Fig 1: RAPSYN is an E3 ligase to neddylate BCR-ABL.(A) Immunoblots of AChR subunits α7, M2, M3, and M4 in Ph+ leukemia cells (K562, KU812, and MEG-01) compared to normal bone marrow stromal cells (HS-5). (B) Immunoblotting analyses of AChR subunit α7, M2, M3, and M4 neddylation levels after immunoprecipitation of NEDD8 in WT and RAPSN KO K562 cells. (C) Heatmap showing the fold change in mRNA level of neddylation-related proteins in chronic myeloid leukemia (CML) patients compared to that in healthy donors. (D) Immunoblots of BCR-ABL and RAPSYN in KU812 cells after co-immunoprecipitation with RAPSYN and BCR-ABL antibodies, respectively. (E) Immunoblotting analyses of BCR-ABL neddylation after immunoprecipitation of BCR-ABL in KU812 and Jurkat cells. (F) Immunoblotting analyses of BCR-ABL neddylation after immunoprecipitation of BCR-ABL in KU812 and Jurkat cells treated with MLN4924 or DMSO for 24 hr. (G) Immunoblotting analyses of BCR-ABL neddylation after immunoprecipitation of BCR-ABL in K562 cells transduced with shAChRα7, shAChRM2, shAChRM3, shAChRM4, or shNC. (H) Immunoblotting analyses of PKC–RAS–ERK and JAK2–AKT changes in K562 cells treated with AChR agonist carbamylcholine chloride (carbachol, 100 μM) and antagonist benzethonium (5 μM) for nAChR or tomatropine bromide (homatropine, 5 μM) for mAChR for 24 hr. (I), Immunoblotting analyses of BCR-ABL neddylation after immunoprecipitation of BCR-ABL in K562 cells treated with AChR agonist carbamylcholine chloride (carbachol, 100 μM) and antagonist benzethonium (5 μM) for nAChR or tomatropine bromide (homatropine, 5 μM) for mAChR for 24 hr. See numerical source data in Figure 2—source data 2. Figure 2—figure supplement 1—source data 1.Original file for the Western blot analysis in Figure 2—figure supplement 1A. Figure 2—figure supplement 1—source data 2.PDF containing Figure 2—figure supplement 1A and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 3.Original file for the Western blot analysis in Figure 2—figure supplement 1B. Figure 2—figure supplement 1—source data 4.PDF containing Figure 2—figure supplement 1B and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 5.Original file for the Western blot analysis in Figure 2—figure supplement 1D. Figure 2—figure supplement 1—source data 6.PDF containing Figure 2—figure supplement 1D and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 7.Original file for the Western blot analysis in Figure 2—figure supplement 1E. Figure 2—figure supplement 1—source data 8.PDF containing Figure 2—figure supplement 1E and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 9.Original file for the Western blot analysis in Figure 2—figure supplement 1F. Figure 2—figure supplement 1—source data 10.PDF containing Figure 2—figure supplement 1F and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 11.Original file for the Western blot analysis in Figure 2—figure supplement 1G. Figure 2—figure supplement 1—source data 12.PDF containing Figure 2—figure supplement 1G and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 13.Original file for the Western blot analysis in Figure 2—figure supplement 1H. Figure 2—figure supplement 1—source data 14.PDF containing Figure 2—figure supplement 1H and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 15.Original file for the Western blot analysis in Figure 2—figure supplement 1I. Figure 2—figure supplement 1—source data 16.PDF containing Figure 2—figure supplement 1I and original scan of the relevant Western blot analysis with highlighted bands and sample labels.
