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Epstein-Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1.

Saha A, Halder S, Upadhyay SK, Lu J, Kumar P, Murakami M, Cai Q, Robertson ES - PLoS Pathog. (2011)

Bottom Line: EBNA3C together with Cyclin D1-CDK6 complex also efficiently ifies the inhibitory effect of pRb on cell growth.Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth.Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Tumor Virology Program, Abramson Comprehensive Cancer Center, University of Pennsylvania Medical School, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130-160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1-50), and C-terminal domain (residues 171-240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently ifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

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EBNA3C bypasses GSK3β dependent nuclear export of Cyclin D1.A) 15 million HEK 293 cells were transiently co-transfected with vectors of flag-tagged Cyclin D1 and myc-tagged EBNA3C and subjected to sub-cellular fractionation. Fractionated proteins were analyzed by probing western blots with flag and myc antibodies. Nuclear protein Sp1 and cytoplasmic protein GAPDH were immuno-detected as control. B) As similar to (A) 15 million HEK 293 cells were transfected with Cyclin D1 and different combinations of expression constructs as indicated and subjected to sub-cellular fractionation. The fractionated proteins were analyzed by using indicated antibodies. The percent distribution of Cyclin D1 is shown as numbers below panel (A) and (B). C) 15 million HEK 293T cells were co-transfected with myc-tagged GSK-3β and flag-tagged EBNA3C expression vectors. Cells proteins were collected 36 h post-transfection and immunoprecipitated (IP) with 1 ug of flag antibody. Lysates and IP complexes were resolved by 10% SDS-PAGE and western blotted (WB) with the indicated antibodies. Asterisk indicates the immunoglobulin bands. D) HEK 293T cells were transfected with myc-tagged GSK-3β and flag-tagged EBNA3C vectors as indicated. Empty vector was used to balance total transfected DNA. At 36 h posttransfction, GSK-3β immunoprecipitates were captured with myc antibody and assayed for in vitro kinase activity toward either recombinant GST-Cyclin D1 (lanes 1 and 2) or GST-Cyclin D1 T286 mutant (lanes 3 and 4) using γP32-ATP. Western blot using whole cell lysates are shown in first three panels and coomassie staining of SDS-PAGE resolved recombinant GST proteins used in this study is shown in panel 4.
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ppat-1001275-g006: EBNA3C bypasses GSK3β dependent nuclear export of Cyclin D1.A) 15 million HEK 293 cells were transiently co-transfected with vectors of flag-tagged Cyclin D1 and myc-tagged EBNA3C and subjected to sub-cellular fractionation. Fractionated proteins were analyzed by probing western blots with flag and myc antibodies. Nuclear protein Sp1 and cytoplasmic protein GAPDH were immuno-detected as control. B) As similar to (A) 15 million HEK 293 cells were transfected with Cyclin D1 and different combinations of expression constructs as indicated and subjected to sub-cellular fractionation. The fractionated proteins were analyzed by using indicated antibodies. The percent distribution of Cyclin D1 is shown as numbers below panel (A) and (B). C) 15 million HEK 293T cells were co-transfected with myc-tagged GSK-3β and flag-tagged EBNA3C expression vectors. Cells proteins were collected 36 h post-transfection and immunoprecipitated (IP) with 1 ug of flag antibody. Lysates and IP complexes were resolved by 10% SDS-PAGE and western blotted (WB) with the indicated antibodies. Asterisk indicates the immunoglobulin bands. D) HEK 293T cells were transfected with myc-tagged GSK-3β and flag-tagged EBNA3C vectors as indicated. Empty vector was used to balance total transfected DNA. At 36 h posttransfction, GSK-3β immunoprecipitates were captured with myc antibody and assayed for in vitro kinase activity toward either recombinant GST-Cyclin D1 (lanes 1 and 2) or GST-Cyclin D1 T286 mutant (lanes 3 and 4) using γP32-ATP. Western blot using whole cell lysates are shown in first three panels and coomassie staining of SDS-PAGE resolved recombinant GST proteins used in this study is shown in panel 4.

Mentions: Based on immuno-fluorescence studies, we observed that Cyclin D1 localization was mainly restricted to the cytoplasmic fraction of asynchronously growing cells. However, on expression of EBNA3C the localization of Cyclin D1 was predominantly nuclear. To further support these data, transiently transfected HEK 293 cells were subjected to sub-cellular fractionation and fractionated proteins were analyzed by immunoblot analysis. The result showed that flag-tagged Cyclin D1 alone was detected approximately 50% in both cytoplasmic and nuclear fractions, respectively (Fig. 6A, compare lanes 1 and 4). However, when co-transfected with EBNA3C, flag-Cyclin D1 was detected predominantly within the nuclear fraction (Fig. 6A, compare lanes 3 and 6), with an approximately 50% increase compared to flag-Cyclin D1 alone (Fig. 6A, compare lanes 1 and 3). EBNA3C was detected completely within nuclear fraction (Fig. 6A, lanes 2 and 3). The efficiency of cytoplasmic and nuclear fractionation was confirmed by localization of nuclear transcription factor Sp1 and cytoplasmic protein GAPDH (Fig. 6A). These observations strongly suggested that the apparent nuclear trans-localization of Cyclin D1 mediated by EBNA3C, as determined by indirect immuno-fluorescence microscopy or sub-cellular fractionation assay may be due to deregulation of the critical regulatory kinase GSK-3β, a negative regulator of Cyclin D1 nuclear retention and protein stability [31]. We thus decided to examine whether EBNA3C can ify the effect of GSK-3β on Cyclin D1 function.


Epstein-Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1.

