Fig 1.
A, Protein sequence alignment of NR4A family members. Conserved SUMOylation consensus motifs among members (green squares) and potential ERK phosphorylation consensus sites in NR4A1 (blue squares) are shown. K558 is conserved in NR4A2 and NR4A1. B, Autophagy is induced during SP/NK1R- induced cell death. Electron microscopy of cells transfected with NK1R and exposed (SP) or not (Control) to SP. Autophagic vesicles accumulate in response to SP (black arrow). A representative image of an autophagosome labeled with immunogold localization of LC3 is shown (white arrows). C, Autophagy inhibition reduced the number of dead cells. Cells were transfected with NK1R and exposed (SP) or not (Control) to SP. Autophagy was inhibited by addition of Spautin 1, otherwise vehicle was added as control. Cell death was estimated by Trypan blue exclusion. Two-way ANOVA with Turkey´s multiple comparison test **** p <0.0001; n = 3. D, NR4A1 is SUMOylated in response to SP. Immunoprecipitation (IP) of NR4A1 from cells transfected with NK1R expression vector and exposed to SP for the indicated times, to detect by Western blot either SUMO1, SUMO2/3 or NR4A1. IgG from IP was detected by the secondary antibody. E, Over-expression of myc-SUMO1 enhances the amount of NR4A1 conjugation to SUMO1 in response to SP. Cells were transfected with NK1R and MYC-tagged SUMO1 expression vectors (Myc-SUMO1) and exposed (+) or not (-) to SP for 3 hr. NR4A1 was immunoprecipitated and developed by Western blot to detect Myc tag or NR4A1. Tubulin was detected in whole extracts as a loading reference (Input). F, Triple mutant NR4A1_K102,558,577R (TriMut) has reduced SUMOylation. Cells were transfected with expression vectors for HA tagged SUMO2, FLAG-NR4A1 wild type or FLAG-NR4A1_TriMut, and NK1R to allow post-translational modifications in response to SP (+). Immunoprecipitation was performed with FLAG antibody (antiFLAG) or IgG as a control of irrelevant antibody. WB was developed with antibodies against indicated proteins. TUBULIN was detected as a loading reference (Input).
Fig 2.
NR4A1 SUMOylation depends on former phosphorylation.
A, SUMOylation on K102, K558 and K577 is not necessary for NR4A1 phosphorylation in response to SP. Cells were transfected with expression vectors for FLAG-NR4A1 wild type or FLAG-NR4A1_TriMut and NK1R, and exposed (+) or not (-) to SP for 3 hr. Total protein extracts were immunoprecipitated with anti-FLAG or IgG and the level of threonine phosphorylation was estimated by WB. TUBULIN was detected for comparison of total protein extraction for IP, and FLAG to show the level of expression of each construct. B, Phosphorylation in T143 in necessary for NR4A1 SUMOylation in response to SP. Cells were transfected with expression vectors for FLAG-NR4A1 wild type or FLAG-NR4A1_T143A, HA-SUMO2 and NK1R, and exposed (+) or not (-) to SP for 3 hr. Total protein extracts were immunoprecipitated with anti-FLAG and the level of SUMOylation was estimated by WB detecting HA. FLAG was detected to show the level of expression of NR4A1 constructs. C, NR4A1 peak of synthesis is at 3 hr after SP addition and has a half-life of less than three hours. Lanes 1–6, total protein extracts were obtained from cells transfected with NK1R and exposed to SP for the indicated times. Lanes 7–12, total protein extracts were obtained from cells transfected with NK1R expression vector, treated for 3 hr with SP to reach maximum expression of NR4A1 (considered time 0 for cycloheximide treatment), and then exposed to cycloheximide (CHX) for the indicated time to inhibit new protein synthesis. Three hr after CHX less than half of the initial amount of NR4A1 protein remains. D, NR4A1 basal stability is enhanced by SUMOylation, but TriMut still becomes stabilized in response to SP signaling. To compare stability of NR4A1 wild type with NR4A1_TriMut, cells were transfected with expression vectors for NK1R and either FLAG-NR4A1 or FLAG-NR4A1_TriMut. 24 hr after of transfection cells were treated with CHX for the indicated times, or treated for 3 hr with SP and then with CHX for the indicated time. Clearly, the amount of FLAG-NR4A1_TriMut was reduced and was degraded faster than wild type, but yet responded to SP induction. E. Quantitative comparison of the degradation rate of NR4A1 WT and TriMut, with or without SP, from densitometric analysis of at least three independent experiments described in D. Each blot was normalized with their corresponding TUBULIN. The measurement obtained for each NR4A1 (WT or TriMut) before CHX treatment (time zero) was arbitrary considered 100 units. Notice that the faster degradation rate of TriMut is overcome in response to SP. Each dot represents the mean and bars represent standard deviation.
