Ras Enhances Myc Protein Stability Rosalie Sears, Gustavo Leone, James DeGregori, Joseph R Nevins  Molecular Cell  Volume 3, Issue 2, Pages 169-179 (February 1999) DOI: 10.1016/S1097-2765(00)80308-1

Figure 1 Cell Growth Stimulation Stabilizes the c-Myc Protein (A) Myc protein accumulation is enhanced by serum stimulation. REF52 cells were made quiescent by serum starvation for 48 hr and then infected with the Ad-Myc virus at a multiplicity of infection (MOI) of 1000 FFU per cell. Infected cells were maintained in starvation medium for an additional 12 hr at which point they were stimulated by the addition of fetal bovine serum (FBS) to 20% (+Serum) for the indicated time. Cells were harvested using a whole cell lysate method (Ikeda et al. 1996), and 25 μg of protein from each time point was analyzed by Western analysis using the monoclonal c-Myc antibody C-33 (Santa Cruz Biotechnology). (B) Myc RNA is constant in Ad-Myc infected cells. REF52 cells were treated as described in (A). Poly A+ RNA was isolated from approximately 300 μg of total RNA from each time point and subjected to Northern analysis. The blot was probed for c-myc and GAPDH (loading control) as indicated. (C) Mitogen stimulation increases the half-life of Myc. Quiescent REF52 cells were infected with Ad-Myc (MOI = 200). Following infection, cells were either maintained in starvation medium for 26 hr as indicated (starved) or after 6 hr in starvation medium, cells were stimulated by the addition of FBS to 20% (+Serum) and incubated for an additional 20 hr. Cells were then labeled in vivo with [35S]methionine for 30 min followed by a chase in medium containing excess unlabeled methionine and 0.25% (Starved) or 20% serum (+Serum) for the indicated times. Labeled c-Myc was immunoprecipitated from equal cell numbers and equal labeled proteins for each sample as visualized by autoradiography. (D) Half-life of Myc in quiescent cells. Quiescent REF52 cells were infected with Ad-Myc virus (MOI = 100). Cells were pulse labeled and chased in starvation medium as described in (C) 18 hr postinfection. Labeled c-Myc was immunoprecipitated as described for (C). A 3 day exposure is shown. (E) Half-life of the endogenous c-Myc protein following serum stimulation. Quiescent REF52 cells were stimulated with 20% FCS for 5 hr before pulse labeling for 30 min in vivo with [35S]methionine and 20% charcoal-stripped serum. Labeled cells were chased as described in (C) (+Serum). Labeled c-Myc was immunoprecipitated as described for (C). A 3 day exposure is shown. (F) Half-lives of Myc protein in quiescent and serum-stimulated cells. 35S-labeled Myc1 and Myc2, expressed from the Ad-Myc virus shown in (C) (right panel), Ad-Myc + serum (filled circle), and (D), Ad-Myc starved (filled triangle), and 35S-labeled endogenous c-Myc, shown in (E), c-Myc + serum (filled square) were quantitated by phosphoimager. Background was calculated from an equivalent area for each lane and subtracted from the value for labeled Myc in that lane. Time zero was set at 100. Data is graphed semilog. Best fit lines were calculated using linear regression with the SigmaPlot program. Molecular Cell 1999 3, 169-179DOI: (10.1016/S1097-2765(00)80308-1)

Figure 2 Ras Activation Enhances Myc Protein Accumulation (A) Ras activation is necessary for serum-induced stabilization of Myc. Quiescent REF52 cells were infected with Ad-Myc (MOI = 100) plus either a control virus with an empty expression cassette (Ad-Con MOI = 200) (Ctrl) or Ad-RasN17 at the indicated MOIs. Infected cells were incubated under starvation conditions for 22 hr and then serum stimulated with 20% FBS for 4 hr where indicated (+Serum). Equal cell numbers for each condition were harvested and Western analysis was performed using the C-33 Myc antibody. Equal protein loading was confirmed by Ponceau S staining of the blot. (B) Activated Ras enhances Myc accumulation in the absence of other growth stimuli. Quiescent REF52 cells were infected with the indicated viruses at multiplicities of infection as follows: (Ctrl) Ad-Con (MOI = 300), Ad-Ras61L (MOI = 200), Ad-Myc (MOI = 100), and Ad-p21 (MOI = 200). Infected cells were maintained in starvation medium for 28 hr and then harvested for Western analysis as described in (A). (C) Quiescent REF52 cells were infected as described in (B). Infected cells were maintained in starvation medium and Poly(A)+RNA was prepared 30 hr postinfection for Northern analysis. c-myc and GAPDH (loading control) mRNAs are indicated. Molecular Cell 1999 3, 169-179DOI: (10.1016/S1097-2765(00)80308-1)

