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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 754-760 |
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doi:10.1111/j.1745-7270.2008.00447.x |
Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells
Wei Song1,2, Haixing Xuan1, and Qishui Lin1*
1 Key Laboratory of Molecular Cell Biology, Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
2 Graduates School of the Chinese Academy of Sciences,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China
Received: April 20,
2008�������
Accepted: May 28,
2008
This work was
supported by a grant from the Knowledge Innovation Program of the Chinese Academy
of Sciences
*Corresponding
author: Tel, 86-21-54921248; Fax, 86-21-54921247; E-mail, [email protected]
The epidermal growth factor receptor (EGFR)
is a cytoskeleton-binding protein. Although purified EGFR can interact with
actins in vitro and normally at least 10% of EGFR exist in the insoluble
cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin
can only be visualized by fluorescence resonance energy transfer when epidermal
growth factor presents in the cell medium. Results indicate that the correct
orientation between EGFR and actin is important in the signal transduction
process.
Keywords��� epidermal growth factor receptor; actin; fluorescence resonance energy transfer; interaction
Binding epidermal growth factor (EGF) with the epidermal growth factor receptor (EGFR) induces receptor dimerization and tyrosine autophosphorylation, and triggers a series of signal transduction processes as well as cytoskeleton rearrangement in cells [1,2]. A number of EGFR downstream signal molecules have been shown to play important roles in actin binding. Activated EGFR can activate phosphatidylinositol 3-kinase and a group of small G-proteins (Rho, cdc42, Rac), which control trafficking and organization of cell cytoskeleton [3]. Heterodimer EGFR-ERBB2 activates phospholipase Cg, which activates cofilin, an actin depolymerizing factor [4]. EGF-activated EGFR also activates non-receptor tyrosine kinase c-Src, while the phosphorylated c-Src activates p190 RhoGAP and regulates the EGF-dependent actin cytoskeleton [5].�
Actin polymerization negatively regulates EGF-induced signal transduction [6]. Binding EGFR to actin deactivates the receptor, reducing the EGFR autophosphorylation activity and enhancing its affinity toward tyrosine phosphatase [7]. It has been proposed that actin filaments act as a scaffold on which the EGF-induced signaling complex assembles, leading to more efficient signal transduction process. In infantile pituitary cells, the EGFR/actin association could structure a microdomain and facilitate the cell signaling pathway related to cell-cell adhesion [8].
Polymerized actin co-localizes with activated EGFR in the A431 cell membrane [9]. Purified EGFR co-sediments with purified actin in vitro [10,11], and interacts with actin via an actin-binding domain (ABD) located at amino acid residues 984-996 [12,13]. It is well known that actin cytoskeleton is crucial to endocytosis. Endocytosed EGFR is sorted and subjected to a degradation pathway, a process that requires the participation of an ABD [14]. EGFR complexes and downstream signal molecules associate with actin cytoskeleton and are involved in receptor endocytosis [15,18]. Phosphorylation of EGFR Tyr992, the tyrosine residue within the ABD, reduces the rate of ligand-induced receptor endocytosis, which eventually increases the lifetime of the activated EGFR in the plasma membrane [19].
In the present study, fluorescence resonance energy transfer (FRET) method was used to investigate the interaction between EGFR and actin in vivo, and the temporal and spatial localization of actin bound EGFR was detected.
Materials and methods
Reagents and antibodies
Human recombinant EGF, AG1478, anti-EGFR antibody (29.1.1), and Triton
X-100 were purchased from Sigma-Aldrich (St. Louis, USA). High-glucose
Dulbecco�s modified Eagle�s medium, fetal bovine serum, and other cell culture
supplies were obtained from Invitrogen (Carlsbad, USA). Anti-EGFR antibody
(1005) and anti-phosphorylated tyrsine antibody (pY99) were obtained from Santa
Cruz Biotechnology (Santa Cruz, USA). Anti-green fluorescent protein
antibodies, pECFP-N1 vectors, and pEYFP-actin plasmid were
purchased from BD Biosciences Clontech (Palo Alto, USA).
Plasmid construction
DNA fragments encoding the full-length EGFR were amplified from
CVN/HERc and ligated into pECFP-N1 vectors using SacII-HindIII
according to previous work [28]. The construct was confirmed by DNA sequencing
analysis.
Cell culture and transfection
A431 cells and COS-7 cells were cultured in Dulbecco�s modified
Eagle�s medium supplemented with 10% fetal bovine serum and were incubated at
37 �C in an atmosphere of 5% CO2. COS-7 cells were plated into
culture dishes 24 h prior to transfection. When the cells� confluency reached
90%, they were co-transfected with pECFP-N1/HERc
and pEYFP-actin using the Lipofectamine 2000 method according to the
manufacturer�s instructions. Cells were passaged onto cover slides 12 h after
transfection. For the EGF or AG1478 treatment experiments, COS-7 cells were
serum starved overnight before either EGF (100 ng/ml) or AG1478 (0.5 mM) was applied.
