Skip to main content
. Author manuscript; available in PMC: 2018 Apr 1.
Published in final edited form as: Cancer Discov. 2017 Feb 7;7(4):424–441. doi: 10.1158/2159-8290.CD-16-0647

Figure 6. Pharmacologically Inhibiting BRAF/ERK and CDK4 Restores the APCFZR1 E3 Ligase Activity.

Figure 6

(A) Protein levels of BRAF and other APCFZR1 substrates decreased upon BRAFV600E and CDK4/6 inhibition in melanoma cells. IB analysis of BRAFV600E and CDK4R24C expressing SK-MEL-28 melanoma cells treated with either 1 μM BRAFV600E inhibitor PLX4032 (V600Ei), 10 μM pan-CDK inhibitor mimosine (pan-CDKi), 1 μM CDK4/6 inhibitor PD0332991 (CDK4i), 1 μM PLX4032+10 μM mimosine, 1 μM PLX4032+1 μM PD0332991 or DMSO as a negative control for 24 h before harvesting. BRAF band intensities were quantified using ImageJ, normalized to corresponding TUBULIN band intensities, and then normalized to DMSO control lane.

(B) Protein levels of BRAF and other APCFZR1 substrates reduced upon MEK and CDK4/6 inhibition in melanoma cells. IB analysis of BRAFWT expressing HBL melanoma cells treated with either 1 μM MEK inhibitor PD0325901 (MEKi), 10 μM pan-CDK inhibitor mimosine (pan-CDKi), 1 μM CDK4/6 inhibitor PD0332991 (CDK4i), 1 μM PD0325901+10 μM mimosine, 1 μM PD0325901+1 μM PD0332991 or DMSO as a negative control for 24 h before harvesting. BRAF band intensities were quantified using ImageJ, normalized to corresponding Tubulin band intensities, and then normalized to DMSO control lane.

(C) Depletion of FZR1 in BRAFV600E-inhibited melanoma cells led to the upregulation of BRAF and PLK1. IB analysis of BRAFV600E expressing A375 melanoma cells, which were, infected with the control (shScr) or the indicated shFZR1 lentiviral shRNA constructs. The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells before harvesting. Prior to the harvest, cells were treated with DMSO (as a negative control) or 1 μM BRAFV600E inhibitor PLX4032 (V600Ei) for 24 hours as indicated. BRAF band intensities were quantified using ImageJ, normalized to corresponding Tubulin band intensities, and then normalized to DMSO control lane (upper row) or normalized to shScr+V600Ei lane (lower row).

(D) A schematic illustration of the proposed model for the putative role of FZR1 in suppressing BRAF dimerization-mediated transactivation of downstream MEK/ERK signaling to bypass PLX4032 triggered BRAFV600E inhibition in melanoma cells, as well as how mechanistically hyperactive ERK and/or CYCLIN D1/CDK4-mediated phosphorylation of FZR1 inhibits APCFZR1 E3 ligase activity in BRAFV600E melanoma cells.