1. Volume 64, Issue 5, Pages 875-887 (December 2016)
Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-Induced Phospho- Regulatory Circuit 
Daniel A. Ritt, María T. Abreu-Blanco, Lakshman Bindu, David E. Durrant, Ming Zhou, Suzanne I. Specht, Andrew G. Stephen, Matthew Holderfield, Deborah K. Morrison 
Molecular Cell 
Volume 64, Issue 5, Pages (December 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 64, 875-887DOI: (10.1016/j.molcel.2016.10.029)
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3. Figure 1 Effects of Rigosertib on Ras/Raf/MEK/ERK Signaling
(A) Biacore SPR analysis of in vitro RafRBD binding: K-Ras-GTP or K-Ras-GDP (10 μM) were injected over WT- or R89L-RafRBD, and binding responses were determined.
(B) A 2-fold dilution series of rigosertib (50–0.39 μM) was injected over WT-RafRBD, and binding was determined by SPR.
(C) WT-RafRBD (5 μM) was mixed with increasing concentrations of rigosertib (0.19–50 μM), following which binding to avi-K-RasGTP was measured (red lines) and compared with a RafRBD calibration series (black lines).
(D) WT- or R89L-GST-RafRBD was incubated for 1 hr with increasing concentrations of rigosertib, and binding to GTP-loaded WT- or G12V-K-Ras was determined by proximity-based energy transfer. As a control for nonspecific inhibition, the effect of rigosertib on Avi-GST was also monitored.
(E) HeLa cells were treated as indicated with DMSO or 2 μM rigosertib (Rig) prior to stimulation with EGF for 5 min (+) and lysis. Raf dimerization was monitored by probing Raf immunoprecipitates for the presence of other Raf family members. Lysates were examined for activated pMEK, pERK, and pAKT levels and for total B-Raf, C-Raf, A-Raf, and tubulin (loading control) levels.
(F) Depiction of the Raf domain structure and location of the rigosertib-induced sites of S/TP phosphorylation.
(G) The indicated cancer lines were treated with DMSO (−) or rigosertib (+) for 18 hr prior to lysis. Endogenous C-Raf was immunoprecipitated and probed for pS642P levels or B-Raf. Shifts in the electrophoretic mobility of B-Raf and C-Raf were also examined.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
JNK Contributes to Raf Hyperphosphorylation in Rigosertib-Treated Cells
(A) HeLa cells were treated with rigosertib for the indicated times prior to lysis. Levels of pS642P-C-Raf, pMEK, pERK, pMKK4, pJNK, and pP38 as well as shifts in B-Raf and C-Raf mobility were determined.
(B) HeLa cells were treated with rigosertib for 2 or 18 hr in the presence or absence of the MEK inhibitor U0126 prior to lysis. C-Raf immunoprecipitates and lysates were examined as indicated.
(C) HeLa cells were treated with rigosertib for 18 hr in the presence or absence of the antioxidant NAC prior to lysis. C-Raf immunoprecipitates and lysates were examined as indicated.
(D) HeLa cells treated for various times with rigosertib were examined for cell-cycle arrest by FACS analysis and for mitochondrial ROS generation by staining with MitoSOX RED.
(E) ROS levels were quantified and the data are represented as mean ± SEM.
(F) HeLa cells were treated with rigosertib for 18 hr in the presence or absence of the JNK inhibitor (JNKi, SP600125) prior to lysis. pS642P-C-Raf levels and Raf mobility shifts were determined.
(G) HeLa, HCT-116, and SW620 were treated with DMSO (−) or rigosertib (+) for 18 hr prior to lysis. pS642P-C-Raf and pJNK levels, MKK4 expression, and Raf mobility shifts were monitored.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Rigosertib-Induced S/TP Phosphorylation Alters Sos1 and Raf Function
(A) HeLa cells were treated for 18 hr with DMSO or rigosertib prior to lysis. Endogenous Sos1 was immunoprecipitated and probed for pS/TP phosphorylation. A similar analysis of tubulin was included as a negative control.
(B) HeLa cells were treated as indicated prior to lysis. Lysates were examined for the electrophoretic mobility of Sos1, C-Raf, and tubulin.
