Differential Modification of Ras Proteins by Ubiquitination Natalia Jura, Elizabeth Scotto-Lavino, Aleksander Sobczyk, Dafna Bar-Sagi  Molecular Cell  Volume 21, Issue 5, Pages 679-687 (March 2006) DOI: 10.1016/j.molcel.2006.02.011 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 HRas Undergoes Mono- and Diubiquitination In Vivo (A) Analysis of HRas ubiquitination in vivo. Lysates from CHOK1 cells expressing T7-HRas and HA-ubiquitin (HA-Ub) alone or in combination were subjected to Ras immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. (B) Ni-NTA affinity chromatography assay for ubiquitination. CHOK1 cells were transfected with T7-HRas and HIS-ubiquitin (HIS-Ub) as indicated. Cell lysates were subjected to Ni-NTA affinity chromatography, and the ubiquitinated species of T7-HRas were detected by immunoblotting with anti-T7 antibodies. T7-HRas expression levels were determined by immunoblotting of whole-cell lysates (WCL) with anti-T7 antibodies. (C) Migration of HRas fusion constructs on SDS-polyacrylamide gel. CHOK1 cells were transfected with T7-HRas, monoUb-T7-HRas fusion (Ub-Ras), and diUb-T7-HRas fusion (diUb-Ras), as indicated. The expression of Ras fusion proteins was detected by immunoblotting of cell lysates with anti-T7 antibodies. (D) Ubiquitination of endogenous Ras. Analysis of whole-cell lysates (WCL) from CHOK1 cells with anti-Ras antibody. Ras proteins were immunoprecipitated from CHOK1 cells with rat monoclonal anti-Ras antibody (Y13-259) crosslinked to protein A-sepharose (Ras). A rat monoclonal antibody of the same isotype crosslinked to protein A was used as a negative control (Ctrl). Immunoblotting was performed with anti-ubiquitin P4D1 or anti-Ras (clone Ras10) antibodies. (E) Diubiquitin chain on HRas is extended through lysine 63. Immunoprecipitation of Ras from CHOK1 cells expressing T7-HRas with HA-Ub, lysine-deficient HA-Ub (HA-UbK0), or HA-UbK0 with a lysine 63 knockin (HA-UbK0R63K). Molecular Cell 2006 21, 679-687DOI: (10.1016/j.molcel.2006.02.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Regulation of HRas Ubiquitination (A) The effect of EGF stimulation on HRas ubiquitination. CHOK1 cells expressing EGFR, T7-HRas, and HA-Ub were serum starved (−) and stimulated (+) for 15 min with 100 ng/ml EGF. Lysates were subjected to Ras immunoprecipitation followed by immunoblotting with the indicated antibodies. As a control for EGF stimulation, the status of ERK phosphorylation was determined by immunoblotting of whole-cell lysates (WCL). (B) Dependence of ubiquitination on the nucleotide loading status of HRas. Lysates from CHOK1 cells expressing T7-HRas wt or G12V alone or together with HA-Ub were subjected to Ras immunoprecipitation followed by immunoblotting. (C) Analysis of the ubiquitination of Ras isoforms. CHOK1 coexpressing HA-Ub and the indicated T7-tagged Ras chimeric constructs were subjected to Ras immunoprecipitation followed by immunoblotting. (D) The effect of membrane attachment on the ubiquitination of HRas. CHOK1 coexpressing HA-Ub and farnesylation-deficient (C186S) or palmitoylation-deficient (C181,184S) T7-HRas constructs were subjected to Ras immunoprecipitation followed by immunoblotting. Molecular Cell 2006 21, 679-687DOI: (10.1016/j.molcel.2006.02.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Ubiquitination Regulates the Intracellular Trafficking of HRas (A) Ubiquitination analysis of HRas 8RK mutant. Lysates from CHOK1 cells expressing T7-HRas wt or T7-HRas8RK alone or together with HA-Ub were subjected to Ras immunoprecipitation followed by immunoblotting. (B) Colocalization of Ras constructs (red) and the Golgi marker, YFP-1,4-β-galactosyltransferase (YFP-β-Gal, green), in COS-1 cells expressing HRas wt, HRas8RK, and Ub-HRas. Blue color indicates nuclear DAPI staining. Selected regions indicated in the merged images by the white rectangle are shown at a higher magnification to the right. Scale bar represents 20 μM. (C) Analysis of the extent of localization of HRas constructs to the Golgi. Pixel intensity for HRas wt, HRas 8RK, or Ub-HRas fluorescence signal was assessed at the Golgi and normalized to the fluorescence intensity of the Golgi marker, YFP-1,4-β-Gal (del Pozo et al., 2004). Values are means of n = 12 cells measured for HRasWT, n = 14 for HRas8RK, and n = 11 for Ub-HRas and are represented as a fold over HRasWT ± SEM. Molecular Cell 2006 21, 679-687DOI: (10.1016/j.molcel.2006.02.