1. Volume 52, Issue 2, Pages 161-172 (October 2013)
Phosphorylation of BRAF by AMPK Impairs BRAF-KSR1 Association and Cell Proliferation 
Che-Hung Shen, Ping Yuan, Rolando Perez-Lorenzo, Yaqing Zhang, Sze Xian Lee, Yang Ou, John M. Asara, Lewis C. Cantley, Bin Zheng 
Molecular Cell 
Volume 52, Issue 2, Pages (October 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2013 52, 161-172DOI: (10.1016/j.molcel.2013.08.044)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1
Activation of AMPK Attenuates RAF-MEK-ERK Signaling in Various Cell Lines
(A) C140 melanocytes or CCD1106 human keratinocytes were treated with or without 2 mM AICAR for 2 hr.
(B) WT and AMPK-null MEFs were treated with or without 100 μM A in DMEM media containing 10% FBS for 2 hr. Cell lysates were used for western blotting with indicated antibodies. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 2 AMPK Phosphorylates BRAF at Ser 729 Both In Vitro and In Vivo
(A) Identification of phosphorylated BRAF peptides containing Ser729 by LC-MS/MS analysis. COS-7 cells transfected with FLAG-BRAF were treated with or without 2 mM AICAR for 2 hr. FLAG-BRAF proteins were immunoprecipitated using anti-FLAG M2 agarose beads and subjected to trypsin or chymotrypsin digestion followed by the LC-MS/MS analysis. Percentages of phospho-Ser 729 peptides versus these peptides in the unphosphorylated states were calculated using total ion intensities over elution peaks.
(B) Alignment of the sequence surrounding Ser729 in BRAF with the analogous sites in CRAF and ARAF and other AMPK phosphorylation sites in selective proteins.
(C) AMPK directly phosphorylates BRAF in vitro. FLAG-BRAF WT or S729A (SA) mutant proteins that immunoprecipitated from HEK293 cells were incubated with active recombinant AMPK proteins in the presence of γ-32P-ATP.
(D) Treatment with AICAR increases phosphorylation of BRAF Ser729, but not CRAF Ser621, in CCD1106 cells. Cells were treated with or without 2 mM AICAR for 2 hr.
(E) AICAR-induced BRAF Ser729 phosphorylation is dependent on the presence of AMPK. WT or AMPK null MEFs were treated with or without 100 μM A for 2 hr.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3 AMPK Associates with BRAF
(A) FLAG-BRAF proteins coimmunoprecipitate with endogenous AMPKα proteins.
(B) Myc-AMPKα2 proteins coimmunoprecipitate with endogenous BRAF proteins.
(C) Endogenous BRAF proteins coimmunoprecipitate with endogenous AMPKα1 proteins in CCD1106 melanocytes. HEK293 cells were transfected with FLAG-BRAF (A), Myc-AMPKα2 WT, or K45R (kinase-dead, KD) mutant (B) as indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 4
Phosphorylation of BRAF Ser729 Is Critical for Attenuation of ERK Signaling by AMPK
(A) Attenuation of MEK-ERK signaling by AMPK activation is mediated by BRAF Ser729 in MEFs. BRAF-null MEFs were stably transfected with control vector or various BRAF constructs as indicated. Cells were treated with 1 mM AICAR for 1 hr.
(B) Attenuation of MEK-ERK signaling by AMPK activation is mediated by BRAF S729 in CCD1106 cells. Cells stably expressing various BRAF constructs were treated with 2 mM AICAR for 2 hr. Quantification analysis was done for three independent experiments. Data are presented as mean ± SEM; ∗p < 0.05;∗∗p < 0.01; N.S., not significant.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