(C) Depiction of the Sos1 domain structure and location of the rigosertib-induced sites of S/P phosphorylation.
(D) HeLa cells were treated for 18 hr with the indicated concentrations of rigosertib prior to EGF stimulation (+) and lysis. pS642P-C-Raf, activated pY1068-EGFR, pMEK, and pMKK4 levels as well as Sos1, C-Raf, and B-Raf mobility shifts were determined.
(E) HeLa cells were treated for 18 hr with DMSO or rigosertib prior to EGF stimulation (+) and lysis. Lysates were incubated with glutathione beads containing GST-RafRBD, and binding of endogenous GTP-Ras to GST-RafRBD was determined by immunoblot analysis. Total Ras and Sos1 levels were also monitored.
(F and G) HeLa cells (F) or those expressing WT-, 5A-, or R401H-C-Raf (G) were treated for 18 hr with DMSO or rigosertib prior to lysis. The lysates were incubated with glutathione beads containing GST-RasV12, and the binding of the Raf proteins to GST-RasV12 was assessed by immunoblot analysis. Total Raf levels were also determined.
(H) HeLa cells were treated for 18 hr with DMSO or rigosertib prior to EGF stimulation (+) and lysis. Raf dimer formation was monitored, and lysates were examined for pMEK levels and Raf mobility shifts.
(I and J) HeLa cells expressing increasing amounts of Halo-G12V-K-Ras (I) or Venus-V600E-B-Raf (J) were treated for 18 hr with DMSO or rigosertib prior to lysis. Lysates were examined as indicated.
(K) A375 and SK-Mel28 melanoma cells, homozygous for V600E-B-Raf, were treated for 18 hr with DMSO or rigosertib prior to lysis. Lysates were examined as indicted.
See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Effects of Other Inducers of Mitotic and Oxidative Stress on Ras/Raf/MEK/ERK Signaling
(A) HeLa cells were treated for 18 hr with the indicated drugs prior to lysis. pS642P-C-Raf levels were determined and lysates were examined for activated pMKK4, pJNK, and pP38 levels and for the shifts in Raf and Sos1 mobility. Cells treated for 18 hr with Taxol or KG5 were also examined for cell-cycle arrest and ROS generation. ROS levels were quantified, and the data are represented as mean ± SEM.
(B) HeLa cells were treated with the indicated drugs in the presence (+) or absence (−) of the JNK inhibitor (SP600125) prior to lysis and analysis.
(C) HeLa cells treated as indicated were lysed and lysates examined as in (A).
(D) HeLa cells were treated for either 2 or 18 hr with the indicated drugs prior to EGF stimulation (+) and lysis. Raf dimer formation, shifts in Raf and Sos1 mobility, and pMEK levels were monitored.
(E) HeLa cells were treated as indicated prior to EGF stimulation (+) and lysis. Lysates were incubated with glutathione beads containing GST-Raf-RBD, and binding of endogenous GTP-Ras to GST-Raf RBD was determined by immunoblot analysis. Total Ras and Sos1 levels are also shown.
(F) Lysates prepared from cells treated as in (A) were incubated with glutathione beads containing GST-RasV12, and the binding of Raf proteins to GST-RasV12 was determined by immunoblot analysis. Raf mobility shifts were also examined.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
TNFα Treatment Induces the S/TP Phosphorylation of the Rafs and Sos1
(A) HeLa cells were treated for various times with TNFα alone or TNFα and cycloheximide (TNF + CHX) prior to lysis. pS642P-C-Raf were determined and lysates were examined for pJNK levels and the shifts in Raf and Sos1 mobility.
(B) HeLa cells were pretreated or not with a JNK inhibitor for 1 hr, following which they were stimulated with TNFα, lysed and analyzed.
(C) ROS levels were quantified in HeLa cells treated as indicated, and the data are represented as mean ± SEM.
(D) HeLa cells were treated as indicated prior to EGF stimulation (+) and lysis. Raf dimer formation, shifts in Raf and Sos1 mobility, and pMEK levels were monitored.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Model for Stress-Induced Phospho-Inhibition of Ras/Raf/MEK/ERK Signaling
See text for details.
Molecular Cell  , DOI: ( /j.molcel )
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