Figures and data
High protein levels of RAPSYN promotes Ph+ leukemia progression.
(A) Immunoblots of RAPSYN and BCR-ABL in the peripheral blood mononuclear cells (PBMCs) of clinical samples.
(B) Immunoblots of RAPSYN and BCR-ABL in Ph+ leukemic cells and normal bone marrow stromal cells (HS-5).
(C) Cytotoxicity induced by shRNA-mediated RAPSYN knockdown in leukemic and HS-5 cells.
(D) Cytotoxicity induced by shRNA-mediated RAPSYN knockdown in the PBMCs of CML patients.
(E) Rescue of leukemic cells from shRAPSN #3-induced toxicity by exogenous expression of RAPSN cDNA.
(F) An in vivo experimental design for testing the effects of RAPSYN on tumor growth and survival.
(G) The growth curve of subcutaneous xenograft tumors was measured every two days from the third day after tumor inoculation for 19 days (five mice in each group).
(H) Photograph and weight quantification of excised tumor xenografts from (I).
(I) Immunoblots of RAPSYN and BCR-ABL in mouse xenograft tumor biopsies from K562 cells transduced with shRAPSN #3 or shNC.
(J) Immunoblots of RAPSYN and BCR-ABL in K562-RAPSYNWT and K562-RAPSYNKO cells.
(K) Kaplan-Meier survival curve of NCG mice following intravenous injection of K562-RAPSYNWT or K562-RAPSYNKO cells, as shown in (H) (ten mice in each group).
All data represent mean ± SD of at least three independent experiments. P values were calculated using unpaired Student’s t-test or log-rank test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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 the PBMCs of CML patients.
(G) Analysis of BCR-ABL neddylation levels in leukemic cells treated with MLN4924 or DMSO for 24 h.
(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) Analysis of BCR-ABL neddylation levels in K562 WT, RAPSYN-KO and RAPSYN-KO with exogenous expression of a RAPSN cDNA cells.
(K) 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.
(L) Assessment of BCR-ABL neddylation by RAPSYN in vitro enzymatic reactions. 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 Fig. 1H.
(N) Verification of BCR-ABL neddylation sites in HEK293T cells transfected with indicated constructs.
RAPSYN attenuates BCR-ABL ubiquitination and degradation.
(A) Immunoblots of BCR-ABL in leukemic cells treated with MLN4924 or DMSO for 24 h and corresponding quantification of three independent replicates.
(B) Immunoblots of BCR-ABL in K562 WT and RAPSYN-KO cells and corresponding quantification of three independent replicates.
(C) Assessment of BCR-ABL protein stability in K562 cells expressing DOX-inducible shRAPSN #3 treated with CHX alone or in combination with DOX at indicated time points by immunoblotting.
(D) Analysis of BCR-ABL neddylation and ubiquitination levels in leukemic cells treated with MLN4924 or DMSO for 24 h.
(E) Analysis of BCR-ABL neddylation and ubiquitination levels in K562 WT and RAPSYN KO cells.
(F) Immunoblots of HA and Myc after His-immunoprecipitation in HEK293T cells transfected with His-tagged BCR-ABL, HA-tagged Ub or without Myc-tagged NEDD8.
(G) Analysis of BCR-ABL ubiquitination and neddylation in leukemic cells treated with MG132 or DMSO for 12 h.
(H) Co-immunoprecipitation of BCR-ABL, c-CBL and RAPSYN in leukemic cells expressing exogenous RAPSN cDNA or empty vector.
(I) Co-immunoprecipitation of BCR-ABL, c-CBL and RAPSYN in K562 WT and RAPSYN KO cells.
All data represent mean ± SD of at least three independent experiments. P values were calculated using unpaired Student’s t-test. ** p < 0.01, *** p < 0.001.
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 h.
(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 RAPSYN WT or Y336F cDNA by immunoblotting.
(J) Immunoblots of RAPSYN in leukemic cells treated with saracatinib or DMSO for 12 h, and subsequently with MG132 or DMSO for another 12 h.
(K) Immunoblots of RAPSYN in leukemic cells transduced with shNC or shSRC and treated with MG132 or DMSO for 12 h.
RAPSYN phosphorylation at Y336 potentiates its E3 ligase activity and promotes BCR-ABL stabilization.
(A) Immunoblots of BCR-ABL neddylation levels in leukemic cells treated with saracatinib or DMSO for 24 h.
(B) Immunoblots of BCR-ABL neddylation levels in leukemic cells transduced with shSRC or shNC.
(C) Immunoblots of BCR-ABL neddylation levels in leukemic cells expressing exogenous SRC cDNA or empty vector.
(D) Effects of RAPSYN phosphorylation on BCR-ABL neddylation levels in HEK293T cells transfected with indicated constructs.
(E) Effects of RAPSYN phosphorylation at Y336 on BCR-ABL neddylation levels in leukemic cells expressing exogenous RAPSYN WT, Y336F cDNA, or empty vector.
(F) Assessment of BCR-ABL protein stability in leukemic cells transduced with exogenous cDNA for RAPSYNWT, RAPSYNY336F or empty vector by immunoblotting.
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 h of incubation with indicated concentrations of saracatinib.
(F) Viability of leukemic cells transduced with either RAPSNY336F cDNA or corresponding empty vector after 72 h of incubation with indicated concentrations of saracatinib.
(G) Viability of leukemic cells transduced with either shNC or shRAPSN #3 after 72 h 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-RAPSYNWT or K562-RAPSYNY336F 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 ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; log-rank test (I-L).
