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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.

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v-E10 is targeted to the plasma membrane via an isoprenylated COOH-terminal Cys–Cys motif. (A) HeLa cells were transfected with expression vectors for wild-type v-E10 (wt) or v-E10 lacking the two COOH-terminal cysteine residues (v-E10ΔCC), and the subcellular localization of both proteins was analyzed by Triton X-114 extraction as described in the legend to Fig. 1 B. (B) HeLa cells were transfected as in A and the subcellular distribution of wild-type v-E10 and its mutants v-E10ΔCC, v-E10-CA, and v-E10-AC was analyzed by confocal staining using anti-VSV– and Cy5-labeled secondary antibody. (C) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and membrane proteins were analyzed by Triton X-114 extraction, SDS-PAGE, and Western blotting with the indicated primary antibodies. (D) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and total cellular extracts or Triton X-114–extracted membrane proteins were analyzed by SDS-PAGE and anti-FLAG Western blotting. Bars, 10 μm.
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Figure 2: v-E10 is targeted to the plasma membrane via an isoprenylated COOH-terminal Cys–Cys motif. (A) HeLa cells were transfected with expression vectors for wild-type v-E10 (wt) or v-E10 lacking the two COOH-terminal cysteine residues (v-E10ΔCC), and the subcellular localization of both proteins was analyzed by Triton X-114 extraction as described in the legend to Fig. 1 B. (B) HeLa cells were transfected as in A and the subcellular distribution of wild-type v-E10 and its mutants v-E10ΔCC, v-E10-CA, and v-E10-AC was analyzed by confocal staining using anti-VSV– and Cy5-labeled secondary antibody. (C) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and membrane proteins were analyzed by Triton X-114 extraction, SDS-PAGE, and Western blotting with the indicated primary antibodies. (D) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and total cellular extracts or Triton X-114–extracted membrane proteins were analyzed by SDS-PAGE and anti-FLAG Western blotting. Bars, 10 μm.

Mentions: v-E10 does not contain a putative transmembrane segment and must therefore be targeted to the plasma membrane in some other manner. Covalent lipid modifications, such as myristylation, palmitoylation, farnesylation, or geranylgeranylation, can mediate membrane targeting of proteins that lack a transmembrane domain (Casey and Seabra 1996; Resh 1999). Indeed, the amino acid sequence of v-E10 terminates in two cysteine residues and thus contains a cognate consensus site for geranylgeranylation by geranylgeranyltransferase II (rab geranylgeranyltransferase), which transfers geranylgeranyl to proteins terminating in CC, CXC, or CCXX (Seabra et al. 1992; Glomset and Farnsworth 1994). To investigate whether the two COOH-terminal cysteine residues of v-E10 are implicated in its membrane localization, we generated mutants of v-E10 lacking both cysteine residues (v-E10ΔCC) or in which either one of the two cysteine residues was replaced by an alanine residue (v-E10-CA and v-E10-AC, respectively). Biochemical fractionation and confocal laser scanning showed that v-E10 lacking both COOH-terminal cysteine residues was localized in the cytoplasm and no longer detectable in the membrane fraction (Fig. 2A and Fig. B). The subcellular distribution of the mutants v-E10-CA and v-E10-AC was similar to the one observed for the deletion mutant, indicating that both Cys residues are required for plasma membrane targeting of v-E10 (Fig. 2 B). The 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor lovastatin inhibits isoprenylation, e.g., farnesylation and geranylgeranylation, by decreasing cellular pools of farnesylpyrophosphate and geranylgeranylpyrophosphate, respectively. Pretreatment of stably v-E10–expressing Jurkat cells with lovastatin significantly reduced the membrane localization of v-E10, but not of the unrelated transmembrane protein Fas, whereas it did not affect the overall expression levels of v-E10 (Fig. 2C and Fig. 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 is targeted to the plasma membrane via an isoprenylated COOH-terminal Cys–Cys motif. (A) HeLa cells were transfected with expression vectors for wild-type v-E10 (wt) or v-E10 lacking the two COOH-terminal cysteine residues (v-E10ΔCC), and the subcellular localization of both proteins was analyzed by Triton X-114 extraction as described in the legend to Fig. 1 B. (B) HeLa cells were transfected as in A and the subcellular distribution of wild-type v-E10 and its mutants v-E10ΔCC, v-E10-CA, and v-E10-AC was analyzed by confocal staining using anti-VSV– and Cy5-labeled secondary antibody. (C) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and membrane proteins were analyzed by Triton X-114 extraction, SDS-PAGE, and Western blotting with the indicated primary antibodies. (D) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and total cellular extracts or Triton X-114–extracted membrane proteins were analyzed by SDS-PAGE and anti-FLAG Western blotting. Bars, 10 μm.
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Figure 2: v-E10 is targeted to the plasma membrane via an isoprenylated COOH-terminal Cys–Cys motif. (A) HeLa cells were transfected with expression vectors for wild-type v-E10 (wt) or v-E10 lacking the two COOH-terminal cysteine residues (v-E10ΔCC), and the subcellular localization of both proteins was analyzed by Triton X-114 extraction as described in the legend to Fig. 1 B. (B) HeLa cells were transfected as in A and the subcellular distribution of wild-type v-E10 and its mutants v-E10ΔCC, v-E10-CA, and v-E10-AC was analyzed by confocal staining using anti-VSV– and Cy5-labeled secondary antibody. (C) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and membrane proteins were analyzed by Triton X-114 extraction, SDS-PAGE, and Western blotting with the indicated primary antibodies. (D) Jurkat cells expressing FLAG-tagged v-E10 or the parental clone (wt) were treated for 60 h with the indicated concentrations of lovastatin, and total cellular extracts or Triton X-114–extracted membrane proteins were analyzed by SDS-PAGE and anti-FLAG Western blotting. Bars, 10 μm.
Mentions: v-E10 does not contain a putative transmembrane segment and must therefore be targeted to the plasma membrane in some other manner. Covalent lipid modifications, such as myristylation, palmitoylation, farnesylation, or geranylgeranylation, can mediate membrane targeting of proteins that lack a transmembrane domain (Casey and Seabra 1996; Resh 1999). Indeed, the amino acid sequence of v-E10 terminates in two cysteine residues and thus contains a cognate consensus site for geranylgeranylation by geranylgeranyltransferase II (rab geranylgeranyltransferase), which transfers geranylgeranyl to proteins terminating in CC, CXC, or CCXX (Seabra et al. 1992; Glomset and Farnsworth 1994). To investigate whether the two COOH-terminal cysteine residues of v-E10 are implicated in its membrane localization, we generated mutants of v-E10 lacking both cysteine residues (v-E10ΔCC) or in which either one of the two cysteine residues was replaced by an alanine residue (v-E10-CA and v-E10-AC, respectively). Biochemical fractionation and confocal laser scanning showed that v-E10 lacking both COOH-terminal cysteine residues was localized in the cytoplasm and no longer detectable in the membrane fraction (Fig. 2A and Fig. B). The subcellular distribution of the mutants v-E10-CA and v-E10-AC was similar to the one observed for the deletion mutant, indicating that both Cys residues are required for plasma membrane targeting of v-E10 (Fig. 2 B). The 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor lovastatin inhibits isoprenylation, e.g., farnesylation and geranylgeranylation, by decreasing cellular pools of farnesylpyrophosphate and geranylgeranylpyrophosphate, respectively. Pretreatment of stably v-E10–expressing Jurkat cells with lovastatin significantly reduced the membrane localization of v-E10, but not of the unrelated transmembrane protein Fas, whereas it did not affect the overall expression levels of v-E10 (Fig. 2C and Fig. 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