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Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism.

Higashino F, Aoyagi M, Takahashi A, Ishino M, Taoka M, Isobe T, Kobayashi M, Totsuka Y, Kohgo T, Shindoh M - J. Cell Biol. (2005)

Bottom Line: We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells.The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA.Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Sapporo 060-8586, Japan. fhigashi@den.hokudai.ac.jp

ABSTRACT
E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

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E4orf6 interacts with pp32/LANP. (A) Identification of pp32/LANP as an associated protein of E4orf6 using 293 cells transfected with expression constructs for FLAG-E4orf6 by M2 (anti-FLAG antibody) affinity column chromatography. Arrows indicate the proteins identified as FLAG-E4orf6 and pp32/LANP by MALDI-TOF/MS analysis. (B) The interaction of E4orf6 with pp32/LANP was confirmed using transformed BRK cells. The expressions of E4orf6, FLAG-E4orf6, and pp32/LANP are shown. (C) Schematic diagram of deletion mutants of E4orf6. NES, nuclear export signal; CCR, conserved cysteine-rich region; Helix, amphipathic α helix region. Bar indicates the oncodomain (top). In vitro–translated E4orf6 mutants were incubated with GST-pp32/LANP, and the associated E4orf6 mutants were confirmed by immunoblotting using the antibody to E4orf6 and HA tag (for dl1-203; bottom). (D) Subcellular localization of pp32/LANP (a and d) and E4orf6 (b and e) in mixture of BRK E1 (arrows) and E1+E4 (top) or BRK dl210-294 (bottom) cells. (c and f) Phase images are shown.
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fig1: E4orf6 interacts with pp32/LANP. (A) Identification of pp32/LANP as an associated protein of E4orf6 using 293 cells transfected with expression constructs for FLAG-E4orf6 by M2 (anti-FLAG antibody) affinity column chromatography. Arrows indicate the proteins identified as FLAG-E4orf6 and pp32/LANP by MALDI-TOF/MS analysis. (B) The interaction of E4orf6 with pp32/LANP was confirmed using transformed BRK cells. The expressions of E4orf6, FLAG-E4orf6, and pp32/LANP are shown. (C) Schematic diagram of deletion mutants of E4orf6. NES, nuclear export signal; CCR, conserved cysteine-rich region; Helix, amphipathic α helix region. Bar indicates the oncodomain (top). In vitro–translated E4orf6 mutants were incubated with GST-pp32/LANP, and the associated E4orf6 mutants were confirmed by immunoblotting using the antibody to E4orf6 and HA tag (for dl1-203; bottom). (D) Subcellular localization of pp32/LANP (a and d) and E4orf6 (b and e) in mixture of BRK E1 (arrows) and E1+E4 (top) or BRK dl210-294 (bottom) cells. (c and f) Phase images are shown.

Mentions: To identify the E4orf6-associated proteins (Higashino et al., 1998), 293 cells were transfected with the expression plasmid of FLAG-tagged E4orf6. The associated proteins were then isolated using M2 affinity column chromatography and the acquired proteins were analyzed with MALDI-TOF/MS. Two proteins were identified as pp32/LANP (Matsuoka et al., 1994; Chen et al., 1996; Fig. 1 A). We expect that one of these proteins is the phosphorylated form of pp32/LANP or APRIL (Mencinger et al., 1998), which has high similarity to pp32/LANP.


Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism.

Higashino F, Aoyagi M, Takahashi A, Ishino M, Taoka M, Isobe T, Kobayashi M, Totsuka Y, Kohgo T, Shindoh M - J. Cell Biol. (2005)

E4orf6 interacts with pp32/LANP. (A) Identification of pp32/LANP as an associated protein of E4orf6 using 293 cells transfected with expression constructs for FLAG-E4orf6 by M2 (anti-FLAG antibody) affinity column chromatography. Arrows indicate the proteins identified as FLAG-E4orf6 and pp32/LANP by MALDI-TOF/MS analysis. (B) The interaction of E4orf6 with pp32/LANP was confirmed using transformed BRK cells. The expressions of E4orf6, FLAG-E4orf6, and pp32/LANP are shown. (C) Schematic diagram of deletion mutants of E4orf6. NES, nuclear export signal; CCR, conserved cysteine-rich region; Helix, amphipathic α helix region. Bar indicates the oncodomain (top). In vitro–translated E4orf6 mutants were incubated with GST-pp32/LANP, and the associated E4orf6 mutants were confirmed by immunoblotting using the antibody to E4orf6 and HA tag (for dl1-203; bottom). (D) Subcellular localization of pp32/LANP (a and d) and E4orf6 (b and e) in mixture of BRK E1 (arrows) and E1+E4 (top) or BRK dl210-294 (bottom) cells. (c and f) Phase images are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171388&req=5

fig1: E4orf6 interacts with pp32/LANP. (A) Identification of pp32/LANP as an associated protein of E4orf6 using 293 cells transfected with expression constructs for FLAG-E4orf6 by M2 (anti-FLAG antibody) affinity column chromatography. Arrows indicate the proteins identified as FLAG-E4orf6 and pp32/LANP by MALDI-TOF/MS analysis. (B) The interaction of E4orf6 with pp32/LANP was confirmed using transformed BRK cells. The expressions of E4orf6, FLAG-E4orf6, and pp32/LANP are shown. (C) Schematic diagram of deletion mutants of E4orf6. NES, nuclear export signal; CCR, conserved cysteine-rich region; Helix, amphipathic α helix region. Bar indicates the oncodomain (top). In vitro–translated E4orf6 mutants were incubated with GST-pp32/LANP, and the associated E4orf6 mutants were confirmed by immunoblotting using the antibody to E4orf6 and HA tag (for dl1-203; bottom). (D) Subcellular localization of pp32/LANP (a and d) and E4orf6 (b and e) in mixture of BRK E1 (arrows) and E1+E4 (top) or BRK dl210-294 (bottom) cells. (c and f) Phase images are shown.
Mentions: To identify the E4orf6-associated proteins (Higashino et al., 1998), 293 cells were transfected with the expression plasmid of FLAG-tagged E4orf6. The associated proteins were then isolated using M2 affinity column chromatography and the acquired proteins were analyzed with MALDI-TOF/MS. Two proteins were identified as pp32/LANP (Matsuoka et al., 1994; Chen et al., 1996; Fig. 1 A). We expect that one of these proteins is the phosphorylated form of pp32/LANP or APRIL (Mencinger et al., 1998), which has high similarity to pp32/LANP.

Bottom Line: We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells.The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA.Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Sapporo 060-8586, Japan. fhigashi@den.hokudai.ac.jp

ABSTRACT
E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

Show MeSH
Related in: MedlinePlus