First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01193
Journal of Cell Science 117, 3221-3231 (2004)
Published by The Company of Biologists 2004
Farnesyltransferase inhibitors disrupt EGF receptor traffic through modulation of the RhoB GTPase
Matthew Wherlock1,
Alexandra Gampel1,
Clare Futter2 and
Harry Mellor1,*
1 Mammalian Cell Biology Laboratory, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK
2 Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK
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Fig. 1. FTI treatment defines two cellular pools of RhoB. (A) (a) HeLa cells were allowed to accumulate fluorescent-labelled transferrin (red) for 1 hour and then fixed and stained for endogenous RhoB (green). (b) Cells were co-stained for endogenous RhoB and LAMP-1 (red). Endogenous RhoB did not colocalise with early or recycling endosomes as marked by transferrin (a) but showed clear colocalization with the late endosome/lysosome marker LAMP-1 (b). Scale bar, 10 µm. (B) Single confocal sections taken through the broadest part of the cells showed that endogenous RhoB (green) has a heterogenous distribution between endocytic and plasma membrane pools (c). FTI treatment caused a complete loss of the plasma membrane pool, with a corresponding gain in endocytic staining (d). Myc-epitope tagged RhoB (green) localised almost entirely to endosomal structures (e), a fraction of which became clustered near the nucleus with FTI treatment (f). Scale bars, 10 µm. (C) The relative distribution of RhoB between plasma membrane and endosomal pools was quantified from confocal sections of FTI-treated and untreated cells using Scion Image. The data represents the mean±s.e.m. of three independent experiments, where 10 c ells were quantified for each condition.
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Fig. 2. FTI blocks the degradation of the EGF receptor in EGF stimulated HeLa cells. Serum-starved HeLa cells incubated without (a), or with (b) FTI were stimulated with 100 ng/ml EGF for the indicated times. Total EGF receptor content at each time point was analyzed by western blot. The positions of the intact EGF receptor and an intermediate degradation product are indicated to the left of the figure, as is the position of a non-specific band (*). Serum starved cells incubated without (c) or with (d) FTI were pulse-labelled with [125I]EGF. Following the removal of surface bound ligand, cells were chased with unlabeled EGF and medium was collected at the indicated times. Medium was analyzed for intact and degraded EGF and after 4 hours the cells were lysed and assayed for retained radio-ligand. Error bars represent standard error of the mean (s.e.m.) based on four independent experiments, with three replicates per experiment. Differences between EGF receptor degradation in control and FTI treated cells were found to be significant by two-way ANOVA (P<0.001).
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Fig. 3. EGF receptor is predominantly retained in CD63+ endosomes in FTI treated cells. HeLa cells were serum starved overnight in the presence or absence of FTI and then incubated with 100 ng/ml EGF for 2 hours. Cells were fixed and stained for endogenous EGF receptor (green), nuclei (blue) and endosomal markers (red). EEA1 was used as a marker for early endosomes, CD63 for late endosomes and LAMP-1 for late endosomes/lysosomes. FTI-treatment led to retention of EGF receptor+ vesicles (compare top and bottom panels). In FTI-treated cells EGF receptor did not colocalise with EEA1 (d), almost completely colocalised with CD63 (e) and partially colocalised with LAMP-1 (f). The merged image in panel (e) is split in panels (g,h) to allow clearer visualisation of the EGF receptor (g) and CD63 (h) signals in FTI treated cells; colocalising vesicles (green arrowheads), non-overlapping signals (red arrowheads). Scale bar, 10 µm.
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Fig. 4. HEp2 cells were incubated overnight in serum-free medium in the absence (a and c) or the presence (b and d) of FTI. Cells were then stimulated with EGF in the presence of anti-EGF receptor-gold (10nm) for 45 minutes (a and b) or 90 minutes (c and d) in the continued absence or presence of inhibitor. Multivesicular late endosomes (MVBs) and lysosomes (Lys) are indicated. Anti-EGF receptor-gold in MVBs is monodisperse while that in lysosomes is aggregated owing to proteolysis of the anti-EGF receptor antibody (arrows). In the absence of FTI, gold can be found in both MVBs and lysosomes 45 minutes after EGF treatment, whereas in the presence of FTI the majority of gold is in MVBs. Ninety minutes after EGF stimulation, the majority of gold is in lysosomes in the absence of FTI, whereas gold is still frequently found in MVBs in the presence of inhibitor. There is no evidence that FTI affects sorting of EGF receptor onto internal vesicles of MVB or internal vesicle formation. Scale bar, 100 nm.
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Fig. 5. HEp2 cells were electroporated with RhoB-G14V cDNA and incubated overnight in medium containing 10% serum before incubation with serum-free medium for 1 hour. Cells were then stimulated with EGF in the presence of anti-EGF receptor-gold (10 nm) for 45 minutes (a) or 90 minutes (b and c). RhoB-G14V-expressing cells were identified by immuno-labelling with anti-myc antibody (5 nm gold). The majority of anti-EGF-receptor gold in RhoB-G14V-expressing cells is found in MVBs, even after 90 minutes of EGF stimulation. RhoB-G14V is found on EGF-receptor-containing MVBs as well as on small vesicles and/or tubules in the vicinity of MVBs. Scale bar, 100 nm.
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Fig. 6. Expression of the RhoB-binding HR1 domain from PRK1 overcomes the FTI-induced block on EGF-receptor degradation. Cells were transiently transfected with a HA-tagged HR1 domain construct from PRK1/PKN. Cells were starved overnight in the absence or presence of FTI and then stimulated with 100 ng/ml EGF for 2 hours. Cells were fixed and stained for endogenous EGF receptor and for expression of the HR1 construct. Cells were imaged by confocal microscopy, and the mean fluorescence signal for the EGF receptor within a fixed area was determined from 30 cells for each condition. Error bars represent standard error of the mean (s.e.m.) (n=3; 30 cells each experiment). The negation of the FTI induced block of EGF receptor by expression of the HR1 domain was found to be significant by the Student's t-test (P<0.001).
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Fig. 7. FTI treatment has no obvious short-term effect on signalling through the EGF receptor. Cells were serum starved overnight in the absence or presence of FTI and then stimulated with 100 ng/ml EGF for the indicated times. Activation of Erk1/2, PKB, JNK and p38 was analyzed by western blot using phospho-specific antibodies. The blots are representative of at least three separate experiments.
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Fig. 8. FTI treatment inhibits cell proliferation. Cells were maintained in DMEM containing 10% FBS (fed) or 0.1% fatty acid free BSA (starved) with 10 µM L-744,832 and/or 25 ng/ml EGF as indicated. The medium, L-744,832 and EGF were refreshed every 24 hours. Trypan blue-excluding cells were counted every 24 hours. Bars represent standard error of the mean (s.e.m.) based on five independent experiments. Differences in cell proliferation induced by FTI treatment in each cell culture condition were found to be significant by two-way ANOVA (P<0.001). FTI treatment had a significant effect on cell growth in serum-starved cells incubated with FTI, with or without EGF (P<0.005).
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© The Company of Biologists Ltd 2004