Fig 2: SRC-mediated phosphorylation of RAPSYN at Y336 promotes Ph+ leukemia progression.(A) Cytotoxicity induced by shSRC #2-mediated SRC knockdown in leukemic cells. (B) Rescue of leukemic cells from shSRC #2-induced toxicity by exogenous expression of SRC cDNA. (C) Rescue of leukemic cells from shSRC #2-induced toxicity by exogenous expression of RAPSNWT cDNA. (D) Failed rescue of leukemic cells from shSRC #2-induced toxicity by exogenous expression of RAPSNY336F cDNA. (E) Viability of leukemic cells transduced with either RAPSNWT cDNA or corresponding empty vector after 72 hr of incubation with indicated concentrations of saracatinib. (F) Viability of leukemic cells transduced with either RAPSNY336F cDNA or corresponding empty vector after 72 hr of incubation with indicated concentrations of saracatinib. (G) Viability of leukemic cells transduced with either shNC or shRAPSN #3 after 72 hr of incubation with indicated concentrations of saracatinib. (H) Experimental design used to test in vivo effects of RAPSYN phosphorylation at Y336 on Ph+ leukemia progression and survival time. (I) Kaplan–Meier survival curve of NCG mice following intravenous injection of K562-RAPSNWT or K562-RAPSNY336F cells and intragastric administration of saracatinib or corresponding vehicle from days 6 to 26 as indicated (ten mice in each group). (J) Kaplan–Meier survival curve of NCG mice following intravenous injection of double-transfected K562 cells (ten mice in each group). Representative results from at least three independent experiments are shown (A–G); error bars, mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; log-rank test (I–J).
Fig 3: SRC-mediated phosphorylation at Y336 promotes RAPSYN stability by repressing its proteasomal degradation.(A) Assessment of RAPSYN phosphorylation levels in leukemic cells treated with saracatinib or DMSO for 24 hr. (B) Assessment of RAPSYN phosphorylation levels in leukemic cells transduced with shSRC or shNC. (C) Assessment of RAPSYN phosphorylation levels in leukemic cells expressing exogenous SRC cDNA or empty vector. (D) Assessment of RAPSYN phosphorylation by SRC in vitro. Purified RAPSYN and SRC were incubated with ATP in the presence or absence of saracatinib for phosphorylation assay. (E) Verification of RAPSYN phosphorylation sites. Purified SRC and RAPSYN WT or indicated mutants were incubated with ATP for phosphorylation assay. (F) Assessment of RAPSYN protein stability in leukemic cells treated with CHX in combination with saracatinib or DMSO at indicated time points by immunoblotting. (G) Assessment of RAPSYN protein stability in leukemic cells transduced with shSRC or shNC by immunoblotting. (H) Assessment of RAPSYN protein stability in leukemic cells transduced with exogenous SRC cDNA or empty vector by immunoblotting. (I) Assessment of RAPSYN protein stability in leukemic cells transduced with exogenous RAPSN WT or Y336F cDNA by immunoblotting. (J) Immunoblots of RAPSYN in leukemic cells treated with saracatinib or DMSO for 12 hr, and subsequently with MG132 or DMSO for another 12 hr. (K) Immunoblots of RAPSYN in leukemic cells transduced with shNC or shSRC and treated with MG132 or DMSO for 12 hr. Figure 4—source data 1.Original file for the Western blot analysis in Figure 4A. Figure 4—source data 2.PDF containing Figure 4A and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 3.Original file for the Western blot analysis in Figure 4B. Figure 4—source data 4.PDF containing Figure 4B and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 5.Original file for the Western blot analysis in Figure 4C. Figure 4—source data 6.PDF containing Figure 4C and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 7.Original file for the Western blot analysis in Figure 4D. Figure 4—source data 8.PDF containing Figure 4D and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 9.Original file for the Western blot analysis in Figure 4E. Figure 4—source data 10.PDF containing Figure 4E and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 11.Original file for the Western blot analysis in Figure 4F. Figure 4—source data 12.PDF containing Figure 4F and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 13.Original file for the Western blot analysis in Figure 4G. Figure 4—source data 14.PDF containing Figure 4G and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 15.Original file for the Western blot analysis in Figure 4H. Figure 4—source data 16.PDF containing Figure 4H and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 17.Original file for the Western blot analysis in Figure 4I. Figure 4—source data 18.PDF containing Figure 4I and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 19.Original file for the Western blot analysis in Figure 4J. Figure 4—source data 20.PDF containing Figure 4J and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 4—source data 21.Original file for the Western blot analysis in Figure 4K. Figure 4—source data 22.PDF containing Figure 4K and original scan of the relevant Western blot analysis with highlighted bands and sample labels.
Fig 4: RAPSYN promotes BCR-ABL stabilization.Immunoblotting analyses of BCR-ABL in KU812 and Jurkat cells treated with MLN4924 or DMSO for 24 hr (n=3). Error bars, mean ± standard deviation (SD); *p < 0.05; ***p < 0.001; Student’s t-test. See numerical source data Figure 4—figure supplement 1—source data 2. Figure 3—figure supplement 1—source data 1.Original file for the Western blot analysis in Figure 3—figure supplement 1. Figure 3—figure supplement 1—source data 2.PDF containing Figure 3—figure supplement 1 and original scan of the relevant Western blot analysis with highlighted bands and sample labels.