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Computational Model of HRas Intracellular Trafficking (A) The intracellular HRas trafficking pathways included in the simulations shown in (B)–(D). PM, plasma membrane; k1, rate of anterograde movement along the secretory pathway; k2, rate of retrograde pathway; k3, rate of endocytosis; k4, rate of endocytic recycling. For details, see the Supplemental Data. (B) The size of Golgi pool is influenced by the size of endosomal pool of HRas. The size of endosomal pool of HRas was changed by varying the rates of endocytosis from k3 = 0.7 min−1 to k3 = 0.01 min−1 and recycling from k4 = 0.01 min−1 to k4 = 0.7 min−1 by 0.01 increments. The plot represents concentrations of HRas in arbitrary units at the endosome and Golgi for varying k3/k4 ratios. The simulation of concentration of HRas in both of these compartments for every k3/k4 with time is shown in Figure S3. (C) The relationship between the concentration of HRas at the Golgi and the rate of endocytosis. In this simulation, intracellular trafficking of HRas was simulated at varying rates of endocytosis (k3) from k3 = 0 min−1 to k3 = 0.72 min−1 by 0.01 increments. Recycling rate was set as k4 = 0.15 min−1 (t1/2 = 4.6 min), which corresponds to a moderate rate of recycling (see Supplemental Data for details). HRas concentration at the Golgi is presented in arbitrary units and is plotted as a function of time. (D) The relationship between the concentration of HRas at the Golgi and the rate of recycling. Intracellular trafficking of HRas was simulated at varying rates of endocytic recycling (k4) from k4 = 0 min−1 to k4 = 0.72 min−1 with step 0.01. Endocytosis rate was set as k3 = 0.15 min−1 (t1/2 = 4.6 min, see Supplemental Data for details). HRas concentration at the Golgi is presented in arbitrary units and is plotted as a function of time. Molecular Cell 2006 21, 679-687DOI: (10.1016/j.molcel.2006.02.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Ubiquitination of HRas Affects Its Ability to Activate the ERK Cascade (A) Analysis of the activation of the ERK signaling pathway. CHOK1 cells expressing the indicated Ras constructs and HA-ERK were analyzed for ERK activation as described in Experimental Procedures. The fold of ERK activation was quantified using Storm Phosphorimager (Molecular Dynamics) and is indicated below each lane in arbitrary units. Results shown are representative of three independent experiments. (B) Raf binding assay. CHOK1 cells expressing HRas or HRas8RK were subjected to GST-RBD pull-down assay. (C) Raf recruitment assay. CHOK1 cells transfected with the indicated plasmids were fractionated, and P100 fractions were immunoblotted for Raf-1 and Ras constructs (note that these experiments necessitate the use of activated Ras [G12V] in order to promote the membrane recruitment of Raf-1 [Leevers et al., 1994]). Bar graphs show the quantitative analysis of Raf recruitment performed using Storm Phosphor Imager. The data are normalized to the amount of Ras in P100 fraction and represent the averages of three independent experiments. Error bars represent SEM; differences between experiments were examined using Student's t test (∗p < 0.05; ∗∗p < 0.01). Molecular Cell 2006 21, 679-687DOI: (10.1016/j.molcel.2006.02.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Ubiquitin-Dependent Targeting of HRas to the Endocytic Pathway Controls the Signaling Potential of HRas (A) Analysis of the activation of the ERK signaling pathway. CHOK1 cells expressing the indicated Ras constructs and HA-ERK were analyzed for ERK activation as described in Experimental Procedures. (B) Raf binding assay. CHOK1 cells expressing HRasV12 or Ub-HRasV12 were subjected to GST-RBD pull-down assay. (C) Raf recruitment assay. CHOK1 cells transfected with the indicated plasmids were fractionated, and P100 fractions were immunoblotted for Raf-1 and Ras constructs. Bar graphs show the quantitative analysis of Raf recruitment performed using Storm Phosphor Imager. The data are normalized to the amount of Ras in P100 fraction and represent the averages of three independent experiments. Error bars represent SEM; differences between experiments were examined using Student's t test (∗p < 0.05). (D) Subcellular localization of Ub-HRasV12 and UbLIV-HRasV12 constructs in COS-1 cells. Arrowheads point to endosome-localized Ub-HRasV12. (E) Analysis of the activation of the ERK signaling pathway. CHOK1 cells expressing the indicated Ras constructs and HA-ERK were analyzed for ERK activation as described in Experimental Procedures. Molecular Cell 2006 21, 679-687DOI: (10.1016/j.molcel.2006.02.011) Copyright © 2006 Elsevier Inc. Terms and Conditions