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Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

Ohtani N, Brennan P, Gaubatz S, Sanij E, Hertzog P, Wolvetang E, Ghysdael J, Rowe M, Hara E - J. Cell Biol. (2003)

Bottom Line: Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression.As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway.These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK.

ABSTRACT
The p16INK4a-RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

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Both CTAR1/2 domains are required for the nuclear export of E2F4. (A) Schematic representation of LMP1 mutants used for assay described in B. (B) Wild-type E2F4 was coexpressed with wild-type and a series of LMP1 mutants in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (C) Wild-type E2F4 was coexpressed with LMP1 in the presence or absence of pharmacological inhibitors (SB203580, Rapamycin, LY294002, and U0126) at 10 μM, 0.1 nM, 20 μM, and 10 μM, respectively. 2 d after the transfection, E2F4 was detected by immunostaining with anti-E2F4 antibody, and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (D) Early passage (40 PDLs) TIG-3 cells were transfected with plasmids shown at the bottom by Nucleofector primary cell transfection system (Amaxa Biosystems). Transfection efficiencies were nearly 100% in all the transfected cells. Relative cell numbers were calculated using the GFP-transfected cells (lane 1) as the reference after 3 d. The experiments were repeated eight times with similar results. (B–D) Error bars indicate SD. (E) Model of LMP1 effects on p16INK4a–pRB pathway. Oncogenic Ras/MAPK signaling activates Ets2 and p16INK4a induction, thereby causing growth arrest/cellular senescence. Expression of the LMP1 oncoprotein blocks this pathway by targeting Ets2 and E2F4/5 for CRM1-dependent intracellular relocation.
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fig7: Both CTAR1/2 domains are required for the nuclear export of E2F4. (A) Schematic representation of LMP1 mutants used for assay described in B. (B) Wild-type E2F4 was coexpressed with wild-type and a series of LMP1 mutants in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (C) Wild-type E2F4 was coexpressed with LMP1 in the presence or absence of pharmacological inhibitors (SB203580, Rapamycin, LY294002, and U0126) at 10 μM, 0.1 nM, 20 μM, and 10 μM, respectively. 2 d after the transfection, E2F4 was detected by immunostaining with anti-E2F4 antibody, and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (D) Early passage (40 PDLs) TIG-3 cells were transfected with plasmids shown at the bottom by Nucleofector primary cell transfection system (Amaxa Biosystems). Transfection efficiencies were nearly 100% in all the transfected cells. Relative cell numbers were calculated using the GFP-transfected cells (lane 1) as the reference after 3 d. The experiments were repeated eight times with similar results. (B–D) Error bars indicate SD. (E) Model of LMP1 effects on p16INK4a–pRB pathway. Oncogenic Ras/MAPK signaling activates Ets2 and p16INK4a induction, thereby causing growth arrest/cellular senescence. Expression of the LMP1 oncoprotein blocks this pathway by targeting Ets2 and E2F4/5 for CRM1-dependent intracellular relocation.

Mentions: LMP1 is composed of six transmembrane domains and a long carboxy-terminal cytoplasmic segment. The region containing the six transmembrane domains mediates its oligomerization in the cytoplasmic membrane, resulting in the constitutive activation of the downstream signals (Eliopoulos and Young, 2001). There are at least two functional domains (CTAR1 and CTAR2) in the cytoplasmic tail of LMP1, which activate multiple signal transduction pathways (Brown et al., 2001; Schultheiss et al., 2001; Thorley-Lawson, 2001). Therefore, we examined the effect of a series of LMP1 mutants lacking CTAR1 and/or CTAR2 domain on the subcellular localization of E2F4 (Fig. 7 A). As shown in Fig. 7 B, LMP1 mutants lacking CTAR1 and/or CTAR2 domain failed to induce cytoplasmic accumulation of E2F4, suggesting that the signaling from both CTAR1 and CTAR2 domains of LMP1 are required for intracellular redistribution of E2F4. This is consistent with a previous observations that the mutant LMP1 lacking CTAR2 failed to immortalize MEFs (Xin et al., 2001), and both CTAR1 and CTAR2 domains are necessary for efficient B cell immortalization (Eliopoulos and Young, 2001).


Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

Ohtani N, Brennan P, Gaubatz S, Sanij E, Hertzog P, Wolvetang E, Ghysdael J, Rowe M, Hara E - J. Cell Biol. (2003)

Both CTAR1/2 domains are required for the nuclear export of E2F4. (A) Schematic representation of LMP1 mutants used for assay described in B. (B) Wild-type E2F4 was coexpressed with wild-type and a series of LMP1 mutants in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (C) Wild-type E2F4 was coexpressed with LMP1 in the presence or absence of pharmacological inhibitors (SB203580, Rapamycin, LY294002, and U0126) at 10 μM, 0.1 nM, 20 μM, and 10 μM, respectively. 2 d after the transfection, E2F4 was detected by immunostaining with anti-E2F4 antibody, and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (D) Early passage (40 PDLs) TIG-3 cells were transfected with plasmids shown at the bottom by Nucleofector primary cell transfection system (Amaxa Biosystems). Transfection efficiencies were nearly 100% in all the transfected cells. Relative cell numbers were calculated using the GFP-transfected cells (lane 1) as the reference after 3 d. The experiments were repeated eight times with similar results. (B–D) Error bars indicate SD. (E) Model of LMP1 effects on p16INK4a–pRB pathway. Oncogenic Ras/MAPK signaling activates Ets2 and p16INK4a induction, thereby causing growth arrest/cellular senescence. Expression of the LMP1 oncoprotein blocks this pathway by targeting Ets2 and E2F4/5 for CRM1-dependent intracellular relocation.
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Related In: Results  -  Collection

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fig7: Both CTAR1/2 domains are required for the nuclear export of E2F4. (A) Schematic representation of LMP1 mutants used for assay described in B. (B) Wild-type E2F4 was coexpressed with wild-type and a series of LMP1 mutants in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (C) Wild-type E2F4 was coexpressed with LMP1 in the presence or absence of pharmacological inhibitors (SB203580, Rapamycin, LY294002, and U0126) at 10 μM, 0.1 nM, 20 μM, and 10 μM, respectively. 2 d after the transfection, E2F4 was detected by immunostaining with anti-E2F4 antibody, and the histograms indicating the percentage of nuclei that were positive (N+) or negative (N−) for E2F4 expression were shown. (D) Early passage (40 PDLs) TIG-3 cells were transfected with plasmids shown at the bottom by Nucleofector primary cell transfection system (Amaxa Biosystems). Transfection efficiencies were nearly 100% in all the transfected cells. Relative cell numbers were calculated using the GFP-transfected cells (lane 1) as the reference after 3 d. The experiments were repeated eight times with similar results. (B–D) Error bars indicate SD. (E) Model of LMP1 effects on p16INK4a–pRB pathway. Oncogenic Ras/MAPK signaling activates Ets2 and p16INK4a induction, thereby causing growth arrest/cellular senescence. Expression of the LMP1 oncoprotein blocks this pathway by targeting Ets2 and E2F4/5 for CRM1-dependent intracellular relocation.
Mentions: LMP1 is composed of six transmembrane domains and a long carboxy-terminal cytoplasmic segment. The region containing the six transmembrane domains mediates its oligomerization in the cytoplasmic membrane, resulting in the constitutive activation of the downstream signals (Eliopoulos and Young, 2001). There are at least two functional domains (CTAR1 and CTAR2) in the cytoplasmic tail of LMP1, which activate multiple signal transduction pathways (Brown et al., 2001; Schultheiss et al., 2001; Thorley-Lawson, 2001). Therefore, we examined the effect of a series of LMP1 mutants lacking CTAR1 and/or CTAR2 domain on the subcellular localization of E2F4 (Fig. 7 A). As shown in Fig. 7 B, LMP1 mutants lacking CTAR1 and/or CTAR2 domain failed to induce cytoplasmic accumulation of E2F4, suggesting that the signaling from both CTAR1 and CTAR2 domains of LMP1 are required for intracellular redistribution of E2F4. This is consistent with a previous observations that the mutant LMP1 lacking CTAR2 failed to immortalize MEFs (Xin et al., 2001), and both CTAR1 and CTAR2 domains are necessary for efficient B cell immortalization (Eliopoulos and Young, 2001).

Bottom Line: Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression.As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway.These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK.

ABSTRACT
The p16INK4a-RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

Show MeSH
Related in: MedlinePlus