Figure 6 Ras-Mediated Stabilization of Myc Coincides with Increased Myc Function (A) Myc and Ras collaborate to activate S phase. Quiescent REF52 cells were infected with Ad-Con (MOI = 200) (Ctrl, Serum), Ad-Myc (MOI = 100), Ad-Ras61L (MOI = 200), and Ad-p21 (MOI = 200) as indicated. Cells were maintained in starvation medium except as indicated (Serum) where FBS was added to the medium to 20% 5 hr postinfection. Cells were labeled with BrdU for 8 hr prior to processing for BrdU incorporation 30 hr postinfection. (B) Myc and Ras collaborate to activate E2F2 transcription. Quiescent REF52 cells were infected with the indicated viruses as follows: Ad-Con (MOI = 300) (Serum, Ctrl), Ad-Myc (MOI = 200), Ad-Ras61L (MOI = 100), and Ad-p21 (MOI = 200). Cells were maintained in starvation medium except as indicated (Serum) where the medium was replaced by D-MEM/10% serum 5 hr postinfection. Poly A+ RNA was isolated from approximately 300 μg of total RNA at 30 hr postinfection and subjected to Northern analysis. E2F2 and GAPDH mRNAs are indicated. (C) Quiescent REF52 cells were treated as described for (B) with the addition of Ad-E2F2 (MOI = 100), which was harvested 18 hr postinfection. Whole cell lysates were prepared as described (Ikeda et al. 1996), and 50 μg protein from each sample was analyzed by Western analysis with an E2F2 polyclonal antibody (Santa Cruz Biotechnology). (D) Ras augments Myc-mediated activation of the E2F2 promoter. REF52 cells were transfected 20 hr after plating with 5 μg of either the wild-type E2F2 promoter driving the luciferase reporter gene (E2F2-luc) (left panel) or a mutant E2F2 promoter containing point mutations in the three E box Myc binding sites (which eliminate Myc binding) [E2F2 (ΔMyc)-luc] (right panel) along with 2 μg of CMV-β-gal as an internal control. After transfection, cells were placed in DMEM containing 0.25% serum. Transfected cells were infected with Ad-Con (MOI = 200) (Ctrl), Ad-Myc (MOI = 100), Ad-Ras61L (MOI = 200), or Ad-Myc + Ad-Ras61L, as indicated, 48 hr later. Cells were harvested, and luciferase and β-galactosidase activities were measured 20 hr postinfection. Luciferase activity was normalized to β-galactosidase, and data are presented as fold activation over control. All transfections were performed at least three times, and a representative experiment is shown. Molecular Cell 1999 3, 169-179DOI: (10.1016/S1097-2765(00)80308-1)

Figure 3 Activation of the Ras/MAPK Pathway Stabilizes Myc Protein (A) Activated Raf1 expression enhances Myc protein levels. Quiescent REF52 cells were infected with Ad-Myc (MOI = 100) and either Ad-Con (Ctrl), Ad-Ras61L, or Ad-RafCAAX as indicated, all at an MOI of 50. Infected cells were maintained in starvation medium for 26 hr, and equal cell numbers were harvested for Western analysis with the C33 antibody. Equal protein loading was confirmed by Ponceau S staining of the blot. (B) MAPK activation is required for Raf-mediated Myc protein accumulation. Quiescent REF52 cells were infected with Ad-Myc (MOI = 100) and, as indicated, either Ad-Con (Ctrl) (MOI = 200) or Ad-RafCAAX (MOI = 200). Infected cells were maintained in starvation medium. The MAPK inhibitor PD 098059 (Calbiochem) was added as indicated 8 hr after infection. Cells were harvested 26 hr postinfection for Western analysis as described for (A). (C) E2F4 protein levels are not affected by serum addition or Ras activity. Quiescent REF52 cells were infected with Ad-E2F4 (MOI = 100) and either Ad-Con (MOI = 200) (Ctrl), Ad-Ras61L (MOI = 200), Ad-p21 (MOI = 200), or Ad-RasN17 (MOI = 25, 100) as indicated, or treated with PD 098059 8 hr after infection with Ad-E2F4 (10, 50 μm). Cells were harvested 26 hr postinfection. Samples shown in lanes 4–8 were serum stimulated with 20% FCS 4 hr before harvesting. Western analysis was performed from equal cell numbers for each condition using the E2F4 polyclonal antibody SC-1082x (Santa Cruz Biotechnology). (D) Expression of activated Raf1 increases Myc protein half-life. Quiescent REF52 cells were infected with Ad-Myc (MOI = 100) and either Ad-Con (MOI = 200) (Myc), or Ad-RafCAAX (MOI = 200) (Myc + Raf) as indicated. Infected cells were maintained in starvation medium for 26 hr and then labeled in vivo with [35S]methionine for 15 min followed by a chase in medium containing excess unlabeled methionine and 0.25% serum for the indicated times. Myc protein was immunoprecipitated from equal cell numbers and equal labeled protein from each sample as visualized by autoradiography. (E) Labeled Myc1 and Myc 2 from each sample shown in (D) was measured by phosphoimager. Ad-Myc + Ad-Con (filled triangles); Ad-Myc + Ad-Raf (filled squares). Background was calculated from an equivalent area for each lane and subtracted from the value for labeled Myc in that lane. Time zero was set at 100. Data is graphed semilog. Best fit lines were calculated by linear regression using the Sigmaplot program. Molecular Cell 1999 3, 169-179DOI: (10.1016/S1097-2765(00)80308-1)

Figure 4 Myc Is Rapidly Degraded by the 26S Proteasome in Quiescent Cells (A) Quiescent REF52 cells were infected with Ad-Myc (MOI = 100). Cells were maintained in starvation medium. Where indicated, the proteasome inhibitor LLnL, 20 μg/ml (Sigma) or Lactacystin, 10 μM (Dr. E. Corey, Harvard) was added for the times shown prior to harvesting. Equal cell numbers were harvested 26 hr postinfection for Western analysis with the C33 antibody. Equal protein loading was confirmed by Ponceau S staining of the blot. (B) Activated Raf1 prevents proteasome-mediated degradation of Myc. Quiescent REF52 cells were infected with Ad-Myc (MOI = 100), or Ad-Myc plus Ad-RafCAAX (MOI = 200). Infected cells were maintained in starvation medium. Where indicated (+L), cells were treated with lactacystin for 2 hr prior to harvesting. At 26 hr postinfection, equal cell numbers from each condition were harvested for Western analysis as described in (A). Molecular Cell 1999 3, 169-179DOI: (10.1016/S1097-2765(00)80308-1)

Figure 5 The Accumulation of Endogenous Myc Protein Is Regulated by the Ras Pathway (A) Proteasome-mediated degradation of c-Myc in quiescent cells. Quiescent REF52 cells were either stimulated with 20% serum for 4 hr (+S), harvested without treatment (-), or treated with lactacystine for 4 hr (+L) before harvesting, as indicated. Myc protein was immunoprecipitated from approximately 1 × 106 cells (lane 1) or 3 × 106 cells (lanes 2 and 3) with the C-33 monoclonal Myc antibody and then subjected to Western blot analysis with the same antibody. c-Myc and IgG heavy chain are indicated. (B) Inhibition of the Ras pathway reduces the accumulation of endogenous Myc protein following growth stimulation. Quiescent REF52 cells were either harvested (−), treated with 20% FCS for 4 hr (+Serum), treated with the indicated concentrations of the MAPK inhibitor PD 098059 for 12 hr prior to the 4 hr serum stimulation, or infected with Ad-RasN17 at the indicated MOI 18 hr prior to serum stimulation. Myc protein was immunoprecipitated from 1 × 106 cells for each condition and analyzed by Western blotting as described above. (C) Effect of MAPK inhibitor and the Ras mutant on the accumulation of Myc RNA. RNA was prepared from aliquots of the cells treated as in (B). Poly A+ RNA was isolated from 200 μg of total RNA from each condition and subjected to Northern blot analysis. Blots were probed with either a c-myc probe or GAPDH as a control. Molecular Cell 1999 3, 169-179DOI: (10.1016/S1097-2765(00)80308-1)