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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.031443

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Plasma-membrane-anchored growth factor pro-amphiregulin binds A-type lamin and regulates global transcription

¹ Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan
² Department of Oral and Maxillofacial Surgery, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan
³ Department of Dermatology, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan
⁴ Department of Bioscience, INCS, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan
⁵ Protein Network Laboratory, CEREM, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan

View larger version (53K): [in this window] [in a new window]   Fig. 1. ProAR translocates from the plasma membrane to the inner nuclear membrane. (A) Schematic representation of human AREG gene products. The AR coding region is translated as a precursor form (pre-proAR) with five structural domains consisting of predicted 252 amino acids (Plowman et al., 1990). The signal sequence is trimmed off, and the resulting protein is expressed on the plasma membrane as proAR whose N-terminal sequence is not yet determined (*). In response to various stimuli, proAR is shed at the juxtamembrane domain, resulting in the production of AR and AR-CTF. Anti-AR-N and anti-AR-C pAbs specifically recognize the extracellular and the cytoplasmic domains of proAR, respectively. The single line is the epitope region of anti-AR-N pAb, and double line is that of anti-AR-C pAb. (B) HeLa cells were transfected with a full-length AREG plasmid, then incubated with or without TPA. Cells were immunostained with anti-AR-N or anti-AR-C pAb. (C) HeLa cells transiently expressing AREG were incubated with 20 µg/ml cycloheximide for 60 minutes. The cells were then treated with TPA and immunostained with anti-AR-N pAb. (D) HeLa cells transiently expressing AREG were permeabilized with digitonin and then fixed. The cells were re-permeabilized with (left panels) or without (right panels) Triton X-100, and immunostained with anti-AR-C (green) or/and anti-lamin B (red) pAbs. The lower panels are high magnification images of the boxed regions labelled 1 and 2. Scale bars: 5 µm. (E) Ultra-thin sections were stained with anti-V5 mAb and 15 nm gold-conjugated secondary antibodies. Right panel, without TPA; middle panel, with TPA; left panel, high magnification image of boxed region in middle panel. PM, plasma membrane; NE, nuclear envelope. Scale bars: 200 nm.

View larger version (56K): [in this window] [in a new window]   Fig. 2. ProAR is targeted to the nuclear envelope. (A) Schematic presentation of AR deletion mutants. All constructs (AR-V5, AR-C1-V5, AR-C2-V5, AR-C3-V5 and AR-C4-V5) were V5-tagged at the C-terminus. (B) Three monoclonal antibodies mAb2, mAb5 and mAb9 against the cytoplasmic domain of proAR were established. The core epitope region for each antibody is underlined. (C) Total lysates of HeLa cells expressing AR-V5 or AR-C-V5 mutants were analyzed by immunoblotting with the three mAbs. An anti-V5 mAb was used as a positive control. (D) Lysates of HeLa cells expressing wild-type AREG were immunoblotted with anti-AR-C mAbs. (E and F) HeLa cells transiently expressing wild-type AREG were incubated with or without TPA for 30 minutes and immunostained with the anti-AR-C mAbs (E) or mAb2 and anti-LAMP-1 mAbs (F). (G) HeLa cells transiently expressing wild-type AREG were treated with 50 nM Bafilomycin (Baf) for 60 minutes and incubated with TPA in the presence of Baf. The cells were then immunostained with mAb2 and anti-LAMP-1 mAb. Scale bars: 5 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 3. ProAR interacts with A-type lamin. (A) HeLa cells transiently expressing wild-type AREG were incubated with or without TPA. Cells were stained with anti-AR-C pAb and anti-lamin A/C mAb. Arrowheads indicate AREG-expressing cells. Scale bars: 5 µm. (B) Lysates of HeLa cells transiently transfected with or without wild-type AREG and treated with TPA for 60 minutes, were subjected to immunoblotting using anti-lamin A/C mAb (left) or anti-lamin B pAb (right). The left lanes show standard proteins. (C) HeLa cells transiently expressing wild-type AREG were incubated with or without TPA for 60 minutes. The cell lysates were subjected to immunoblotting with an anti-AR-C pAb (lane 1 and lane 2). The cell lysates were immunoprecipitated with an anti-lamin A/C mAb or normal mouse IgG, followed by western blotting using anti-AR-C pAb and anti-lamin A/C mAb. Arrows indicate the molecular mass of lamin A/C-interacted (upper) and AR-CTF (lower). (D) Schematic representation of GST-fused lamin A deletion mutants. (E) The cell lysates from cells transiently expressing wild-type AREG were incubated with GST or GST-lamin A derivatives, which were pulled down by glutathione-Sepharose beads. Bound proteins were analyzed by western blotting using the anti-AR-C pAb (upper panel). GST fusion proteins were analyzed by SDS-PAGE and stained with CBB (lower panel).

