Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins

Abstract

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

FIGURE 1.

NUP98-HOXA9 causes nuclear retention of CRM-1 export substrates. K562 cells were nucleofected with EGFP-NPMc (A) or GFP-Rev (B) in combination with either empty pcDNA3 vector (control) or vector expressing NUP98-HOXA9 or NUP98-HOXA9 without the NUP98 portion (NUP98-HOXA9ΔNUP). The upper panels show fluorescent images, and the lower panels show corresponding phase contrast images. The arrows in the upper middle panel in A point to cells with nuclear-retained NPMc. The images were viewed using a Nikon Eclipse 80i microscope with a Nikon 40×, 0.75 numerical aperture CFI Plan Fluor DLL objective and were acquired with a Nikon Coolsnap ES camera using MetaMorph 6.3r2 software.

FIGURE 2.

NUP98-HOXA9 binds CRM1 through the FG motif in a Ran-GTP-dependent manner. A, schematic of NUP98-HOXA9 and its variants used in the binding assays. GLEBS, Gle2p-binding motif; HD, homeodomain. B, ³⁵S-labeled NUP98-HOXA9 and its variants were incubated with GST (Control) or GST-CRM1 (CRM1) immobilized on glutathione-Sepharose 4B beads in the presence or absence of RanGDP or the RanGTP analog, RanGMPPNP. Approximately 30% of the total bound material and 5% of total unbound material for each reaction were analyzed as shown. Uncropped gels with their corresponding Coomassie-stained images are shown in supplemental Fig. S2. C, recombinant GFP-NUP98-HOXA9 protein was incubated with GST (Control) or GST-CRM1 (CRM1) immobilized on glutathione-Sepharose 4B beads in the presence of RanGTP. Approximately 0.67% of the total bound material and 0.13% of total unbound material for each reaction were analyzed by immunoblotting against GFP. D, ³⁵S-labeled NUP98-HOXA9, NUP98, NPMc, and Rev were incubated with GST-CRM1 immobilized on glutathione-Sepharose 4B beads in the presence of RanGMPPNP with or without the indicated amount of LMB. Approximately 30% of the total bound material and 3% of total unbound material for each reaction were analyzed as shown. Uncropped gels with their corresponding Coomassie-stained images are shown in supplemental Fig. S3. Binding of Rev to CRM1 compared with control beads is shown in supplemental Fig. S4.

FIGURE 3.

NUP98-HOXA9 and NUP98-DDX10 co-immunoprecipitate with endogenous CRM1. A, K562 cells were nucleofected with either empty pcDNA3 vector (Control) or vector expressing FLAG-tagged NUP98-HOXA9 or NUP98-DDX10. Anti-NUP98 immunoblot of nuclear lysates shows the transduced fusion proteins as well as endogenous NUP98. B, nuclear lysates and immunoprecipitates (IP) prepared using anti-FLAG antibody from control cells or cells expressing the indicated FLAG-tagged NUP98 fusions were analyzed by anti-CRM1 immunoblotting.

FIGURE 4.

NUP98 fusion proteins and NUP98 interact differently with NUP214. ³⁵S-Labeled NUP98-HOXA9, NUP98-DDX10, and NUP98 were incubated with GST-CRM1 immobilized on glutathione-Sepharose 4B beads in the presence or absence of RanGDP or RanGMPPNP in combination with or without NUP214C or NUP358ZFD proteins as shown. Approximately 30% of the total bound material and 10% of total unbound material for each reaction were analyzed as shown. Uncropped gels with their corresponding Coomassie-stained images are shown in supplemental Fig. S5.

FIGURE 5.

NUP98 fusion proteins co-localize with CRM1 in nuclear subdomains. A and B, K562 cells were retrovirally transduced with either empty vector (Control) or a vector expressing HA-NUP98-HOXA9 or HA-NUP98-DDX10. The cells were immunostained with anti-HA (A) or anti-CRM1 (B) antibodies in combination with rhodamine-conjugated secondary antibody. The upper panels show fluorescent images, and the lower panels show corresponding phase contrast images. The images were viewed using a Nikon Eclipse 80i microscope with a Nikon 40×, 0.75 numerical aperture CFI Plan Fluor DLL objective and were acquired with a Nikon Coolsnap ES camera using MetaMorph 6.3r2 software. C, K562 cells were retrovirally transduced to express HA-NUP98-HOXA9 and were immunostained with anti-HA antibody (top panels), anti-CRM1 antibody (middle panels), or both antibodies (bottom panels) in combination with both Alexa Fluor 647-conjugated and rhodamine-conjugated secondary antibodies. The images were acquired with a Zeiss LSM510 Meta laser scanning confocal microscope equipped with a Zeiss 63×, 1.4 numerical aperture Plan Apochromat oil objective using Zeiss LSM510 software. D, K562 cells were retrovirally transduced to express HA-NUP98-DDX10 and were immunostained with anti-HA and anti-CRM1 antibodies in combination with Alexa Fluor 647-conjugated and rhodamine-conjugated secondary antibodies. The images were acquired with a Zeiss LSM510 Meta laser scanning confocal microscope equipped with a Zeiss 63×, 1.4 numerical aperture Plan Apochromat oil objective using Zeiss LSM510 software. E, human primary CD34+ cells were retrovirally transduced to express HA-NUP98-HOXA9 or HA-NUP98-DDX10 and were immunostained with anti-HA and anti-CRM1 antibodies in combination with Alexa Fluor 647-conjugated and rhodamine-conjugated secondary antibodies. The images were acquired with a Zeiss LSM510 Meta laser scanning confocal microscope equipped with a Zeiss 63×, 1.4 numerical aperture Plan Apochromat oil objective using Zeiss LSM510 software. F, cell lysates of human primary CD34+ cells retrovirally transduced to express HA-NUP98-HOXA9 or HA-NUP98-DDX10 were analyzed by anti-HA immunoblotting.

FIGURE 6.

NUP98 fusions enhance transcription by NFAT and NFκB by nuclear entrapment. A and B, K562 cells were nucleofected with GFP-NFAT (A) or EGFP-NFκB(p65) (B) in combination with either empty pcDNA3 vector (Control) or vector expressing NUP98-HOXA9 or NUP98-DDX10. The upper panels show fluorescent images, and the lower panels show corresponding phase contrast images. The images were viewed using a Nikon Eclipse 80i microscope with a Nikon 40×, 0.75 numerical aperture CFI Plan Fluor DLL objective and were acquired with a Nikon Coolsnap ES camera using MetaMorph 6.3r2 software. C, ³⁵S-labeled NUP98-HOXA9, NUP98-DDX10, NFAT, and NFκB were incubated with GST (Control) or GST-CRM1 (CRM1) immobilized on glutathione-Sepharose 4B beads in the presence of RanGMPPNP. Approximately 30% of the total bound material and 2.1% of total unbound material for each reaction were analyzed as shown. The positions of molecular mass markers (expressed in kilodaltons) are shown. Uncropped gels with their corresponding Coomassie-stained images are shown in supplemental Fig. S6E. D, K562 cells were transfected by electroporation with either NFAT-pGL3 or NFκB-pTransLucent luciferase reporter vector in combination with either empty pcDNA3 vector (Control) or vector expressing NUP98-HOXA9 or NUP98-DDX10. The pRL-TK vector expressing Renilla luciferase was included in all samples to control for transfection efficiency, and the results were normalized to Renilla luciferase activities. The results shown are averages of three or four experiments ± standard deviations. The p value was obtained by comparing to empty pcDNA3 vector control using a paired two-tailed distribution t test. *, p < 0.05; **, p < 0.01.