Figure 1.
MYC SUMOylation is observed following proteasomal inhibition.
(A) MYC was immunoprecipitated under denaturing conditions from samples either treated with vehicle control (DMSO) or MG132 for 4 hours. Immunoblotting for SUMO2/3 revealed the accumulation of high molecular weight SUMO signal following proteasomal inhibition. (B) 293Tv cells were transfected with MYC and either empty vector (EV) control or SUMO paralogs 1–3 (S1, S2, S3). After 24 hours, cells were harvested and subjected to an immunoprecipitation for MYC followed by immunoblotting for the presence of SUMO paralogs.
Figure 2.
Identification of a SUMO site on MYC.
(A) 293Tv cells were transfected with MYC and Flag-SUMO1. Cells were then lysed under denaturing conditions and sequential pulldowns were performed first for MYC and then for FlagSUMO1. The resulting eluate was subjected to mass spectrometry analysis. (B) Observed sequence of the detected peptide fragments for both the modification (SUMO1) and the target (MYC). (C) Mass spectrometry fragmentation spectra for MYC (target) and SUMO (modification) demonstrating MYC SUMOylation at K326. (D) Diagrammatic representation of MYC and the post-translational modifications observed by mass spectrometry. P: phosphorylation, Ub: ubiquitylation, B: basic region, HLH-LZ: helix-loop-helix leucine zipper, NLS: nuclear localization signal. Grey boxes indicate regions of homology among MYC family proteins, termed MYC Boxes (MBs).
Figure 3.
K326R mutant has no effect on cell proliferation and death in MCF10A cells.
(A) Western blot depicting the relative expression levels of MYC in empty vector control (EV), wild-type (WT) and K326R cells following 24 hours of 4-hydroxytamoxifen (4OHT) treatment. (B) MCF10A cells were treated with 4OHT to induce transgene expression. 24 hours post-treatment, cells were treated with cycloheximide and protein harvests were collected and analyzed for relative MYC expression and half-life (n = 3). (C) MCF10A cells were subjected to starvation media for 24 hours, and then treated with 4OHT for 24 hours to induce MYC expression. Cells were stained for ethynyl-2′-deoxyuridine (EdU) incorporation, revealing that cells expressing WT- and K326R-MYC could stimulate proliferation to a similar extent and were significantly different than EV expressing cells (n = 3, ***p<0.001, one-way ANOVA with Bonferroni's post-test). (D) MCF10A cells were starved for 24 hours and then treated with 4OHT and tunicamycin for an additional 24 hours. Cells were fixed and stained with propidium iodide and percent pre-G1 population was determined, revealing that both WT- and K326R-MYC could potentiate apoptosis to the same extent and significantly different compared to EV control (n = 3; *p<0.05, **p<0.01, one-way ANOVA with Bonferroni's post-test).
Figure 4.
K326R mutant has no observed phenotypic effect in SH-EP cells.
(A) Western blot depicting relative expression levels of EV control, WT-MYC and K326R-MYC. Actin was used as a loading control. (B) Soft agar colony formation assay revealed no significant difference between the ability of WT and K326R MYC to potentiate colony formation (n = 4; *p<0.05;**p<0.01; one-way ANOVA with Bonferroni post-test). (C) Left panel: Proliferation assay was performed demonstrating that EV is compromised in activity compared to WT- and K326R-MYC (n = 3). Right panel: Doubling time for EV, WT and K326R expressing cells. Both WT- and K326R-MYC had significantly different doubling times than EV control (n = 3, **p<0.01, one-way ANOVA with Bonferroni post-test). (D) Cellular fractionations revealed that WT- and K326R-MYC both localize to the nuclear compartment. Lamin B1 was used as a marker of the nuclear compartment (N), and tubulin was used as a marker of the cytosolic compartment (C).
Figure 5.
K326R mutant drives transcriptional activation and repression to the same extent as WT-MYC.
(A) Western blotting demonstrated the expression of WT- and K326R-MYC following 24 hours of treatment with 100 nM 4OHT. Actin was used as a loading control. (B) Dual luciferase assay for nucleolin (NCL), an activated MYC target, revealed that both WT and K326R MYC can regulate its expression to the same extent (n = 3, p<0.001, one-way ANOVA with Bonferroni post-test). (C) Dual luciferase assays for growth arrest and DNA damage 45 (GADD45) and cyclin dependent kinase inhibitor 1A (CDKN1A), two MYC repressed targets, revealed that both WT- and K326R-MYC repressed these targets to the same extent (n = 3, *p<0.05, one-way ANOVA with Bonferroni post-test).
Figure 6.
WT- and K326R-MYC exhibit similar subcellular localization.
SH-EP cells were stained for MYC localization with either DMSO (vehicle) treatment or MG132 treatment for 4 hours. MYC signal is shown in red and nuclei in blue (DAPI). Empty vector expressing cells had minimal signal for MYC due to low endogenous expression of MYC in this cell line. Ectopic expression of WT- and K326R-MYC revealed nuclear signal. This signal was retained in the nucleus following proteasomal inhibition but gained a punctate staining pattern.
Figure 7.
MCF10A cells were treated with 4OHT for 24 hours to stimulate transgene expression. Cells were then either treated with vehicle (DMSO) or MG132 for 4 hours prior to harvest and immunoprecipitation for MYC. Immunoblotting was performed for SUMO1 and SUMO2/3 revealing that SUMOylation of MYC was enhanced following proteasomal inhibition. This effect was independent of the amount of MYC present in the pulldown (lower panel).