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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 enhances functional activity of Cyclin D1/CDK6 complex to negatively regulate pRb protein stability.A–B) Analysis of Cyclin D1/CDK6 mediated phosphorylation of Histone H1 and pRb. HEK 293T cells were transfected with flag-tagged Cyclin D1 and CDK6 vectors and 0, 5, or 15 µg of myc-tagged EBNA3C vector. 36 h post-transfection, flag immunoprecipitates were captured and assayed for in vitro kinase activity on (A) Histone H1 or (B) recombinant GST-pRb (residues 792-928) as similar to figure 6D. C) Stability assay of pRb. Saos-2 (pRb-/-) cells were co-transfected with expression plasmids for myc- tagged pRb, flag-tagged Cyclin D1 and CDK6, and EBNA3C. 36 h post-transfection, cells were treated with 40 µg/ml cycloheximide (CHX) for the indicated times. Samples were resolved by SDS-PAGE. GAPDH was immunodetected to normalize protein levels. Western blots were probed with the indicated antibodies. D) Ubiquitination of pRb. HEK 293T cells were transfected with expression plasmids for myc-tagged pRb, HA-tagged ubiquitin (Ub), and EBNA3C (E3C), and flag-tagged Cyclin D1/CDK6 as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with myc-specific antibody. Samples were resolved by SDS-PAGE. Western blots were probed with the indicated antibodies.
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ppat-1001275-g007: EBNA3C enhances functional activity of Cyclin D1/CDK6 complex to negatively regulate pRb protein stability.A–B) Analysis of Cyclin D1/CDK6 mediated phosphorylation of Histone H1 and pRb. HEK 293T cells were transfected with flag-tagged Cyclin D1 and CDK6 vectors and 0, 5, or 15 µg of myc-tagged EBNA3C vector. 36 h post-transfection, flag immunoprecipitates were captured and assayed for in vitro kinase activity on (A) Histone H1 or (B) recombinant GST-pRb (residues 792-928) as similar to figure 6D. C) Stability assay of pRb. Saos-2 (pRb-/-) cells were co-transfected with expression plasmids for myc- tagged pRb, flag-tagged Cyclin D1 and CDK6, and EBNA3C. 36 h post-transfection, cells were treated with 40 µg/ml cycloheximide (CHX) for the indicated times. Samples were resolved by SDS-PAGE. GAPDH was immunodetected to normalize protein levels. Western blots were probed with the indicated antibodies. D) Ubiquitination of pRb. HEK 293T cells were transfected with expression plasmids for myc-tagged pRb, HA-tagged ubiquitin (Ub), and EBNA3C (E3C), and flag-tagged Cyclin D1/CDK6 as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with myc-specific antibody. Samples were resolved by SDS-PAGE. Western blots were probed with the indicated antibodies.

Mentions: To address the functional consequences as a result of the association of Cyclin D1 and EBNA3C, we tested the activity of Cyclin D1/CDK6 complexes for the ability to phosphorylate histone H1 or recombinant GST-pRb (residues 792-928). HEK 293T cells were transiently transfected with increasing amounts of a myc-tagged EBNA3C expression construct. Flag-tagged Cyclin D1/CDK6 immunoprecipitated complexes were assayed for in vitro kinase activity as determined by histone H1 or GST-pRb phosphorylation (Fig. 7A and B, respectively). The results showed that Cyclin D1-dependent kinase activity increased in a dose-responsive manner with increased expression of EBNA3C (Fig. 7A and B). Phosphorimager analysis revealed 1.6-times more P32-Histone H1 and 2.3-times more P32-GST-pRb (Fig. 7A and B). Parallel blots showed the expressed protein levels (Fig. 7A and B, top two panels) and the amount of substrates (histone H1 or GST-pRb) used in this study (Fig. 7A and B).


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 enhances functional activity of Cyclin D1/CDK6 complex to negatively regulate pRb protein stability.A–B) Analysis of Cyclin D1/CDK6 mediated phosphorylation of Histone H1 and pRb. HEK 293T cells were transfected with flag-tagged Cyclin D1 and CDK6 vectors and 0, 5, or 15 µg of myc-tagged EBNA3C vector. 36 h post-transfection, flag immunoprecipitates were captured and assayed for in vitro kinase activity on (A) Histone H1 or (B) recombinant GST-pRb (residues 792-928) as similar to figure 6D. C) Stability assay of pRb. Saos-2 (pRb-/-) cells were co-transfected with expression plasmids for myc- tagged pRb, flag-tagged Cyclin D1 and CDK6, and EBNA3C. 36 h post-transfection, cells were treated with 40 µg/ml cycloheximide (CHX) for the indicated times. Samples were resolved by SDS-PAGE. GAPDH was immunodetected to normalize protein levels. Western blots were probed with the indicated antibodies. D) Ubiquitination of pRb. HEK 293T cells were transfected with expression plasmids for myc-tagged pRb, HA-tagged ubiquitin (Ub), and EBNA3C (E3C), and flag-tagged Cyclin D1/CDK6 as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with myc-specific antibody. Samples were resolved by SDS-PAGE. Western blots were probed with the indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037348&req=5

ppat-1001275-g007: EBNA3C enhances functional activity of Cyclin D1/CDK6 complex to negatively regulate pRb protein stability.A–B) Analysis of Cyclin D1/CDK6 mediated phosphorylation of Histone H1 and pRb. HEK 293T cells were transfected with flag-tagged Cyclin D1 and CDK6 vectors and 0, 5, or 15 µg of myc-tagged EBNA3C vector. 36 h post-transfection, flag immunoprecipitates were captured and assayed for in vitro kinase activity on (A) Histone H1 or (B) recombinant GST-pRb (residues 792-928) as similar to figure 6D. C) Stability assay of pRb. Saos-2 (pRb-/-) cells were co-transfected with expression plasmids for myc- tagged pRb, flag-tagged Cyclin D1 and CDK6, and EBNA3C. 36 h post-transfection, cells were treated with 40 µg/ml cycloheximide (CHX) for the indicated times. Samples were resolved by SDS-PAGE. GAPDH was immunodetected to normalize protein levels. Western blots were probed with the indicated antibodies. D) Ubiquitination of pRb. HEK 293T cells were transfected with expression plasmids for myc-tagged pRb, HA-tagged ubiquitin (Ub), and EBNA3C (E3C), and flag-tagged Cyclin D1/CDK6 as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with myc-specific antibody. Samples were resolved by SDS-PAGE. Western blots were probed with the indicated antibodies.
Mentions: To address the functional consequences as a result of the association of Cyclin D1 and EBNA3C, we tested the activity of Cyclin D1/CDK6 complexes for the ability to phosphorylate histone H1 or recombinant GST-pRb (residues 792-928). HEK 293T cells were transiently transfected with increasing amounts of a myc-tagged EBNA3C expression construct. Flag-tagged Cyclin D1/CDK6 immunoprecipitated complexes were assayed for in vitro kinase activity as determined by histone H1 or GST-pRb phosphorylation (Fig. 7A and B, respectively). The results showed that Cyclin D1-dependent kinase activity increased in a dose-responsive manner with increased expression of EBNA3C (Fig. 7A and B). Phosphorimager analysis revealed 1.6-times more P32-Histone H1 and 2.3-times more P32-GST-pRb (Fig. 7A and B). Parallel blots showed the expressed protein levels (Fig. 7A and B, top two panels) and the amount of substrates (histone H1 or GST-pRb) used in this study (Fig. 7A and B).

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