Fig 3.
SUMOylation regulates NR4A1 transcriptional activity and its intracellular distribution.
A, NR4A1 mutant in Lys residues located in SUMOylation motifs (TriMut) have increased transcriptional activity compared to wild type in response to SP. Cells were transfected with a reporter containing Luciferase cDNA under the control of NuRE/POMC and the indicated plasmids to assess basal transcriptional activity (left panel). Then cells were co- transfected with NK1R and treated or not with SP for 3 hr. TriMut showed enhanced transcriptional activity in response to SP and at a higher level than NR4A1 WT. A mean of 5 independent experiments (each with duplicate wells) is plotted. Error bars represent the standard error. ** p<0.01; n = 5. A representative Western blot showing the level of expression of each construct, with or without SP, is shown below. Both anti-FLAG and anti-TUBULIN were incubated simultaneously. A densitometry analysis of three Western blots normalized with TUBULIN is plotted, bars represent standard deviation. T student analysis showed no significance. B, SUMOylation and phosphorylation regulate NR4A1 intracellular distribution. Cells were transfected with indicated plasmids and the intracellular localization of the proteins was determined by immunofluorescence to detect NR4A1 (green). Two-hundred cells from two independent experiments for each construct were counted. The percentage of cells with only nuclear, or nuclear-cytoplasmic localization of NR4A1, is plotted. Error bars represent error standard. Representative confocal microscopy images are shown below; nuclei were stained with DAPI (blue).
Fig 4.
NR4A1 SUMOylation is necessary for SP-induced autophagic cell death.
A, The viral protein Gam1 reduced SUMOylation of NR4A1. Cells were transfected with NK1R expression vector and the indicated plasmids, and were treated or not with SP for 3 hr. Then, NR4A1 was immunoprecipitated and developed with anti-SUMO2/3 or antiNR4A1. Notice that only in the presence of GAM1, and not with inactive mutant GAM1, there was a reduction in SUMOylated NR4A1. GAPDH was detected in total extract (input) as a reference of initial similar amount of protein. B, Inhibiting SUMOylation by the expression of the viral protein GAM1 prevents SP-induced autophagic cell death. Cells were transfected with NK1R expression vector and an empty vector or Gam1 expression vector and treated or not with SP for 24 hr. Cell death was estimated by Trypan blue exclusion. A mean of three independent experiments is plotted. Error bars represent the standard deviation. *** p<0.0001 2way ANOVA. The Western blot below shows the expression of Myc-Gam1 or Myc-Gam mutant. C, SUMOylation defective mutant (TriMut) is not able to induce autophagic cell death in response to SP. Cells were co-transfected with NK1R expression vector and either NR4A1 WT, TriMut or empty vector as indicated. Cells were also transfected with a control siRNA targeting a viral sequence not present in mammals or a siRNA targeting the 3’ UTR, only present in endogenous mRNA. Cells were exposed or not to SP for 24 hr and cell death was estimated by Trypan blue exclusion or LDH activity released. Every experiment was performed in triplicate and averaged. The mean of three independent experiments is plotted. Error bars represent the standard deviation. ***, p<0.0001. Total protein extracts were obtained from replica wells taken at 3hr after SP addition to estimate the content of NR4A1 by WB. Tubulin was detected as a loading reference.