Figure 1.
Upregulation of Pim-1 expression by EBNA3C.
A) 10×106 human PBMC (Peripheral blood mononuclear cells) were infected with wild type or ΔEBNA3C mutant BAC-GFP EBV for 6 hrs at 37°C. Cells were harvested after 0, 2, 4, 7, 15 days of post-infection. Total RNA was isolated and subjected to quantitative real-time PCR analysis to detect Pim-1 mRNA level. B) Wild type or ΔEBNA3C mutant BAC-GFP EBV infected cells were lysed in RIPA buffer. Western blot analysis was performed with indicated antibodies to detect specific endogenous proteins. C) 50 million EBV negative BJAB, EBNA3C expressing BJAB7, BJAB10, EBV transformed LCL1, LCL2 cells were harvested and total cell lysates were subjected to Western blot analysis (WB) using indicated antibodies. D) 10 million HEK-293 cells and E) 50 million EBV negative DG75 cells were transfected with increasing amount of EBNA3C expressing construct (0, 5, 10, 15 µg) and Western blot analysis was performed to detect Pim-1, EBNA3C, GAPDH proteins. F) Total RNA was isolated from BJAB, BJAB7, LCL1, sh-Ctrl LCL1, sh-E3C LCL1 cells and subjected to quantitative real-time PCR analysis to detect Pim-1 mRNA levels. G) Lentivirus mediated stable EBNA3C knockdown (sh-E3C) or scramble control (sh-Ctrl) LCL1 cells were subjected to Western blot analysis with indicated antibodies. Protein bands from Western blot analysis were analyzed by the Odyssey imager software and represented as bar diagrams based on internal loading control GAPDH.
Figure 2.
EBNA3C physically associates with Pim-1.
A–B) 10 million HEK- 293 cells were co-transfected with Myc-tagged Pim-1, untagged-EBNA3C or Flag-tagged EBNA3C expression vectors. Control samples were balanced by using empty vector. Transfected cells were harvested at 36 hrs of post-transfection and approximately 5% of the lysates were used as input and the residual lysate was immunoprecipitated (IP) with 1 µg of anti-Flag (M2) or A10 antibodies. Lysates and Immunoprecipitated samples were resolved by 10% SDS-PAGE and western blot (WB) analysis was performed with the indicated antibodies. C) 50 million BJAB, BJAB7, LCL1, were harvested and lysed in RIPA buffer. Cell lysates were incubated with either GST control or GST-Pim-1 beads. EBNA3C protein was detected by western blot analysis using EBNA3C specific monoclonal antibody (A10). D) Purified control GST and GST-Pim-1 proteins used in this experiment were resolved by 10% SDS-PAGE and stained with Coomassie Blue. 50 million E–F) BJAB, BJAB7, BJAB10, LCL1, and LCL2 cells were lysed and immunoprecipitation was performed by Pim-1 specific antibody. Immunoprecipitated samples were resolved by 10% SDS-PAGE and endogenous EBNA3C, Pim-1 proteins were detected by their specific antibodies.
Figure 3.
Binding of Pim-1 to the N-terminal EBNA3C domain.
A–B) 10 million HEK-293 cells were transfected with either control vector or Full length and different truncated mutants of Myc-tagged EBNA3C with GFP-tagged Pim-1 plasmid constructs. After 36 hours of post-transfection, cells were harvested and immunoprecipitation performed with 1 µg of GFP or anti-Myc antibodies. IP samples were resolved in 10% SDS-PAGE. Western blot was performed with anti-Myc and anti-GFP antibodies. C) Full length and different domains truncated mutant constructs of EBNA3C were in vitro translated using a T7-TNT translation kit. After pre-clearing with GST-beads, all S35-radiolabeled in vitro translated proteins incubated with either GST control or GST-Pim-1 beads. Reaction samples were washed with Binding Buffer and resolved by 10% SDS-PAGE, exposed to phosphoimager plate and scanned by Typhoon Scanner. D) Myc-Pim-1 construct were used for in vitro translation and S35-radiolabeled in vitro translated proteins were incubated with either GST control or different GST-EBNA3C truncated mutant beads. Coomassie staining of SDS-PAGE resolved purified GST proteins is shown in the bottom panel of D). In each case, 5% of IVT input was used for the comparison. E) The diagram shows EBNA3C 130–159 amino acids motif important binding sites for different cellular proteins. F) The schematic diagram represents various structural and interactive domains of EBNA3C and summarizes the binding affinities between different domains of EBNA3C with Pim-1. +, binding; −, no binding.
Figure 4.
EBNA3C co-localizes with Pim-1 and facilitates its nuclear translocation.
