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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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LMP1 induces dissociation of E2F4 from pRB family proteins. (A and B) LMP1-inducible cell line (A) and Svts8 cells (B) were treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with anti-E2F4 antibody (IP). (C) E2F4-WT or E2F4-WT fused to NLS sequence (NLS–E2F4) or E2F4 ΔNES (68, 70A E2F4s1) was coexpressed with or without GFP-tagged LMP1 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. Error bars indicate SD. (D and E) LMP1-inducible cell line was treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with antibodies shown left (IP).
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fig6: LMP1 induces dissociation of E2F4 from pRB family proteins. (A and B) LMP1-inducible cell line (A) and Svts8 cells (B) were treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with anti-E2F4 antibody (IP). (C) E2F4-WT or E2F4-WT fused to NLS sequence (NLS–E2F4) or E2F4 ΔNES (68, 70A E2F4s1) was coexpressed with or without GFP-tagged LMP1 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. Error bars indicate SD. (D and E) LMP1-inducible cell line was treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with antibodies shown left (IP).

Mentions: To seek mechanistic insight into how LMP1 promotes intracellular redistribution of Ets2 and E2F4/5 from the nucleus to the cytoplasm, we decided to focus our attention on E2F4, because E2F4 contains typical nuclear export signal (NES) sequences and is well established as a nuclear shuttling protein (Gaubatz et al., 2001; Trimarchi and Lees, 2002). Because E2F4/5 lack an NLS, it has been suggested that association with an NLS-containing protein, such as pRB family proteins or with DP2, plays important roles in the nuclear localization of E2F4 (Muller et al., 1997; Verona et al., 1997). Furthermore, a recent report from Rayman and co-workers has shown that E2F4 is localized only in the cytoplasm of MEFs lacking both p107 and p130 (Rayman et al., 2002), suggesting that the association with p107 or p130 are required for the nuclear localization of E2F4. Therefore, we tested if LMP1 blocks the interaction between endogenous E2F4 and endogenous pRB family proteins. As shown in Fig. 6 A, the interaction between endogenous E2F4 and endogenous p107 was significantly reduced if LMP1 expression was induced in the LMP1-inducible cell line. Similarly, the interaction between endogenous E2F4 and endogenous pRB was also inhibited by LMP1 expression (Fig. 6 A, lanes 1 and 2). These effects were not observed in the control cells, which do not induce LMP1 expression by the addition of Ecdyson homologue, Ponasteron A (Fig. 6 B, lanes 1 and 2), precluding the possibility that these effects were caused by the Ponasteron A treatment. We were unable to see interaction between E2F4 and p130 in this cell line (unpublished data). These effects were not due to the phosphorylation of pRB family proteins by Cdks, because we were unable to see any difference of the phosphorylation pattern of pRB and p107 in the presence or absence of LMP1 expression (Fig. 6 A, lanes 1 and 2). To examine whether or not dissociation of E2F4 from the NLS-containing protein is required for intracellular redistribution of E2F4 from the nucleus to the cytoplasm, E2F4 was fused to the NLS sequence and coexpressed with LMP1. As shown in Fig. 6 C, the NLS–E2F4 fusion protein is predominantly expressed in the nucleus and is resistant to LMP1-induced cytoplasmic accumulation. This is suggesting that dissociation of E2F4 from the NLS-containing protein is required for the LMP1-induced intracellular redistribution of E2F4. However, it is still possible that dissociation of E2F4 from pRB family proteins is a consequence of the cytoplasmic accumulation of E2F4 and does not have a causal role in promoting nuclear export of E2F4. Indeed, LMB treatment abolished LMP1-induced cytoplasmic accumulation of E2F4 (Fig. 4 A). Moreover, the mutation of NES sequences (Gaubatz et al., 2001) accumulated E2F4 in the nucleus and made E2F4 less sensitive to LMP1-induced intracellular redistribution (Fig. 6 C). These results suggest the possibility that activation of nuclear export machinery could be involved in the LMP1-induced intracellular redistribution of E2F4 from the nucleus to the cytoplasm.


