Figure 6. Identification of the MEK5-ERK5 Pathway as a Regulator of MYC Protein Stability in PDAC Cells.
(A) Histograms of EGFP/DsRed ratio in MIA PaCa-2 cells stably expressing the GPSMYC reporter and infected with control or two lentivirus expression vectors encoding constitutively-activated variants of MEK5 (Myr-FLAG-MEK5 or MEK5S311/T315D/DD). In a well-by-well format, MIA PaCa-2 cells expressing the GPS-MYC reporter were infected with individual lentiviral vectors encoding activated signaling components from the Cancer Toolkit). Forty-eight hr after infection, EGFP and DsRed fluorescence were measured by FACS and EGFP/DsRed ratio histograms were generated using Summit 5.2.
(B) Chemical structure of the compound UNC10225170 (GW284543), a 4-anilinoquin(az)oline chemotype, from the PKIS2 library.
(C) MIA PaCa-2 cells were treated with the indicated doses of UNC10225170 for 6 hr. Cell lysates were immunoblotted as indicated.
(D) MIA PaCa-2 cells were transfected with expression vectors encoding ubiquitin and HA epitope-tagged WT or kinase-deficient ERK5 (K84R) for 48 hr. Following 6 hr MG132 treatment, MYC protein was isolated by immunoprecipitation and immunoblotted for ubiquitination using anti-ubiquitin antibody. MYC protein precipitates and heavy chain as loading control are also shown. Immunoblotting of cell lysates was also done to determine total MYC and HA-ERK5 input protein levels.
(E) 293T cells were transiently transfected with expression vectors encoding GFP (control), or HA epitope-tagged ERK5. After 48 hr, the cultures were treated with CHX for the indicated times. Cell lysates were then immunoblotted with the indicated antibodies (left panel). Following quantitation of blot data to the left, MYC half-life was calculated using Prism (right panel).
(F) PDAC cell lines were transfected with siRNA oligos targeting ERK5 for 42 hr, then treated with 5 μM MG132 for an additional 6 hr. Cell lysates were immunoblotted as indicated.
(G) PDAC cells were transfected with NS or ERK5 siRNAs for 48 hr and RNA was extracted to measure gene expression. Following cDNA synthesis, real-time qPCR was performed using MYC or ERK5 specific primers. MYC or ERK5 mRNA expression levels were normalized to GAPDH mRNA levels. Data are presented as the mean of three replicates with error bars representing SEM.
(H) PDAC cell lines were treated with the indicated doses of ERK5-selective inhibitor XMD8–92 (ERK5i) or MEK5-selective inhibitor BIX02189 (MEK5i) for 8 hr. Cell lysates were immunoblotted as indicated.
See also Figure S6.