View larger version (50K): [in this window] [in a new window]   Fig. 4. A-type lamin is essential for targeting of proAR to the nuclear envelope. (A) HeLa cells were transfected with 20 nM or 50 nM of lamin A/C-targeting duplex siRNA (si-lamin A/C) or control siRNA (si-control). After 72 hours, the cells lysates were analyzed by western blotting with anti-lamin A/C mAb or anti-lamin B pAb. (B) HeLa cells were transfected twice: first with 20 nM of control siRNA (upper) or lamin A/C-siRNA (lower); and, after 48 hours, with wild-type AREG plasmid. After 24 hours, cells were stimulated with or without TPA, and then fixed. Cells were stained with anti-AR-C pAb and anti-lamin A/C mAb. The lower panels (1 and 2) are high magnification images of the boxed areas above. Scale bars: 5 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Un-shed proAR that interacts with A-type lamin is truncated at the C-terminus. (A) HeLa cells transiently expressing wild type AREG were incubated with or without TPA. Cell lysates were immunoprecipitated with anti-lamin A/C mAb and analyzed by western blot using the mAb5 and mAb2. (B) HeLa cells transiently expressing AR-V5 were incubated with or without TPA. Cell lysates were immunoprecipitated with anti-lamin A/C mAb. Cell lysates (upper) and precipitated proteins (lower) were analyzed with indicated mAbs. (C) Cell lysates of HeLa cells expressing AR-V5, AR-C1-V5 (lane 1), AR-C2-V5 (lane 2) or AR-C3-V5 (lane 3) were immunoprecipitated with anti-lamin A/C mAb. Cell lysates and precipitated proteins were analyzed with β-actin, anti-V5 mAb or mAb5.

View larger version (93K): [in this window] [in a new window]   Fig. 6. Lysine239 and/or lysine240 in the cytoplasmic domain of proAR are essential for targeting to the nuclear envelope. (A) Amino acid sequence of the cytoplasmic domain of proAR. (B) Schematic representation of mutations in K239 and K240 in the cytoplasmic domain (AR-m2). AR-m2 p indicates AR-m2 precursor protein. HeLa cells transfected with AR-m2 were incubated with or without TPA and immunostained with anti-AR-N pAb (red) and anti-TGN46 pAb (green). (C) Schematic representation of V5-tag inserted AR and its deletion mutants. AR-V5-C p, AR-V5-C10 p and AR-V5-C11 p indicate precursor proteins. (D,E) HeLa cells transiently expressing AR-V5-C, AR-V5-C10 or AR-V5-C11 were incubated with or without TPA, then immunostained with anti-V5 mAb and anti-AR-N pAb (D) or with anti-AR-N pAb and anti-lamin A/C mAb (E). Arrowheads indicate AR-expressing cells. (F) Cell lysates of HeLa cells expressing AR-V-C5, AR-V5-C10 or AR-V5-C11 were immunoprecipitated with anti-lamin A/C mAb. Cell lysates (upper) and precipitated proteins (lower) were analyzed with anti-V5 mAb. Scale bars: 5 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 7. Targeting of proAR to the nuclear envelope causes heterochromatin assembly. (A) HeLa cells transiently expressing wild-type AR were incubated with TPA and treated with Triton X-100, DNase I and/or NaCl. Cells were then stained with anti-AR-N pAb and anti-lamin A/C mAb. Arrowheads indicate AR-expressing cells. (B) HeLa cells transiently expressing wild-type AR were incubated with TPA and then stained with anti-AR-N pAb, anti-dimethylated H3K9 (H3K9Me2) mAb, anti-trimethylated H3K9 (H3K9Me3) pAb and anti-HP1b pAb. Cells were counterstained with Hoechst 33342. Each panel shows representative cells that are untransfected (top row) and transfected (bottom row). (C) Cell lysates of HeLa cells transiently expressing wild-type AR or AR-C11 treated with or without TPA were analyzed by western blotting with anti-AR-N pAb, anti-H3K9Me3 mAb and anti-β-actin mAb. Scale bars: 5 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 8. Targeting of proAR to the nuclear envelope induces transient suppression of global transcription. (A,C) 5 mM Br-U was added in the presence or absence of TPA to the culture medium of HeLa cells transiently expressing AR-V5-C (A), AR-V5-C10 or AR-V5-C11 (C) followed by a 30 minute incubation. Cells were fixed and immunostained with anti-BrdU mAb, anti-V5 mAb and Hoechst 33342. Asterisks indicate AR-expressing cells. (B) HeLa cells transiently expressing AR-V5-C were incubated with or without TPA for 30 minutes and re-grown in fresh medium for the duration indicated. 5 mM Br-U was added to the culture medium 30 minutes prior to fixation. After fixation, Br-U incorporated in mRNA was stained with anti-BrdU mAb and fluorescent intensity in the AR-expressing cells was quantified (n=200). Statistical analysis was carried out as described in Materials and Methods. Scale bars: 5 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 9. Schematic representation of proAR intracellular trafficking. Without TPA stimulation, proAR primarily localizes at the plasma membrane. TPA induces partial proAR-ectodomain shedding, resulting in the production of a soluble growth factor (AR) and a C-terminal fragment (AR-CTF). In addition, TPA causes the endocytosis of both AR-CTF and the residual un-shed proAR. AR-CTF then translocates to the lysosome (black arrow) and un-shed proAR trafficks to the INM (red arrow). During translocation to the INM, proAR is truncated at the C-terminus, and targeted to the ER. Subsequently ER-localized proAR diffuses or is actively transported to the INM, where it is tethered via interaction with A-type lamin and downregulates global transcription. PM, plasma membrane.

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