7. Figure 5
AMPK-Dependent Phosphorylation of BRAF Ser729 Promotes Its Binding to and Disrupts Its Association with KSR1
(A) Treatment with AICAR does not affect the kinase activity of BRAF. C140 cells were treated with or without 2 mM AICAR for 2 hr. Endogenous BRAF proteins were immunoprecipitated and the kinase activities of BRAF were measured using the BRAF kinase cascade assay. Data are represented as mean ± SEM.
(B) Activation of AMPK by AICAR promotes the BRAF/ association in WT MEFs but not Ampk-null MEFs. Lysates from WT or Ampk-null MEFs were immunoprecipitated with anti antibodies, followed by immunoblotting.
(C) Activation of AMPK by AICAR promotes the association between and BRAF, but not CRAF. CCD1106 cells lysates were incubated with GST in GST pull-down assays.
(D) Activation of AMPK by AICAR disrupts the association between FLAG-KSR1 and BRAF. CCD1106 cells were transiently infected with FLAG-KSR1-expressing retrovirus. Cells were treated with 2 mM AICAR for 2 hr.
(E) Activation of AMPK by AICAR disrupts the endogenous BRAF-KSR1 dimer formation.
(F) Ser729 of BRAF is critical for the disruption of BRAF-KSR1 association upon activation of AMPK. CCD1106 cells stably expressing HA-KSR1 were transiently infected by various FLAG-BRAF-expressing retroviruses, as indicated.
(G) Phosphorylation of BRAF Ser729 is critical for the association between BRAF and Braf-null MEFs stably expressing various BRAF constructs were subjected to immunoprecipitation. Cells were treated with 1 mM AICAR for 1 hr, as indicated.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

8. Figure 6
Phosphorylation of BRAF at Ser729 Regulates Keratinocyte Cell Proliferation and Cell-Cycle Progression
(A) Expression of BRAF S729A mutant (SA) in CCD1106 keratinocytes promotes cell proliferation. Cells were seeded at 3 × 104 cells per well, and cells were counted daily. Cell media were changed daily. Cell proliferation of CCD1106 cells stably expressing WT BRAF or S729A (SA) mutant was measured. One representative from three independent experiments is shown. Data are represented as mean ± SEM.
(B) Treatment of CCD1106 cells expressing BRAF SA mutant with AICAR leads to more dramatic decrease of cells in the G2 phase and increase of cells in S phase, compared to that of CCD1106 cells expressing WT BRAF. Cells were stained with propidium iodide and analyzed by FACS. Data are presented as mean ± SEM (n = 3); ∗∗p < 0.01; N.S. indicates not significant. Histograms from one representative experiment are shown.
(C) CCD1106 cells expressing BRAF S729A mutant are less sensitive to the inhibitory effects of AICAR on cell proliferation than those expressing WT BRAF. Cells were treated with 0.3 mM AICAR for the indicated time. Cell numbers were counted daily and expressed as percentage of cell numbers of each stable line at day 0 before AICAR treatment. Differences in cell numbers between cell lines at each time point were assessed by paired t test. Data are represented as mean ± SEM. ANOVA: time X cell, F (2,48) = 3.97, p < 0.05.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

9. Figure 7
Activation of AMPK Prevents PLX4720-Induced Hyperactivation of ERK and Proliferative Response in Keratinocytes
(A) Activation of AMPK by AICAR attenuates PLX4032-induced hyperactivation of ERK. CCD1106 keratinocytes were treated with 2 mM AICAR for 1 hr before adding 10 μM PLX4032 for an additional hour, as indicated.
(B) Schematic diagram of drug treatment. FVB/n mice were given PLX4720, 100 mg/kg (bid) via oral gavage for 2 days. In an additional group, mice were treated twice daily with 100 mg phenformin/Kg (bid) via oral gavage, one day before PLX4720 treatment and during the 2-day PLX4720 treatment.
(C and D) Phenformin prevents the PLX4720-induced epidermal hyperplasia (C) and increase of proliferation index (D) in mouse skin. Five mice were included in each group. Epidermal thickness and proliferation index were measured as described in Experimental Procedures. Paired t test was used for comparisons between groups. Data are represented as mean ± SEM.
(E) Treatment with phenformin leads to activation of AMPK in mouse skin and prevents PLX4720-induced activation of ERK and increase of Ki67 staining. Representative images of mouse skin samples subjected for various immunohistochemical analyses are shown. Bar indicates 50 μm.
(F) A schematic model for regulation of BRAF-MEK-ERK signaling pathway by AMPK.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions