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⁻.