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)

LMP1 induces dissociation of E2F4 from pRB family proteins. (A and B) LMP1-inducible cell line (A) and Svts8 cells (B) were treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with anti-E2F4 antibody (IP). (C) E2F4-WT or E2F4-WT fused to NLS sequence (NLS–E2F4) or E2F4 ΔNES (68, 70A E2F4s1) was coexpressed with or without GFP-tagged LMP1 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. Error bars indicate SD. (D and E) LMP1-inducible cell line was treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with antibodies shown left (IP).
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fig6: LMP1 induces dissociation of E2F4 from pRB family proteins. (A and B) LMP1-inducible cell line (A) and Svts8 cells (B) were treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with anti-E2F4 antibody (IP). (C) E2F4-WT or E2F4-WT fused to NLS sequence (NLS–E2F4) or E2F4 ΔNES (68, 70A E2F4s1) was coexpressed with or without GFP-tagged LMP1 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. Error bars indicate SD. (D and E) LMP1-inducible cell line was treated with (lane 2) or without (lane 1) 0.5 μg/ml of Ponasteron A for 2 d. Next, cell lysates were immunoblotted directly with antibodies shown right (lysate) or after immunoprecipitated with antibodies shown left (IP).
Mentions: To seek mechanistic insight into how LMP1 promotes intracellular redistribution of Ets2 and E2F4/5 from the nucleus to the cytoplasm, we decided to focus our attention on E2F4, because E2F4 contains typical nuclear export signal (NES) sequences and is well established as a nuclear shuttling protein (Gaubatz et al., 2001; Trimarchi and Lees, 2002). Because E2F4/5 lack an NLS, it has been suggested that association with an NLS-containing protein, such as pRB family proteins or with DP2, plays important roles in the nuclear localization of E2F4 (Muller et al., 1997; Verona et al., 1997). Furthermore, a recent report from Rayman and co-workers has shown that E2F4 is localized only in the cytoplasm of MEFs lacking both p107 and p130 (Rayman et al., 2002), suggesting that the association with p107 or p130 are required for the nuclear localization of E2F4. Therefore, we tested if LMP1 blocks the interaction between endogenous E2F4 and endogenous pRB family proteins. As shown in Fig. 6 A, the interaction between endogenous E2F4 and endogenous p107 was significantly reduced if LMP1 expression was induced in the LMP1-inducible cell line. Similarly, the interaction between endogenous E2F4 and endogenous pRB was also inhibited by LMP1 expression (Fig. 6 A, lanes 1 and 2). These effects were not observed in the control cells, which do not induce LMP1 expression by the addition of Ecdyson homologue, Ponasteron A (Fig. 6 B, lanes 1 and 2), precluding the possibility that these effects were caused by the Ponasteron A treatment. We were unable to see interaction between E2F4 and p130 in this cell line (unpublished data). These effects were not due to the phosphorylation of pRB family proteins by Cdks, because we were unable to see any difference of the phosphorylation pattern of pRB and p107 in the presence or absence of LMP1 expression (Fig. 6 A, lanes 1 and 2). To examine whether or not dissociation of E2F4 from the NLS-containing protein is required for intracellular redistribution of E2F4 from the nucleus to the cytoplasm, E2F4 was fused to the NLS sequence and coexpressed with LMP1. As shown in Fig. 6 C, the NLS–E2F4 fusion protein is predominantly expressed in the nucleus and is resistant to LMP1-induced cytoplasmic accumulation. This is suggesting that dissociation of E2F4 from the NLS-containing protein is required for the LMP1-induced intracellular redistribution of E2F4. However, it is still possible that dissociation of E2F4 from pRB family proteins is a consequence of the cytoplasmic accumulation of E2F4 and does not have a causal role in promoting nuclear export of E2F4. Indeed, LMB treatment abolished LMP1-induced cytoplasmic accumulation of E2F4 (Fig. 4 A). Moreover, the mutation of NES sequences (Gaubatz et al., 2001) accumulated E2F4 in the nucleus and made E2F4 less sensitive to LMP1-induced intracellular redistribution (Fig. 6 C). These results suggest the possibility that activation of nuclear export machinery could be involved in the LMP1-induced intracellular redistribution of E2F4 from the nucleus to the cytoplasm.

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