Saha A, Halder S, Upadhyay SK, Lu J, Kumar P, Murakami M, Cai Q, Robertson ES - PLoS Pathog. (2011)

EBNA3C bypasses GSK3β dependent nuclear export of Cyclin D1.A) 15 million HEK 293 cells were transiently co-transfected with vectors of flag-tagged Cyclin D1 and myc-tagged EBNA3C and subjected to sub-cellular fractionation. Fractionated proteins were analyzed by probing western blots with flag and myc antibodies. Nuclear protein Sp1 and cytoplasmic protein GAPDH were immuno-detected as control. B) As similar to (A) 15 million HEK 293 cells were transfected with Cyclin D1 and different combinations of expression constructs as indicated and subjected to sub-cellular fractionation. The fractionated proteins were analyzed by using indicated antibodies. The percent distribution of Cyclin D1 is shown as numbers below panel (A) and (B). C) 15 million HEK 293T cells were co-transfected with myc-tagged GSK-3β and flag-tagged EBNA3C expression vectors. Cells proteins were collected 36 h post-transfection and immunoprecipitated (IP) with 1 ug of flag antibody. Lysates and IP complexes were resolved by 10% SDS-PAGE and western blotted (WB) with the indicated antibodies. Asterisk indicates the immunoglobulin bands. D) HEK 293T cells were transfected with myc-tagged GSK-3β and flag-tagged EBNA3C vectors as indicated. Empty vector was used to balance total transfected DNA. At 36 h posttransfction, GSK-3β immunoprecipitates were captured with myc antibody and assayed for in vitro kinase activity toward either recombinant GST-Cyclin D1 (lanes 1 and 2) or GST-Cyclin D1 T286 mutant (lanes 3 and 4) using γP32-ATP. Western blot using whole cell lysates are shown in first three panels and coomassie staining of SDS-PAGE resolved recombinant GST proteins used in this study is shown in panel 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037348&req=5

ppat-1001275-g006: EBNA3C bypasses GSK3β dependent nuclear export of Cyclin D1.A) 15 million HEK 293 cells were transiently co-transfected with vectors of flag-tagged Cyclin D1 and myc-tagged EBNA3C and subjected to sub-cellular fractionation. Fractionated proteins were analyzed by probing western blots with flag and myc antibodies. Nuclear protein Sp1 and cytoplasmic protein GAPDH were immuno-detected as control. B) As similar to (A) 15 million HEK 293 cells were transfected with Cyclin D1 and different combinations of expression constructs as indicated and subjected to sub-cellular fractionation. The fractionated proteins were analyzed by using indicated antibodies. The percent distribution of Cyclin D1 is shown as numbers below panel (A) and (B). C) 15 million HEK 293T cells were co-transfected with myc-tagged GSK-3β and flag-tagged EBNA3C expression vectors. Cells proteins were collected 36 h post-transfection and immunoprecipitated (IP) with 1 ug of flag antibody. Lysates and IP complexes were resolved by 10% SDS-PAGE and western blotted (WB) with the indicated antibodies. Asterisk indicates the immunoglobulin bands. D) HEK 293T cells were transfected with myc-tagged GSK-3β and flag-tagged EBNA3C vectors as indicated. Empty vector was used to balance total transfected DNA. At 36 h posttransfction, GSK-3β immunoprecipitates were captured with myc antibody and assayed for in vitro kinase activity toward either recombinant GST-Cyclin D1 (lanes 1 and 2) or GST-Cyclin D1 T286 mutant (lanes 3 and 4) using γP32-ATP. Western blot using whole cell lysates are shown in first three panels and coomassie staining of SDS-PAGE resolved recombinant GST proteins used in this study is shown in panel 4.
Mentions: Based on immuno-fluorescence studies, we observed that Cyclin D1 localization was mainly restricted to the cytoplasmic fraction of asynchronously growing cells. However, on expression of EBNA3C the localization of Cyclin D1 was predominantly nuclear. To further support these data, transiently transfected HEK 293 cells were subjected to sub-cellular fractionation and fractionated proteins were analyzed by immunoblot analysis. The result showed that flag-tagged Cyclin D1 alone was detected approximately 50% in both cytoplasmic and nuclear fractions, respectively (Fig. 6A, compare lanes 1 and 4). However, when co-transfected with EBNA3C, flag-Cyclin D1 was detected predominantly within the nuclear fraction (Fig. 6A, compare lanes 3 and 6), with an approximately 50% increase compared to flag-Cyclin D1 alone (Fig. 6A, compare lanes 1 and 3). EBNA3C was detected completely within nuclear fraction (Fig. 6A, lanes 2 and 3). The efficiency of cytoplasmic and nuclear fractionation was confirmed by localization of nuclear transcription factor Sp1 and cytoplasmic protein GAPDH (Fig. 6A). These observations strongly suggested that the apparent nuclear trans-localization of Cyclin D1 mediated by EBNA3C, as determined by indirect immuno-fluorescence microscopy or sub-cellular fractionation assay may be due to deregulation of the critical regulatory kinase GSK-3β, a negative regulator of Cyclin D1 nuclear retention and protein stability [31]. We thus decided to examine whether EBNA3C can ify the effect of GSK-3β on Cyclin D1 function.

Bottom Line: EBNA3C together with Cyclin D1-CDK6 complex also efficiently ifies the inhibitory effect of pRb on cell growth.Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth.Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Tumor Virology Program, Abramson Comprehensive Cancer Center, University of Pennsylvania Medical School, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130-160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1-50), and C-terminal domain (residues 171-240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently ifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

Show MeSH
Related in: MedlinePlus