A) 0.3 million HEK-293 cells plated on coverslips and transiently transfected with control vector, GFP-EBNA3C and Myc-Pim-1 expression vectors by using Lipofectamine 2000 transfection reagent. B) 10 million HEK-293 cells were transfected with Myc-Pim-1 and Flag-EBNA3C and subjected to sub-cellular fractionation assay. C) Bar diagram represents quantitation of co-localization in panel A. D) BJAB, BJAB10, LCL1 cells were plated on slides and air-dried. Ectopic and endogenous expressions of Pim-1 was detected using anti-Myc (9E10)-antibody (1∶200 dilution) and Pim-1 specific antibody (1∶50 dilution) respectively, followed by anti-Rabbit Alexa Fluor 594 and anti-goat Alexa Fluor 555 (red) as secondary antibodies. Endogenous EBNA3C was detected using A10 ascites (1∶150 dilution) followed by anti-mouse Alexa Fluor 488 (green). DAPI (49, 69-diamidino-2-phenylindole) was used (1∶500 dilution) to stain nuclei. The images were captured by Olympus Fluoview confocal microscope.
Figure 5.
EBNA3C contributes to Pim-1 stabilization.
A) 10 million HEK-293 cells were co-transfected with Myc-Pim-1 and either vector control (lanes 1 and 3) or Flag-EBNA3C (lanes 2 and 4) expression constructs. After 36 hrs of post-transfection, transfected cells were treated with either 20 µM MG132 (+ lanes) or DMSO (− lanes) for additional 6 hrs and cell lysates were resolved by 10% SDS-PAGE and Western blot was performed with the indicated antibodies. B) HEK-293 cells were transfected with above mentioned expression vectors and At 36 hrs of post-transfection, cells were treated with 40 µg/ml cyclohexamide (CHX) for 0, 3, 6 hrs. Cells were lysed and protein samples were resolved by 10% SDS-PAGE. Western blot was performed by specific antibodies shown. C–D) BJAB, BJAB10, LCL1, sh-Ctrl and sh-EBNA3C cells were treated with 40 µg/ml cyclohexamide (CHX) for indicated time periods. Cell lysates were resolved by 10% SDS-PAGE. Western blot analysis was performed with indicated antibodies. GAPDH blot was shown for internal loading control.
Figure 6.
EBNA3C suppresses poly-ubiquitination of Pim-1.
A–B) 10 million HEK-293 cells were transiently transfected with different combinations of expression vectors as indicated. Cells were harvested after 36 hrs of post-transfection by incubating with proteasome inhibitor MG132 drug for additional 6 hrs. Total protein was immunoprecipitated (IP) with indicated antibodies and protein samples were resolved by 10% SDS-PAGE. Western blots were performed by stripping and re-probing the same membrane. C–D) 50 million EBV negative BJAB cells, BJAB10, LCL1, sh-Ctrl, sh-Pim-1 LCL1 cells were incubated with proteasome inhibitor MG132 drug (20 µM) for 6 hrs. Cells were harvested and lysed with RIPA buffer. IRF4 was immunoprecipitated (IP) by using specific antibodies. Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was performed by stripping and re-probing the same membrane.
Figure 7.
EBNA3C enhances Pim-1 kinase mediated phosphorylation of p21 Thr145 residue.
A–C) HEK-293 cells were transfected with Myc-tagged Pim-1 (wild type or kinase dead mutant) and Flag-tagged EBNA3C vectors as indicated. Empty vector was used to balance total transfected DNA. At 36 h post-transfection, Pim-1 immunoprecipitates were captured with anti-Myc antibody and assayed for in vitro kinase activity toward GST-p21 (wild type or T145A mutant) using γP 32-ATP. Western blot using whole cell lysates and Coomassie staining of SDS-PAGE resolved GST proteins used in this study is shown here.
Figure 8.
EBNA3C competes with p21 for Pim-1 binding.
A–B) 10 million HEK-293 cells were transfected with different combinations of Myc-tagged Pim-1, Flag-tagged p21, untagged-EBNA3C expression vectors as indicated by electroporation. A) IP was performed with anti-Myc antibody and IP complexes were resolved by 10% SDS-PAGE. Western blot was performed with indicated antibodies. B) Above mentioned transfected cells were treated with MG132 drug for 6 hrs and protein lysates were prepared by RIPA buffer. Western blot analysis was performed by indicated antibodies.
Figure 9.
EBNA3C destabilizes p21 through Pim-1 independent of the DNA damage response.
A) 10 million HEK-293 cells were transfected with combinations of Myc-tagged Pim-1, Flag-tagged p21, untagged-EBNA3C expression by electroporation. After 36 hrs of post-transfection, cells were treated with cyclohexamide for indicated time points in DMEM medium containing either serum/DMSO or 0.1% FBS with 5 µM etoposide. Protein samples were resolved by 10% SDS-PAGE and Western blots were performed with indicated antibodies. B–C) 50 million EBV negative BJAB cells, EBNA3C expressing BJAB10, EBV transformed LCL1, sh-Ctrl, sh-E3C LCL1 cells were incubated with cyclohexamide for specific time points in RPMI medium containing serum/DMSO or 0.1% FBS with 5 µM etoposide. Samples were resolved by 10% SDS-PAGE and Western blot analysis was performed with specific antibodies.
