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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 blocks downstream mediators of the p16INK4a pathway. (A) The p16INK4a inducible cell line, EH1 cells (McConnell et al., 1999), were transfected with an expression plasmid encoding GFP-tagged LMP1 or GFP-tagged CRM1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, cells were labeled with BrdU for 1 h, fixed, and stained with an anti-BrdU antibody. Transfected cells were identified by their green fluorescence, and the percentages of cells that incorporated BrdU compared with the cells without induction of p16INK4a were determined. (B) EH1 cells were transfected with an expression plasmid encoding GFP-tagged LMP1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, GFP-positive cells were isolated by FACS® and cell lysates were immunoblotted with antibodies shown right. Error bars indicate SD.
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fig3: LMP1 blocks downstream mediators of the p16INK4a pathway. (A) The p16INK4a inducible cell line, EH1 cells (McConnell et al., 1999), were transfected with an expression plasmid encoding GFP-tagged LMP1 or GFP-tagged CRM1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, cells were labeled with BrdU for 1 h, fixed, and stained with an anti-BrdU antibody. Transfected cells were identified by their green fluorescence, and the percentages of cells that incorporated BrdU compared with the cells without induction of p16INK4a were determined. (B) EH1 cells were transfected with an expression plasmid encoding GFP-tagged LMP1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, GFP-positive cells were isolated by FACS® and cell lysates were immunoblotted with antibodies shown right. Error bars indicate SD.

Mentions: If repression of p16INK4a expression is the major function of LMP1 in blocking the p16INK4a–RB pathway, ectopic expression of p16INK4a should be dominant over the LMP1 function. To test this idea, we used the U2OS cells that have been engineered to induce p16INK4a expression by addition of IPTG (EH1 cells; McConnell et al., 1999). As shown previously (McConnell et al., 1999), IPTG treatment significantly blocks entry into S-phase (Fig. 3 A, lanes 1 and 2). However, surprisingly, the ability of p16INK4a to induce a G1 arrest was significantly attenuated when LMP1 was coexpressed as seen in CRM1-expressing cells (Fig. 3 A, lanes 3–6). Induction of p16INK4a is similarly observed in both LMP1-expressing cells and in control cells expressing GFP (Fig. 3 B, lanes 2 and 4). Moreover, phosphorylation of the pRB family proteins was blocked by the induction of the p16INK4a in both cases (Fig. 3 B, lanes 2 and 4), showing that p16INK4a is effectively functioning as a Cdk inhibitor in LMP1 expressing cells. These results led us to hypothesize that LMP1 also targets downstream mediator(s) of the p16INK4a-induced growth arrest pathway.


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 blocks downstream mediators of the p16INK4a pathway. (A) The p16INK4a inducible cell line, EH1 cells (McConnell et al., 1999), were transfected with an expression plasmid encoding GFP-tagged LMP1 or GFP-tagged CRM1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, cells were labeled with BrdU for 1 h, fixed, and stained with an anti-BrdU antibody. Transfected cells were identified by their green fluorescence, and the percentages of cells that incorporated BrdU compared with the cells without induction of p16INK4a were determined. (B) EH1 cells were transfected with an expression plasmid encoding GFP-tagged LMP1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, GFP-positive cells were isolated by FACS® and cell lysates were immunoblotted with antibodies shown right. Error bars indicate SD.
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Related In: Results  -  Collection

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fig3: LMP1 blocks downstream mediators of the p16INK4a pathway. (A) The p16INK4a inducible cell line, EH1 cells (McConnell et al., 1999), were transfected with an expression plasmid encoding GFP-tagged LMP1 or GFP-tagged CRM1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, cells were labeled with BrdU for 1 h, fixed, and stained with an anti-BrdU antibody. Transfected cells were identified by their green fluorescence, and the percentages of cells that incorporated BrdU compared with the cells without induction of p16INK4a were determined. (B) EH1 cells were transfected with an expression plasmid encoding GFP-tagged LMP1 or a control vector expressing GFP alone. 12 h later, cells were treated with (+) or without (−) 1 mM IPTG for p16INK4a induction. 48 h after IPTG treatment, GFP-positive cells were isolated by FACS® and cell lysates were immunoblotted with antibodies shown right. Error bars indicate SD.
Mentions: If repression of p16INK4a expression is the major function of LMP1 in blocking the p16INK4a–RB pathway, ectopic expression of p16INK4a should be dominant over the LMP1 function. To test this idea, we used the U2OS cells that have been engineered to induce p16INK4a expression by addition of IPTG (EH1 cells; McConnell et al., 1999). As shown previously (McConnell et al., 1999), IPTG treatment significantly blocks entry into S-phase (Fig. 3 A, lanes 1 and 2). However, surprisingly, the ability of p16INK4a to induce a G1 arrest was significantly attenuated when LMP1 was coexpressed as seen in CRM1-expressing cells (Fig. 3 A, lanes 3–6). Induction of p16INK4a is similarly observed in both LMP1-expressing cells and in control cells expressing GFP (Fig. 3 B, lanes 2 and 4). Moreover, phosphorylation of the pRB family proteins was blocked by the induction of the p16INK4a in both cases (Fig. 3 B, lanes 2 and 4), showing that p16INK4a is effectively functioning as a Cdk inhibitor in LMP1 expressing cells. These results led us to hypothesize that LMP1 also targets downstream mediator(s) of the p16INK4a-induced growth arrest pathway.

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