Fig 5: RAPSYN neddylates BCR-ABL.(A) Co-immunoprecipitation of BCR-ABL and RAPSYN in leukemic cells. (B) Immunoblots of GST and His after immunoprecipitation of His or GST in HEK293T cells transfected with His-tagged BCR-ABL and GST-tagged RAPSYN. (C) Immunoblots of GST and His following GST pull-down after in vitro incubation of purified His-tagged BCR-ABL and GST or GST-tagged RAPSYN. (D) His-immunoblots of GST immunoprecipitates from HEK293T cells transfected with GST-tagged RAPSYN alone or in combination with His-tagged full-length or truncated BCR-ABL (Δ1: aa 1–927, Δ2: aa 928–2047). (E) Analysis of BCR-ABL neddylation levels in leukemic cells. (F) Analysis of BCR-ABL neddylation levels in primary chronic myeloid leukemia (CML) peripheral blood mononuclear cells (PBMCs). (G) Analysis of BCR-ABL neddylation levels in leukemic cells treated with MLN4924 or dimethyl sulfoxide (DMSO) for 24 hr. (H) HA-immunoblots of His-immunoprecipitate from HEK293T cells transfected with His-tagged BCR-ABL and HA-tagged NEDD8 or NEDD8 ΔGG. (I) HA-immunoblots of His-immunoprecipitate from HEK293T cells transfected with indicated constructs. (J) HA-immunoblots after immunoprecipitation of His-antibody in HEK293T cells transfected with His-tagged BCR-ABL, HA-tagged NEDD8, GFP-tagged WT RAPSYN, or RAPSYN-C366A. (K) Analysis of BCR-ABL neddylation levels in K562 WT, RAPSN KO, and RAPSN KO with exogenous expression of a RAPSN cDNA cells. (L) Assessment of BCR-ABL neddylation by RAPSYN in vitro. Recombinantly expressed and purified RAPSYN and BCR-ABL were incubated with APPBP1/UBA3, UBE2M, or NEDD8 for in vitro neddylation assay. (M) Analysis of BCR-ABL neddylation levels in excised tumor xenografts from Figure 1H. (N) Verification of BCR-ABL neddylation sites in HEK293T cells transfected with indicated constructs. Figure 2—source data 1.Original file for the Western blot analysis in Figure 2A. Figure 2—source data 2.PDF containing Figure 2A and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 3.Original file for the Western blot analysis in Figure 2B. Figure 2—source data 4.PDF containing Figure 2B and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 5.Original file for the Western blot analysis in Figure 2C. Figure 2—source data 6.PDF containing Figure 2C and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 7.Original file for the Western blot analysis in Figure 2D. Figure 2—source data 8.PDF containing Figure 2D and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 9.Original file for the Western blot analysis in Figure 2E. Figure 2—source data 10.PDF containing Figure 2E and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 11.Original file for the Western blot analysis in Figure 2F. Figure 2—source data 12.PDF containing Figure 2F and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 13.Original file for the Western blot analysis in Figure 2G. Figure 2—source data 14.PDF containing Figure 2G and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 15.Original file for the Western blot analysis in Figure 2H. Figure 2—source data 16.PDF containing Figure 2H and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 17.Original file for the Western blot analysis in Figure 2I. Figure 2—source data 18.PDF containing Figure 2I and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 19.Original file for the Western blot analysis in Figure 2J. Figure 2—source data 20.PDF containing Figure 2J and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 21.Original file for the Western blot analysis in Figure 2K. Figure 2—source data 22.PDF containing Figure 2K and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 23.Original file for the Western blot analysis in Figure 2L. Figure 2—source data 24.PDF containing Figure 2L and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 25.Original file for the Western blot analysis in Figure 2M. Figure 2—source data 26.PDF containing Figure 2M and original scan of the relevant Western blot analysis with highlighted bands and sample labels. Figure 2—source data 27.Original file for the Western blot analysis in Figure 2N. Figure 2—source data 28.PDF containing Figure 2N and original scan of the relevant Western blot analysis with highlighted bands and sample labels.
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