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First published online 27 May 2003
doi: 10.1242/jcs.00480

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BTG2 antiproliferative protein interacts with the human CCR4 complex existing in vivo in three cell-cycle-regulated forms

¹ Unité INSERM U590, Centre Léon Bérard, 69373 Lyon Cedex 08, France
² Laboratoire de Cytogénétique Moléculaire, Hôpital Edouard Herriot, 69373 Lyon Cedex 03, France

View larger version (23K): [in a new window]   Fig. 1. hCCR4, hCAF1 and hPOP2 form common complexes in mammalian cells. (A) HeLa cell lysate was analyzed by gel filtration chromatography using a Superose 6 (HR10/30) column. Protein extracts were pre-cleared by centrifugation at 22,000 g for 20 minutes, then 300 µl of the sample (4 mg) were loaded onto the column. The flow rate was 0.4 ml/minute, and 250 µl were collected in each fraction, from which 50 µl were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot using anti-hCCR4, -hCAF1 and -hPOP2 antibodies. The numbers indicate fractions and the control lines represent extracts from HeLa cells transfected respectively with hCCR4FLAG-, hCAF1FLAG- and hPOP2FLAG-expressing plasmids. (B) hCAF1 was associated with hCCR4 in high molecular weight complex. Cellular extracts from HeLa-expressing Flag-tagged CCR4 were fractionated by gel filtration chromatography using a Superose 6 column as described before. Fractions 6-9 were pooled and incubated with anti-Flag M2 affinity gel at 4°C for 8-12 hours. Bound proteins were eluted with the sample buffer and boiled. Western blots were performed using anti-hCCR4 and anti-hCAF1 antibodies. Molecular size markers are given in kilodaltons.

View larger version (16K): [in a new window]   Fig. 2. hCAF1 and hPOP2 act as bridges between hCCR4 and BTG2. (A) Ni2+-NTA agarose column. A cellular extract from HeLa cells transfected with CCR4His-FLAG and BTG2FLAG expression constructs was bound with Ni2+NTA agarose. After intensive washes, bound proteins were eluted and analyzed by western blotting using anti-Flag and anti-hCAF1 antibodies. Molecular size markers are given in kilodaltons. (B) Mammalian two-hybrid assay. HeLa cells were seeded at 104 cells/well in 96-well microtiter plates, then transfected after 8 hours using Exgen 500. The transfected DNA included 100 ng of pG4-TK-Luc reporter plasmid together with 50 ng of GAL4 and/or VP16 fusion vectors in the presence or absence of 50 ng of pSG5FlagCAF1, pSG5FlagPOP2 or pSG5Foll. In all experiments, luciferase activity was normalized with the renilla luciferase activity expressed by the pTK-RL vector. Reporter activity was expressed as a ratio of fold induction to the activity of the reporter vector alone.

View larger version (14K): [in a new window]   Fig. 3. Co-elution of BTG2 protein and CCR4 complexes. Cellular extracts from HeLa cells transfected at low efficacy with CCR4His-FLAG- and BTG2FLAG-expressing plasmids were fractionated by gel filtration chromatography using a Superose 6 column, and subjected to immunoblot analysis as described in Fig. 1 using anti-Flag, anti-hCAF1 and anti-hPOP2 antibodies.

View larger version (29K): [in a new window]   Fig. 4. BTG2, hCAF1 and hCCR4 act as ER coactivators. HeLa cells were transiently transfected with 50 ng of pERE-Luc plasmid in the presence of 10 ng of pSG5ER and indicated amounts (ng) of BTG2-, hCAF1- or hCCR4-expressing vectors or control plasmids (empty pSG5). After 24 hours, the cells were washed, and then treated for 24 hours with a medium containing 10 nM 17ß-estradiol. Transfected cells were washed and collected 48 hours after transfection, then assayed for Luciferase activity. Normalized values are expressed as in Fig. 2B.

View larger version (45K): [in a new window]   Fig. 5. Localization and fractionation profile of endogenous hCAF1 over the course of the cell cycle. (A) Subcellular localization of endogenous hCAF1. HeLa cells were immunostained with anti-CAF1 polyclonal antibody. (B) MRC5 cells were synchronized by serum starvation (t0). After re-addition of serum, cells were analyzed at indicated time points for either immunofluorescence or fluorescence-activated cell sorting (FACS) analysis. (B1) Cell-cycle distribution, as determined by FACS analysis. The cells were fixed in 70% ethanol and treated with RNase. The DNA was stained with propidium iodide. (B2) Immunofluorescence of endogenous hCAF1. MRC5 cells were fixed in 4% PAF at the times indicated and stained for detection of endogenous hCAF1 with polyclonal antibody anti-hCAF1, followed by fluoresceine isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody. (C) Distinct steady-state fractionation profiles of hCAF1 during the course of the cell cycle. Lysates of MRC5 cells arrested in G0 by serum starvation or in G1-S 17 hours after serum restimulation were fractionated on a Superose 6 column (cell lysates and Superose 6 column preparations are described in Fig. 1A). The elution profiles of endogenous hCAF1 were analyzed by western analysis using anti-hCAF1-specific antibody.