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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 stabilizes Cyclin D1 through inhibiting its poly-ubiquitination.A) 50 million EBV negative BJAB cells, BJAB cells stably expressing EBNA3C (BJAB_E3C#7) and an EBV transformed cell, LCL2 were harvested after 6h incubation with proteasome inhibitor MG132 (20 µM). Cells were lysed and Cyclin D1 was immunoprecipitated (IP). Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was done by stripping and reprobing the same membrane. B-E) 15 million HEK 293T cells were transiently transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36h, and total protein was immunoprecipitated (IP) with indicated antibody and samples were resolved by 10% SDS-PAGE. Western blotting was done by stripping and reprobing the same membrane. Asterisks (*) indicate the immunoglobulin bands and poly-(ub) indicates poly-ubiquitination.
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ppat-1001275-g002: EBNA3C stabilizes Cyclin D1 through inhibiting its poly-ubiquitination.A) 50 million EBV negative BJAB cells, BJAB cells stably expressing EBNA3C (BJAB_E3C#7) and an EBV transformed cell, LCL2 were harvested after 6h incubation with proteasome inhibitor MG132 (20 µM). Cells were lysed and Cyclin D1 was immunoprecipitated (IP). Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was done by stripping and reprobing the same membrane. B-E) 15 million HEK 293T cells were transiently transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36h, and total protein was immunoprecipitated (IP) with indicated antibody and samples were resolved by 10% SDS-PAGE. Western blotting was done by stripping and reprobing the same membrane. Asterisks (*) indicate the immunoglobulin bands and poly-(ub) indicates poly-ubiquitination.

Mentions: Recently we have shown that ectopic expression of EBNA3C leads to stabilization of an important cellular oncoprotein, Mdm2 by inhibiting its poly-ubiquitination [18]. The increased stability of Cyclin D1 in the presence of EBNA3C, prompted us to examine whether EBNA3C similarly inhibits poly-ubiquitination of Cyclin D1 and so enhances its stability. To explore this possibility, three cell lines were selected, the EBV negative cell line BJAB, BJAB stably expressing EBNA3C (E3C #7) and an EBV positive lymphoblastoid cell line (LCL2). Immnuprecipitation using specific antibody against Cyclin D1 resulted in formation of high molecular weight species of Cyclin D1 migrating at a slower rate in BJAB cells while in BJAB cells stably expressing EBNA3C or in LCL2 significantly less of these high molecular weight bands were observed (Fig. 2A). Re-probing of the same membrane with Ub specific antibody showed a similar pattern (Fig. 2A). This result indicates that the activity responsible for the change in Cyclin D1 bands is present in EBV positive cells (LCL2) and EBNA3C expressing cell line (E3C #7) when compared to the EBV negative BJAB cells.


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 stabilizes Cyclin D1 through inhibiting its poly-ubiquitination.A) 50 million EBV negative BJAB cells, BJAB cells stably expressing EBNA3C (BJAB_E3C#7) and an EBV transformed cell, LCL2 were harvested after 6h incubation with proteasome inhibitor MG132 (20 µM). Cells were lysed and Cyclin D1 was immunoprecipitated (IP). Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was done by stripping and reprobing the same membrane. B-E) 15 million HEK 293T cells were transiently transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36h, and total protein was immunoprecipitated (IP) with indicated antibody and samples were resolved by 10% SDS-PAGE. Western blotting was done by stripping and reprobing the same membrane. Asterisks (*) indicate the immunoglobulin bands and poly-(ub) indicates poly-ubiquitination.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037348&req=5

ppat-1001275-g002: EBNA3C stabilizes Cyclin D1 through inhibiting its poly-ubiquitination.A) 50 million EBV negative BJAB cells, BJAB cells stably expressing EBNA3C (BJAB_E3C#7) and an EBV transformed cell, LCL2 were harvested after 6h incubation with proteasome inhibitor MG132 (20 µM). Cells were lysed and Cyclin D1 was immunoprecipitated (IP). Samples were resolved by 10% SDS-PAGE. Western blotting (WB) was done by stripping and reprobing the same membrane. B-E) 15 million HEK 293T cells were transiently transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36h, and total protein was immunoprecipitated (IP) with indicated antibody and samples were resolved by 10% SDS-PAGE. Western blotting was done by stripping and reprobing the same membrane. Asterisks (*) indicate the immunoglobulin bands and poly-(ub) indicates poly-ubiquitination.
Mentions: Recently we have shown that ectopic expression of EBNA3C leads to stabilization of an important cellular oncoprotein, Mdm2 by inhibiting its poly-ubiquitination [18]. The increased stability of Cyclin D1 in the presence of EBNA3C, prompted us to examine whether EBNA3C similarly inhibits poly-ubiquitination of Cyclin D1 and so enhances its stability. To explore this possibility, three cell lines were selected, the EBV negative cell line BJAB, BJAB stably expressing EBNA3C (E3C #7) and an EBV positive lymphoblastoid cell line (LCL2). Immnuprecipitation using specific antibody against Cyclin D1 resulted in formation of high molecular weight species of Cyclin D1 migrating at a slower rate in BJAB cells while in BJAB cells stably expressing EBNA3C or in LCL2 significantly less of these high molecular weight bands were observed (Fig. 2A). Re-probing of the same membrane with Ub specific antibody showed a similar pattern (Fig. 2A). This result indicates that the activity responsible for the change in Cyclin D1 bands is present in EBV positive cells (LCL2) and EBNA3C expressing cell line (E3C #7) when compared to the EBV negative BJAB cells.

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