Preparation of the detergent-insoluble cytoskeleton fraction
A431 cells were washed with phosphate-buffered saline (PBS) and extracted for 15 min with extraction buffer (10 mM HEPES, 1 mM phenylmethylsulphonyl fluoride, 1 mM MgCl2) containing 0.5% Triton X-100. The supernatant, containing solubilized EGFR, was collected, and the sediments were gently washed twice with extraction buffer (without Triton X-100), homogenized and centrifuged. The supernatant, which contained the cytoskeleton, was then collected.
Immunoprecipitation and Western blotting
For the immunoprecipitation study of Triton X-100 insoluble fraction
in A431 cells, the supernatants were incubated with anti-EGFR antibody (1005)
for 1 h and protein A Sepharose for 4 h at 4 �C.
Immunoprecipitates were washed five times with PBS and then resuspended in
2�sodium dodecyl sulfate-sample buffer. The lysates in Triton X-100 soluble
fraction were analyzed using Western blotting. COS-7 cells were lysed for 20
min on ice in a 20 mM HEPES with pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM
EGTA, 10% glycerol and protease inhibitor cocktail (Roche Diagnostics,
Rotkreuz, Switzerland). Lysate protein concentrations were quantified using
Bio-Rad Protein Assay (Bio-Rad Lab, Hercules, USA). Samples were analyzed by
SDS-PAGE, transferred to a Nitrcellulose membrane (Millipore, Billerica, USA),
and probed with antibodies against EGFR, phoshorylated tyrosine, and the
appropriate horseradish peroxidase-conjugated secondary antibodies and
chemiluminescence reagent. Band intensity was quantified by densitometry using
UVP image analysis software (UVP Inc, Upland, USA).
Fluorescence resonance energy transfer
The FRET signal with acceptor photobleaching was measured using cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) as the acceptor [29,30]. Co-transfected cells were grown on cover slides for 48 h, washed with PBS three times, and fixed with 4% paraformaldehyde for 20 min at room temperature. The cells were subsequently washed with PBS and mounted onto slides with non-quenching mounting solution (Sigma-Aldrich). FRET analysis was done by using Leica TCS SP2 confocal laser scanning microscope (Bensheim, Germany) and its software. Fluorescence recovery after photobleaching of the FRET donor (EGFR-CFP) was observed using a 63�/1.32 numerical aperture oil immersion objective. A full-intensity 514-nm laser light (200 pulses) was used to bleach the region of interest. The donor spectrum was measured again after recovery, and the FRET efficiency was calculated using the following formula:
Eq. 1
where Dpost is the fluorescence intensity of the
donor after acceptor bleaching and Dpre is the fluorescence intensity of the donor prior to acceptor
bleaching. In all experiments, Dpost>Dpre.
Results
Influences of EGF stimulation on the FRET signal between EGFR and actin
The FRET method was used to determine whether EGFR and actin co-localized in intact cells. The full-length cDNA of EGFR fused to the C-terminal of CFP (HERc-CFP) and actin fused with YFP (YFP-actin) were used. HERc-CFP was expressed transiently in COS-7 cells (Fig. 1). Western blotting analysis showed that the phosphorylation levels of EGFR increased significantly 30 min after the addition of EGF and were identical to that of endogenous WT EGFR in A431 cells [Fig. 1(A)]. Fig. 1(B) shows the co-localization of HERc-CFP and YFP-actin in COS-7 cells, which exists predominantly in the perinuclear and plasma membrane regions.
Double fluorescence images (Merge) showed that HERc-CFP co-localized
with YFP-actin in COS-7 cells. The location where EGFR interacted with actin in
the fixed whole cell was measured by FRET. The FRET signal can hardly be
detected in serum-starved, co-transfected COS-7 cells [Fig. 2(a-d)]. However, after EGF treatment, the FRET signal appeared [Fig.
2(e-p)], and after the cells were treated with
EGF for 5 min, the FRET signal appeared in the plasma membrane area [Fig.
2(e-h)]. After treatment for 30 min, a much
higher FRET signal appeared in the plasma membrane and perimembrane areas [Fig.
2(i-l)]. Although EGFR and actin still
co-localized in the perinuclear region when EGF was present, it was difficult
to visualize FRET signals in this region. HERc-CFP in a suitable conformation
could interact with YFP-actin by FRET assay. It seems likely that those EGFR
located in the perinuclear region and in the perimembrane after EGF treatment
would have different conformation, the former incompetent to the energy
transfer between HERc-CFP and YFP-actin.