Figure 10.
EBNA3C augments Pim-1 dependent degradation of p21.
A–C) 10 million HEK-293 cells were transiently transfected with different combinations of expression vectors as indicated. Cells were harvested after 36 hrs of post-transfection by incubating with MG132 drug for additional 6 hrs and total protein was immunoprecipitated (IP) with indicated antibodies and protein samples were resolved by 10% SDS-PAGE. Western blots were performed by stripping and re-probing the same membrane. D–F) 50 million EBV negative BJAB, BJAB cells stably expressing BJAB10 and EBV transformed LCL1, sh-Ctrl, sh-E3C, sh-Pim-1 LCL1 cells were incubated with proteasome inhibitor MG132 drug (20 µM) for 6 hrs. Treated cells were harvested and lysed with RIPA buffer. p21 was immunoprecipitated (IP) by using specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was performed by stripping and re-probing the same membrane.
Figure 11.
Pim-1 silencing by sh-RNA sensitizes EBV transformed lymphoblastoid cells by induction of the intrinsic apoptosis signaling pathway.
A) Wild type LCL1, Lentivirus mediated and puromycin selected stable sh-Ctrl vector transfected and different clones of Pim-1 knocked down cells were observed by fluorescence microscope for monitoring GFP fluorescence B) 50 million different clones of stable sh-Pim-1, sh-Ctrl LCL1, and LCL1 cells were harvested and cell lysates were prepared by RIPA buffer. Western blot analysis was performed to show the expression levels of EBNA3C, Pim-1 and GAPDH. C) sh-Ctrl, sh-Pim-1 LCL1 cells were grown in RPMI medium for 12 hrs with or without serum starvation. Cells were stained with Propidium iodide and subjected to Flowcytometric analysis. D) Bar diagram represents the fold change of apoptosis observed by apoptosis assay using FACS. The results represented here are representative of three independent experiments. E–F) 50 million sh-Ctrl and sh-Pim-1 LCL1 cells were harvested and cell lysates were used for Western blot analysis with indicated antibodies. G) 1×106 cells were plated and allowed them to grow at 37°C in complete medium without puromycin antibiotic. Viable cells were counted by trypan blue dye exclusion technique at indicated time points. The results shown here are representative of three independent experiments.
Figure 12.
EBNA3C mediated oncogenic Pim-1 activation promotes cell proliferation by impeding the growth suppressive properties of p21.
A–B) 10 million HEK-293 and MEF cells were transfected with different combinations of expression plasmids as indicated (A, P21+Ctrl vector; B, P21+Pim-1; C, P21+EBNA3C; D, P21+Pim-1+EBNA3C; E, P21+Pim-1KD; F, P21+Pim-1KD+EBNA3C; G, P21T145A+Pim-1; H, P21T145A+Pim-1+EBNA3C). Transfected cells were selected for 2 weeks with G418 antibiotic. Cells were incubated for 12 hrs in DMEM medium containing 0.1% FBS with 5 µM etoposide. 0.1×106 cells from each set of selected samples were plated and cultured for 6 days. Viable cells were counted at indicated time points by trypan blue dye exclusion technique. B) G418 selected stable cells were harvested, lysed in RIPA buffer and subjected to immunoblot analyses with indicated antibodies. C) 10×106 HEK-293 were transfected with different combinations of expression vectors and transiently transfected cells were subjected to BrdU incorporation assay by immunofluorescence study with etoposide treatment in DMEM containing 0.1% FBS. D) Bar diagram represents number of BrdU foci per cell. The results shown here are representative of three independent experiments. E) 30 million sh-Ctrl and sh-Pim-1 Ramos cells were co-transfected with p21 and increasing amount of EBNA3C expression vectors. Transfected cells were subjected to Western Blot analysis by using indicated antibodies.
Figure 13.
The schematic diagram illustrates the potential contribution of EBNA3C to oncogenic transformation of B-cells through stabilization of Pim-1 and proteasome mediated degradation of p21 which results in inhibition of the intrinsic apoptotic pathway.
EBNA3C interacts with oncogenic serine/threonine Pim-1 kinase. This molecular association stabilizes Pim-1 by inhibiting its Poly-ubiquitination. EBNA3C also promotes Pim-1 mediated p21 degradation. EBNA3C contributes to B-cell proliferation by activating oncogenic Pim-1 which leads to inhibition of the growth suppressive property of p21 as well as impeding intrinsic apoptosis signaling. This molecular strategy for oncogenic Pim-1 kinase targeted by EBNA3C for inhibition of p21 function, and it can be a potential therapeutic target for EBV-associated malignancies.