Fig 1: PKC-θ-mediated sumoylation of RanGAP1 requires its phosphorylation on Ser504 and Ser506.(A) Immunoblot analysis of serine-phosphorylated RanGAP1 in Jurkat E6.1 T cells transfected with siNC or siPKC-θ, and left unstimulated or stimulated with anti-CD3 plus anti-CD28. (B) In vitro PKC-θ kinase assay using PKC-θ immunoprecipitated from Jurkat E6.1 cells and recombinant GST-RanGAP1 as substrate. (C, D) Immunoblot analysis of the phosphorylation of transfected wild-type or Ser-mutated HA-tagged RanGAP1 using a mixture of p-Ser- and p-Thr-specific antibodies in Jurkat-TAg cells, which were left unstimulated or stimulated for 15 min with anti-CD3 (C). Immunoblotting of the indicated proteins in WCL is shown at bottom. The ratio of phospho-RanGAP1 to immunoprecipitated RanGAP1 is shown in (D). Analysis is based on three biological replicates. n.s., not significant, *p<0.05, **p<0.01 (one-way ANOVA with post hoc test). (E) In vitro PKC-θ kinase assay as in (B), using purified truncated GST-RanGAP1 or GST-RanGAP1 AA (S504A/S506A) as substrate. (F) Immunoblot analysis of the sumoylation of wild-type or the indicated HA-tagged RanGAP1 mutants in Jurkat-TAg cells using an anti-HA antibody (top panel). The ratios of RanGAP1-SUMO1 to RanGAP1 are shown at the bottom panel. Analysis is based on three biological replicates. n.s., not significant, *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA with post hoc test). Data are representative of three biological replicates.Figure 4—source data 1.Uncropped western blot for Figure 4.Figure 4—source data 2.Row data for Figure 4 and for Figure 4—figure supplement 1.
Fig 2: Schematic model of nuclear import regulation by the TCR-PKC-θ-RanGAP1 axis.Upon TCR stimulation, PKC-θ phosphorylates RanGAP1 to increase its association with Ubc9, thereby enhancing the sumoylation of RanGAP1, which is required for assembly of the RanBP2/RanGAP1-SUMO1/Ubc9 subcomplex. This complex then promotes the nuclear import of NF-ATc1, NF-κB, and AP-1.
Fig 3: High content imaging can detect mislocalization of lamin-B1, RAN and RANGAP1 in human HD PSC-derived striatal neurons. (A) Neuronal cultures were stained for lamin-B1, RAN and RANGAP1 and were counterstained with Hoechst and MAP2. In HD neurons, lamin-B1, RAN and RANGAP1 are detected much more widely in soma and processes indicated by the arrows and outline of the nuclear area, compared to control cultures where expression is highly localized to the nucleus. Scale bar = 20 μM. (B) HD Family line 58Q, 69Q, and 75Q neurons exhibited significantly lower lamin-B1 mean intensity nuclear cytoplasmic (N/C) ratios than control 22Q neurons (C) The N/C ratio was significantly lower in IsoHD 81Q, compared to their respective control (IsoHD 30Q). (D) The RAN N/C ratio of the HD Family line 58Q, 69Q, and 75Q neurons was significantly lower than in control 22Q neurons, (E) as was the N/C ratio in IsoHD 81Q neurons compared to in IsoHD 30Q neurons, confirming that the RAN gradient is altered and RAN is mislocalized. (F) Neurons from the HD Family line 58Q, 69Q, and 75Q cultures all exhibited lower RANGAP1 N/C ratios than their respective control. (G) No significant differences were detected in the N/C ratio between control and IsoHD45Q or IsoHD 81Q neurons. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Data are represented as mean ± SEM of at least one differentiation of each clone of the HD Family iPSC lines, or three differentiations of each of the IsoHD ESC lines, subject to one-way ANOVA followed by Bonferroni post hoc test.
Fig 4: PKC-θ-mediated phosphorylation of RanGAP1 is required for its association with Ubc9 and RanBP2.(A, B) Reciprocal IP analysis of the association between HA-tagged wild-type or K524-mutated RanGAP1 and endogenous Ubc9. RanGAP1 expression was analyzed by anti-HA antibody immunoblotting in WCL (bottom panels). RanGAP1AA (S504A/S506A); RanGAP1EE (S504E/S506E). (C–E) Immunoblot analysis of HA-RanGAP1 IPs (C) and Ubc9 IPs or RanBP2 IPs (D) from Jurkat-TAg cells transfected with HA-RanGAP1 and HA-RanGAP1 mutants. The ratio of RanGAP1-SUMO1 to RanGAP1 in the WCL of (C) and (D) is quantified in (E); quantification is based on three biological replicates. *p<0.05 (one-way ANOVA with post hoc test). Data are representative of three biological replicates.Figure 5—source data 1.Uncropped western blot for Figure 5.Figure 5—source data 2.Row data for Figure 5.
Fig 5: RanGAP1 is a target of mitochondrial OXPHOS in erythroid differentiation(A) The suppressed expression of RanGAP1 was evaluated in the DBA patient samples compared to expression in control samples. The x axis was the count of reads for the gene, and the significance was added by stat_compare_means in the R package ggpubr.(B) Western blotting assay revealing the knockdown of RanGAP1 protein expression by two independent shRNA in differentiated HSPCs on Day 7. The expression of representative erythroid-specific proteins, including HBG and GATA1, was detected. Lysates for differentiated cells were loaded into SDS-PAGE gel. The expression of the SUMO1/RanGAP1 complex was simultaneously detected using the RanGAP1 antibody. Actin was used as a loading control.(C) Representative result indicating RanGAP1 knockdown delays erythroid differentiation as revealed by the color of the cell pellets on Day 7. Differentiated cells containing the control vector appeared as a red pellet, whereas pelleted cells with RanGAP1 knockdown did not.(D) Representative flow cytometry analysis indicating decreased number of CD71+GlyA+ double-positive erythroid cells differentiated from human cord blood HSPCs with RanGAP1 knockdown on Day 7. Three independent experiments were performed in this study.(E) A decrease was observed in the expression of globin genes in differentiated cells with RanGAP1 knockdown on Day 14 by real-time PCR analysis. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.(F) Real-time PCR analysis revealing a decrease in the expression of ribosomal representative DBA causative genes in differentiated cells with RanGAP1 knockdown on Day 14. HSPCs were derived from human cord blood, and GAPDH was used as a reference gene in real-time PCR assays. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗∗p < 0.001.(G) Western blotting detection of RanGAP1 overexpression in differentiated erythroid cells on day 7.(H) Overexpression of RanGAP1 rescues erythroid defects caused by OXPHOS suppression resulting from knockdown of NDUFA2. The percentage of CD71+GlyA + erythroid cells on day 7 in GFP-positive cells was compared using flow cytometry.(I) Overexpression of RanGAP1 rescues erythroid defects caused by OXPHOS suppression resulting from other mitochondrial OXPHOS complex inhibitors including Rot (0.1 μM), CAI (12 μM), Atovaquone (Ato, 40 μM) and mIBG (50 μM). The detection of CD71+GlyA+ double-positive erythroid cells (day 7) in GFP-positive cells with RanGAP1 overexpression using flow cytometry was conducted. See also Figures S3 and S4.
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