Limits...
Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

Thome M, Gaide O, Micheau O, Martinon F, Bonnet D, Gonzalez M, Tschopp J - J. Cell Biol. (2001)

Bottom Line: Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization.Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6.Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, CH-1066 Epalinges, Switzerland. margot.thomemiazza@ib.unil.ch

ABSTRACT
v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

Show MeSH

Related in: MedlinePlus

v-E10 induces hyperphosphorylation and plasma membrane recruitment of bcl-10. (A) Bcl-10 is localized in the cytoplasm of HeLa cells. HeLa cells were transfected with 0.5 μg of an expression vector for FLAG-tagged bcl-10 together with 4.5 μg of mock vector; the subcellular distribution of the transfected bcl-10 protein was analyzed by confocal staining using rabbit anti-FLAG antibody and Alexa-488–labeled anti–rabbit secondary antibody. (B) v-E10 and bcl-10 colocalize at the plasma membrane. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (0.5 μg), VSV-tagged v-E10 (2 μg), and mock vector (2.5 μg); the subcellular distribution of the transfected proteins was analyzed by confocal staining using rabbit anti-FLAG and mouse anti-VSV antibody followed by Alexa-488–labeled anti–rabbit and Cy5-labeled anti–mouse secondary antibody. (C) v-E10 induces hyperphosphorylation of bcl-10. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (1 μg) and VSV-tagged v-E10 (3 μg), and cell extracts of transfected cells were incubated for 1 h in the presence or absence of λ-phosphatase, as indicated. Cell lysates were analyzed by Western blotting using affinity-purified anti–bcl-10 antibody (AL114). Arrowheads indicate the migration position of the nonphosphorylated and the hyperphosphorylated forms of bcl-10. (D) v-E10 recruits hyperphosphorylated bcl-10 to the plasma membrane. HeLa cells were transfected with 3 μg of an expression vector for FLAG-tagged v-E10 and the subcellular distribution of the transfected v-E10 and the endogenous bcl-10 proteins was analyzed by cellular fractionation and Western blotting using anti-FLAG and anti–bcl-10 (AL114) antibodies, respectively. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198798&req=5

Figure 4: v-E10 induces hyperphosphorylation and plasma membrane recruitment of bcl-10. (A) Bcl-10 is localized in the cytoplasm of HeLa cells. HeLa cells were transfected with 0.5 μg of an expression vector for FLAG-tagged bcl-10 together with 4.5 μg of mock vector; the subcellular distribution of the transfected bcl-10 protein was analyzed by confocal staining using rabbit anti-FLAG antibody and Alexa-488–labeled anti–rabbit secondary antibody. (B) v-E10 and bcl-10 colocalize at the plasma membrane. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (0.5 μg), VSV-tagged v-E10 (2 μg), and mock vector (2.5 μg); the subcellular distribution of the transfected proteins was analyzed by confocal staining using rabbit anti-FLAG and mouse anti-VSV antibody followed by Alexa-488–labeled anti–rabbit and Cy5-labeled anti–mouse secondary antibody. (C) v-E10 induces hyperphosphorylation of bcl-10. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (1 μg) and VSV-tagged v-E10 (3 μg), and cell extracts of transfected cells were incubated for 1 h in the presence or absence of λ-phosphatase, as indicated. Cell lysates were analyzed by Western blotting using affinity-purified anti–bcl-10 antibody (AL114). Arrowheads indicate the migration position of the nonphosphorylated and the hyperphosphorylated forms of bcl-10. (D) v-E10 recruits hyperphosphorylated bcl-10 to the plasma membrane. HeLa cells were transfected with 3 μg of an expression vector for FLAG-tagged v-E10 and the subcellular distribution of the transfected v-E10 and the endogenous bcl-10 proteins was analyzed by cellular fractionation and Western blotting using anti-FLAG and anti–bcl-10 (AL114) antibodies, respectively. Bars, 10 μm.

Mentions: This led us to investigate whether the subcellular distribution of bcl-10 was altered in the presence of v-E10. Bcl-10 has been reported to be localized in the cytoplasm in filamentous stress fiber–like structures (Yan et al. 1999; Guiet and Vito 2000). However, in our hands bcl-10 shows a granular staining pattern in the cytoplasm of transfected HeLa cells (Fig. 4 A). Filamentous structures were observed only when considerably higher concentrations of the bcl-10 expression vector were used (data not shown). In cells cotransfected with v-E10, a significant portion of bcl-10 was now redistributed to the plasma membrane (Fig. 4 A). Coimmunostaining of HeLa cells transfected with both v-E10 and bcl-10 expression constructs and analysis by confocal laser scanning microscopy revealed a clear colocalization of the two proteins at the plasma membrane in cotransfected cells (Fig. 4 B). Consistent with this observation, cellular (endogenous) bcl-10 was found to partially redistribute to the membrane fraction in v-E10–transfected HeLa cells (Fig. 4 D).


Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

Thome M, Gaide O, Micheau O, Martinon F, Bonnet D, Gonzalez M, Tschopp J - J. Cell Biol. (2001)

v-E10 induces hyperphosphorylation and plasma membrane recruitment of bcl-10. (A) Bcl-10 is localized in the cytoplasm of HeLa cells. HeLa cells were transfected with 0.5 μg of an expression vector for FLAG-tagged bcl-10 together with 4.5 μg of mock vector; the subcellular distribution of the transfected bcl-10 protein was analyzed by confocal staining using rabbit anti-FLAG antibody and Alexa-488–labeled anti–rabbit secondary antibody. (B) v-E10 and bcl-10 colocalize at the plasma membrane. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (0.5 μg), VSV-tagged v-E10 (2 μg), and mock vector (2.5 μg); the subcellular distribution of the transfected proteins was analyzed by confocal staining using rabbit anti-FLAG and mouse anti-VSV antibody followed by Alexa-488–labeled anti–rabbit and Cy5-labeled anti–mouse secondary antibody. (C) v-E10 induces hyperphosphorylation of bcl-10. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (1 μg) and VSV-tagged v-E10 (3 μg), and cell extracts of transfected cells were incubated for 1 h in the presence or absence of λ-phosphatase, as indicated. Cell lysates were analyzed by Western blotting using affinity-purified anti–bcl-10 antibody (AL114). Arrowheads indicate the migration position of the nonphosphorylated and the hyperphosphorylated forms of bcl-10. (D) v-E10 recruits hyperphosphorylated bcl-10 to the plasma membrane. HeLa cells were transfected with 3 μg of an expression vector for FLAG-tagged v-E10 and the subcellular distribution of the transfected v-E10 and the endogenous bcl-10 proteins was analyzed by cellular fractionation and Western blotting using anti-FLAG and anti–bcl-10 (AL114) antibodies, respectively. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198798&req=5

Figure 4: v-E10 induces hyperphosphorylation and plasma membrane recruitment of bcl-10. (A) Bcl-10 is localized in the cytoplasm of HeLa cells. HeLa cells were transfected with 0.5 μg of an expression vector for FLAG-tagged bcl-10 together with 4.5 μg of mock vector; the subcellular distribution of the transfected bcl-10 protein was analyzed by confocal staining using rabbit anti-FLAG antibody and Alexa-488–labeled anti–rabbit secondary antibody. (B) v-E10 and bcl-10 colocalize at the plasma membrane. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (0.5 μg), VSV-tagged v-E10 (2 μg), and mock vector (2.5 μg); the subcellular distribution of the transfected proteins was analyzed by confocal staining using rabbit anti-FLAG and mouse anti-VSV antibody followed by Alexa-488–labeled anti–rabbit and Cy5-labeled anti–mouse secondary antibody. (C) v-E10 induces hyperphosphorylation of bcl-10. HeLa cells were cotransfected with expression vectors for FLAG-tagged bcl-10 (1 μg) and VSV-tagged v-E10 (3 μg), and cell extracts of transfected cells were incubated for 1 h in the presence or absence of λ-phosphatase, as indicated. Cell lysates were analyzed by Western blotting using affinity-purified anti–bcl-10 antibody (AL114). Arrowheads indicate the migration position of the nonphosphorylated and the hyperphosphorylated forms of bcl-10. (D) v-E10 recruits hyperphosphorylated bcl-10 to the plasma membrane. HeLa cells were transfected with 3 μg of an expression vector for FLAG-tagged v-E10 and the subcellular distribution of the transfected v-E10 and the endogenous bcl-10 proteins was analyzed by cellular fractionation and Western blotting using anti-FLAG and anti–bcl-10 (AL114) antibodies, respectively. Bars, 10 μm.
Mentions: This led us to investigate whether the subcellular distribution of bcl-10 was altered in the presence of v-E10. Bcl-10 has been reported to be localized in the cytoplasm in filamentous stress fiber–like structures (Yan et al. 1999; Guiet and Vito 2000). However, in our hands bcl-10 shows a granular staining pattern in the cytoplasm of transfected HeLa cells (Fig. 4 A). Filamentous structures were observed only when considerably higher concentrations of the bcl-10 expression vector were used (data not shown). In cells cotransfected with v-E10, a significant portion of bcl-10 was now redistributed to the plasma membrane (Fig. 4 A). Coimmunostaining of HeLa cells transfected with both v-E10 and bcl-10 expression constructs and analysis by confocal laser scanning microscopy revealed a clear colocalization of the two proteins at the plasma membrane in cotransfected cells (Fig. 4 B). Consistent with this observation, cellular (endogenous) bcl-10 was found to partially redistribute to the membrane fraction in v-E10–transfected HeLa cells (Fig. 4 D).

Bottom Line: Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization.Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6.Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, CH-1066 Epalinges, Switzerland. margot.thomemiazza@ib.unil.ch

ABSTRACT
v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

Show MeSH
Related in: MedlinePlus