Limits...
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.

Show MeSH

Related in: MedlinePlus

LMP1 blocks Ets2 binding to DNA. (A) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding H-RasV12 and sequentially infected with a retrovirus encoding LMP1 (lane 2) or control vector (lane 1). 4 d after superinfection, levels of a series of endogenous proteins were examined by immunoblotting using antibodies shown right. MEK was used here as a loading control. (B) Dose-dependent ability of LMP1 and E2DBD to block activation of the p16INK4a promoter (left) or E36 promoter (right) by Ets2 and activated MEK in SVts8 cells. Expression plasmids encoding proteins shown bottom were introduced into SVts8 cells along with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. Error bars indicate SD. (C) ChIP assays were performed using cells described in A and antibody against Ets2 or SEI-1 (control). The p16INK4a-promoter was recovered by PCR using primers flanking the Ets binding sites in the human p16INK4a promoter.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172795&req=5

fig1: LMP1 blocks Ets2 binding to DNA. (A) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding H-RasV12 and sequentially infected with a retrovirus encoding LMP1 (lane 2) or control vector (lane 1). 4 d after superinfection, levels of a series of endogenous proteins were examined by immunoblotting using antibodies shown right. MEK was used here as a loading control. (B) Dose-dependent ability of LMP1 and E2DBD to block activation of the p16INK4a promoter (left) or E36 promoter (right) by Ets2 and activated MEK in SVts8 cells. Expression plasmids encoding proteins shown bottom were introduced into SVts8 cells along with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. Error bars indicate SD. (C) ChIP assays were performed using cells described in A and antibody against Ets2 or SEI-1 (control). The p16INK4a-promoter was recovered by PCR using primers flanking the Ets binding sites in the human p16INK4a promoter.

Mentions: We have demonstrated previously that the activation of Ets2, which is a downstream mediator of the MAPK cascade, is responsible for the up-regulation of p16INK4a in Ras-induced senescence, whereas Ets1 seems to play a role in replicative senescence (Ohtani et al., 2001; Huot et al., 2002). Thus, we first tested whether LMP1 prevents p16INK4a expression by blocking Ets2 activity. As shown previously (Yang et al., 2000b), coexpression of LMP1 significantly blocked the induction of p16INK4a by oncogenic Ras in human diploid fibroblasts (HDFs; Fig. 1 A, lane 2). Like a dominant negative form of Ets2 (E2DBD; Foos et al., 1998), expression of LMP1 inhibited transcriptional activity of Ets2 on the human p16INK4a promoter and on an artificial promoter (E36) containing tandem repeats of the Ets-binding sequence (Fig. 1 B). Moreover, chromatin-immunoprecipitation (ChIP) analysis indicated that Ets2 was not bound to the p16INK4a promoter in HDFs expressing LMP1 (Fig. 1 C, lanes 2 and 5). However, the level of Ets2 protein was unaffected by LMP1 expression in HDFs (Fig. 1 A). These results strongly suggest that LMP1 blocks Ets2 binding to DNA without affecting the expression level of Ets2.


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 Ets2 binding to DNA. (A) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding H-RasV12 and sequentially infected with a retrovirus encoding LMP1 (lane 2) or control vector (lane 1). 4 d after superinfection, levels of a series of endogenous proteins were examined by immunoblotting using antibodies shown right. MEK was used here as a loading control. (B) Dose-dependent ability of LMP1 and E2DBD to block activation of the p16INK4a promoter (left) or E36 promoter (right) by Ets2 and activated MEK in SVts8 cells. Expression plasmids encoding proteins shown bottom were introduced into SVts8 cells along with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. Error bars indicate SD. (C) ChIP assays were performed using cells described in A and antibody against Ets2 or SEI-1 (control). The p16INK4a-promoter was recovered by PCR using primers flanking the Ets binding sites in the human p16INK4a promoter.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172795&req=5

fig1: LMP1 blocks Ets2 binding to DNA. (A) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding H-RasV12 and sequentially infected with a retrovirus encoding LMP1 (lane 2) or control vector (lane 1). 4 d after superinfection, levels of a series of endogenous proteins were examined by immunoblotting using antibodies shown right. MEK was used here as a loading control. (B) Dose-dependent ability of LMP1 and E2DBD to block activation of the p16INK4a promoter (left) or E36 promoter (right) by Ets2 and activated MEK in SVts8 cells. Expression plasmids encoding proteins shown bottom were introduced into SVts8 cells along with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. Error bars indicate SD. (C) ChIP assays were performed using cells described in A and antibody against Ets2 or SEI-1 (control). The p16INK4a-promoter was recovered by PCR using primers flanking the Ets binding sites in the human p16INK4a promoter.
Mentions: We have demonstrated previously that the activation of Ets2, which is a downstream mediator of the MAPK cascade, is responsible for the up-regulation of p16INK4a in Ras-induced senescence, whereas Ets1 seems to play a role in replicative senescence (Ohtani et al., 2001; Huot et al., 2002). Thus, we first tested whether LMP1 prevents p16INK4a expression by blocking Ets2 activity. As shown previously (Yang et al., 2000b), coexpression of LMP1 significantly blocked the induction of p16INK4a by oncogenic Ras in human diploid fibroblasts (HDFs; Fig. 1 A, lane 2). Like a dominant negative form of Ets2 (E2DBD; Foos et al., 1998), expression of LMP1 inhibited transcriptional activity of Ets2 on the human p16INK4a promoter and on an artificial promoter (E36) containing tandem repeats of the Ets-binding sequence (Fig. 1 B). Moreover, chromatin-immunoprecipitation (ChIP) analysis indicated that Ets2 was not bound to the p16INK4a promoter in HDFs expressing LMP1 (Fig. 1 C, lanes 2 and 5). However, the level of Ets2 protein was unaffected by LMP1 expression in HDFs (Fig. 1 A). These results strongly suggest that LMP1 blocks Ets2 binding to DNA without affecting the expression level of Ets2.

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