GDP in the absence (black) and presence (red) of ten–fold excess free Ubiquitin.

Figure 2

Monoubiquitinated Ras retains intrinsic stability and activity. (a) Intrinsic nucleotide dissociation rates for Ras, Ras^K147C, and mUbRas loaded with MANT–GDP. Dissociation was monitored following the addition of unlabeled GDP by the decrease in fluorescence emission over time. Data were fit to an exponential dissociation curve, and the results are the mean ± s.d. (n=4). (b) Intrinsic single–turnover GTP hydrolysis for Ras, Ras^K147C, and mUbRas. Hydrolysis was initiated by the addition of Mg²⁺ and monitored by the change in fluorescence of Flippi when bound to free phosphate. Data were converted to a phosphate concentration using a standard curve. The concentration of phosphate equal to 100% GTP hydrolyzed was determined in the presence of GAP. Results are the mean ± s.d. (n=6). (c) Thermal stability of Ras, Ras^K147A, and mUbRas measured by 4–Fluoro–7–aminosulfonylbenzofurazan (ABD–F) incorporation as a function of temperature. The data were normalized using the maximum fluorescence intensity. Results are the mean ± s.d. (n=4).

Figure 3

Rosetta model of native and chemical ubiquitination of Ras. (a) The ten lowest scoring Rosetta models of the native linkage of Ras monoubiquitination at position 147 lacking constraints to bias the model. Ras (5P21) is shown in grey with switch regions (SWI and SWII) highlighted in black. Ubiquitin (1UBQ) conformers shown in colors. Inset: native linkage between Ras Lys147 and Ubiquitin G76. (b) The ten lowest scoring Rosetta models of the chemical linkage of Ras monoubiquitination at position 147 lacking any constraints to bias the model. Ras and Ubiquitin colored as in panel a. Inset: chemical linkage between Ras K147C and Ubiquitin G76C. (c) The distribution of Ubiquitin orientations relative to Ras plotted against Rosetta energy scores for the native linkage. The Y axis shows the dihedral angle, in degrees, of the torsional angle between the center of mass of Ubiquitin, the linking Ras residue (147), the center of mass of Ras and an arbitrary Ras reference atom. (d) The distribution of Ubiquitin orientations relative to Ras plotted against Rosetta energy scores for the chemical linkage. Axes are the same as described in panel c.

Figure 4

Surfaces of Ras and Ubiquitin affected by monoubiquitination. (a) HSQC spectra of ¹⁵N–Ubiquitin^G76C free (black) or ligated to Ras^K147C (blue). Residues that broaden are labeled based on previous assignments. (b) Space filling model of the structure of Ubiquitin (1UBQ) with residues that show decreased intensity when ligated to Ras (blue). Residues with no information are colored black. (c) HSQC spectra of ¹⁵N–Ras^K147C bound to Mg–GDP alone (black) and when monoubiquitinated (green). Inset (Top): enhancement of one expanded region showing residues that broaden and disappear. Inset (Bottom Left): SDS–PAGE gel showing integrity of mUbRas sample after HSQC analysis. Inset (Bottom Right): close up of Arg135, which exhibits multiple populations. (d) Mapping of Ras backbone amides that disappear upon monoubiquitination onto the structure of Ras. Darker green indicates more appreciable broadening (primarily in the SW I and SW II). Residues with no information are colored black.

Figure 5

Monoubiquitination decreases the sensitivity of Ras to downregulation by GAPs. (a) Nucleotide dissociation reaction for Ras, Ras^K147C, and mUbRas loaded with MANT–GDP in the presence of a 1:1 molar ratio of Ras to Sos^cat. Data were fit to an exponential dissociation curve, and the results are the mean ± s.d. (n=4). (b) Single–turnover GTP hydrolysis for Ras, Ras^K147C, mUbRas, mUbRas ubiquitinated with Ub^77C (mUb^77CRas), and Ras^G12V in the presence of NF1³³³ or GAP–334 at a molar ratio of 1:500 GAP:Ras. Results are the mean ± s.d. (n=6). (c) Immunoblotting of GTP-bound Ras and GTP-bound mUbRas in cell extract in the presence of increasing concentrations of RasGAP. Anti–Flag and anti–HA antibodies reveal the relative fraction of total Ras and mUbRas, respectively. (d) Titration of Ras with Sos^cat. Experiments were performed as described panel a, except the concentration of Sos^cat was varied while Ras was held constant at 0.2 μM. Data plotted as a function of the Sos^cat concentration. Results are the mean ± s.d. (n=3). (e) Gel filtration of Ras and NF1³³³ in the absence (dotted line) and presence (solid line) of AlF₄⁻. (f) Gel filtration of mUbRas and NF1³³³ in the absence (dotted line) and presence (solid line) of AlF₄⁻.

Figure 6

Modification of Ras with PDZ2 resembles modification with Ubiquitin. (a) Rosetta model of Ras (5P21) in grey modified at position 147 with Ubiquitin (1UBQ) in green and PDZ^UL (3LNX) in purple. (b) The distribution of PDZ^UL orientations relative to Ras plotted against Rosetta energy scores for the chemical linkage. This plot follows the scheme of Figure 3B. (c) Thermal stability of Ras and RasPDZ2 with the Ubiquitin linker (RasPDZ2^UL) measured by ABD–F incorporation as a function of temperature. Results are the mean ± s.d. (n=4). (d) Nucleotide dissociation reaction for RasPDZ2^UL and mUbRas loaded with MANT–GDP in the absence and presence of a 1:1 molar ratio of Ras to Sos^cat. Results are the mean ± s.d. (n=4). (e) Single–turnover GTP hydrolysis for Ras, RasPDZ2^UL, and mUbRas in the presence of GAP–334 at a molar ratio of 1:500 and 1:200 GAP:Ras. Results are the mean ± s.d. (n=6).

Figure 7

The impaired GAP–sensitivity of mUbRas is site–specific. (a) Ribbon diagram of Ras–GDP (1CRR) with the switch regions highlighted in black and the side chains of Lys147, Lys88, and Lys101 represented as spheres in green, fuchsia, and blue, respectively. (b) Single–turnover GTP hydrolysis for Ras mutated and ubiquitinated at position 147, 88, or 101 in the absence and presence of GAP–334 at a molar ratio of 1:200 GAP:Ras. Results are the mean ± s.d. (n=4).