EGF treatment enhanced the interaction of EGFR associated with cytoskeleton
The cell cytoskeleton fraction was isolated with Triton X-100. The results showed that EGFR was hardly detected in the Triton X-100 insoluble fraction of serum-starved A431 cells, but was easily detected in that of the EGF-treated cells (Fig. 3). Many proteins could be associated with actin cytoskeleton, the results further demonstrated that activated EGFR was the species which associated actin cytoskeleton, but not inactivated EGFR. EGFR and proteins bound to actin cytoskeleton were sedimented together in the Triton X-100 insoluble fraction. More EGFR was detected with EGF durative stimulation in the actin cytoskeleton fraction, and this was reflected in the results of the FRET assay. When COS-7 cells were cultured with serum in DMEM/10% FBS medium, less EGFR existed in the actin cytoskeleton fraction, and the degree of EGFR phosphorylation in Triton X-100 soluble fraction was lower (fig. 3, control).
Inhibition of tyrosine kinase activity prevents interaction between intracellular EGFR and actin
AG1478, an inhibitor of EGFR tyrosine kinase, can completely block the activation of EGFR [26,27]. EGFR could not be detected in the Triton X-100 insoluble cytoskeleton fraction of A431 cells treated with AG1478 [Fig. 4(A)].
When the HERc-CFP and YFP-actin co-expressed COS-7 cells were serum starved overnight and treated with EGFR tyrosine kinase inhibitor AG1478, no FERT signal was detected in cytosol, regardless whether it was treated with EGF treatment or not [Fig. 4(B)]. The results clearly showed that the binding of EGFR to actin could be visualized by acceptor bleaching FRET, but only after EGF activated EGFR.
Discussion
FRET images collected at different time intervals after EGF treatment indicated that appropriate conformation of EGFR was essential to its functional interaction with actin, which had a special temporal and spatial location possibly involving receptor internalization and the trafficking process. EGF bound with EGFR, and internalized by accompanying with EGFR. EGFR entered into the early endosome near the cell�s periphery [20], where it autophosphorylated and induced downstream signaling [21]. In A431 cells, the EGFR autophosphorylation process continued up to 20 min after EGF treatment [22]. Activated EGFR combined with other proteins, moved to late endosomes, and finally, degraded in lysosomes. FRET signal images showed that EGFR interaction with actin occurred at a specific time period after EGF activation and took place at specific spatial locations in intact cells. It was postulated that interaction between EGFR and actin would be involved in receptor internalization process. The binding of EGF to its receptor resulted in receptor dimerization and in the activation of receptor�s protein tyrosine kinase, and EGF induced EGFR internalization quickly occurred. Some reports showed that, after treatment with EGF for 30 min, part of the EGF/EGFR complex at the perinuclear region, where late endosomes and lysosomes are located [23,24]. ABD is an essential domain for EGF-induced EGFR movement from late endosomes to lysosomes [14]. Our FRET results showed that the efficiency of FRET was much lower in the perinuclear region than in the perimembrane. During incubation with EGF for 30 min, EGFR appeared in two regions of HeLa cells, mostly in the endosomal region and, to a lesser extent, in the lysosomal compartment [25], indicating that some EGFR had entered into lysosomes.
The binding of EGF with cell surface EGFR leads to the activation of the receptor tyrosine kinase. Tyrosine residues in the cytoplasmic region of the activated EGFR are autophosphorylated and then phosphorylate downstream signal molecules and actin binding proteins. The EGF-induced conformational changes to EGFR facilitate the interaction between EGFR and actin. The Tyr992 within the ABD is a major autophosphorylation site and serves as the binding site for docking proteins, such us phospholipase Cg and Shc, that associate with cytoskeleton. However, in vitro experiments showed that there was no effect on the binding of EGFR to actin when Tyr992 mutated into Phe. It is not yet clear whether proteins participate in or regulate the binding of EGFR to actin.
After binding to F-actin, the EGFR was deactivated as EGFR autophosphorylation activity diminished and the tyrosine phosphatase activity enhanced. If the ABD of EGFR were deleted, the mutated EGFR would hardly be degraded from early endosome to lysosome. As a result, the activation phase of mutated EGFR was prolonged. Both the FRET assay and detergent insoluble cytoskeleton experiments indicated that the amount of EGFR bound to actin increased after EGF stimulation, and it was a more effective negative feedback control than the activation. The extracellular signal-related kinase (ERK) 1/2 signaling cascade pathway is important in regulating cell proliferation [31], and ERK1/2 is normally activated through the autophosphorylated Tyr1068 of EGFR. Comparison of the ERK1/2 phosphorylation level in COS 7 cells expressing full-length EGFR and ABD1 deletion mutation respectively. After EGF treatment, the phopharylated ERK1/2 in the cells expressing ABD deletion mutant was lower than that in the cells expressing WT EGFR, the result is not coincide with the phosphorylation level of ADB mutant increased than that of WT EGFR (data not show). It is reasonable to suggest that, in addition to the negative control of EGFR/actin binding, direct signal transduction might also be involved.
Acknowledgments
We would like to thank Dr. Mei Jiang for her helpful discussions in preparing this article and Dr. Wei Bian for his continued support and technical assistance.
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