1 Laboratory for Toxicopathology, Institute of Pathology, The National Hospital,
University of Oslo, N-0027 Oslo, Norway
2 Neurochemical Laboratory, Department Group of Basic Medical Sciences,
University of Oslo, N-0317 Oslo, Norway
3 Center for Cellular Stress Responses, University of Oslo, N-0027 Oslo,
Norway
* Author for correspondence (e-mail: m.p.oksvold{at}labmed.uio.no )
Accepted 1 November 2001
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Summary |
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Our work indicates that UV induces internalisation of EGFR independent of its phosphorylation or receptor tyrosine kinase activation, and altered EGFR trafficking compared with ligand-activated receptor. In addition, MAPK activation by UV does not appear to be mediated by EGFR activation.
Key words: UV, EGF receptor, Receptor internalisation, Intracellular trafficking
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Introduction |
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The epidermal growth factor receptor (EGFR) is dimerised and becomes
tyrosine phosphorylated on five tyrosine autophoshorylation sites in response
to growth factors (Moghal and Sternberg,
1999). In their phosphorylated state the five cytosolic C-terminal
autophosphorylation sites (Y992, Y1068, Y1086, Y1148 and Y1173)
(Downward et al., 1984
;
Margolis et al., 1989
;
Walton et al., 1990
) serve as
binding sites for a large group of signal proteins, including Shc and Grb2,
which initiate the Ras-Raf-MEK-MAPK signalling pathway
(Carpenter, 2000
;
Moghal and Sternberg,
1999
).
Increased EGFR tyrosine phosphorylation after UV-exposure has previously
been reported in HeLa and A431 cells
(Sachsenmaier et al., 1994),
and HER14 and A431 cells (Coffer et al.,
1995
). Also, UV-induced EGFR complex formation with Shc, Grb2, Sos
and PLC
has been reported in HC11 cells
(Huang, R. P. et al., 1996
).
The mechanism for the UV-induced activation of the EGFR is not known. Huang
and co-workers have suggested that UV-induced EGFR tyrosine phosphorylation
involves formation of reactive oxygen species (ROS)
(Huang, R. P. et al., 1996
).
In favour of this hypothesis, they demonstrated that the ROS scavenger
N-acetyl cysteine (NAC) blocked UV-mediated receptor tyrosine phosphorylation.
It was recently reported that ROS disturb specific cysteine-containing
sequences of tyrosine phosphatases, thereby inhibiting their function
(Gross et al., 1999
). It has
also been suggested that UV induces post-translational modifications of EGFR
by direct physical perturbation of the plasma membrane, or by a conformational
change caused by absorption of UV energy
(Rosette and Karin, 1996
;
Huang, R. P. et al.,
1996
).
After growth factor binding to EGFR, the receptor is internalised through
clathrin-coated pits, distributed to early endosomes and either recycled back
to the cell surface, or transported to late endosomes and lysosomes for
degradation. Whereas internalisation of EGFR induced by UV has been reported
(Rosette and Karin, 1996), the
trafficking of UV-exposed EGFR compared with ligand-activated EGFR has never
been analysed in detail. The function of EGFR tyrosine kinase activity in
receptor internalisation and routing is still not fully understood. It has
been reported that the EGFR kinase activity is required for ligand-induced
receptor internalisation (Wiley et al.,
1991
). However, the role of induced internalisation versus
lysosomal targeting for EGFR downregulation is still unclear. Whereas Wiley
and co-workers have suggested that receptor degradation is primarily regulated
by endocytosis (Wiley et al.,
1991
), others have proposed that inhibition of receptor recycling
is the mechanism that regulates receptor degradation
(Felder et al., 1990
). It has
been suggested that EGFR autophosphorylation results in exposure of
trafficking sequences directly (Cadena et
al., 1994
), or indirectly through formation of a
receptor-substrate complex (Wiley et al.,
1991
; Chang et al.,
1993
). The aim of this study was to compare EGF- and UV-induced
EGFR activation and trafficking.
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Materials and Methods |
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Cells
The human HeLa epidermal carcinoma cell line was obtained from American
Tissue Type Collection (Rockville, MD). Cells were maintained in DMEM
(BioWhittaker, Walkersville, MD) supplemented with 5% (v/v) fetal calf serum
(BioWhittaker), 2 mM L-glutamine and 50-ng/ml gentamycin (Gibco BRL). Cells
were grown to 80% confluency in petri dishes or flasks (Costar Corp.,
Cambridge, MA), and starved in serum-free medium overnight before the start of
the experiment. UV exposure of cells was performed in a Vilber Lourmat BLX-254
(Marne La Vallée, France) with 10-200
J/m2 UV 254 nm. The medium was removed before UV irradiation, and
replaced before further incubations. Tyrosine phosphorylation of EGFR was
induced with EGF (5 nM in Hanks balanced salt solution, pH 7.4 (Gibco BRL)) on
ice for 15 minutes. Cells were then washed in ice cold PBS, incubated in
pre-warmed medium and chased at 37°C for different time intervals.
Western blot analysis
Cells were lysed in Tris lysis buffer, pH 7.4 (10 mM Tris-HCl, 10% (v/v)
glycerol, 2% (w/v) SDS, 5 mM EDTA, 2.5 µg/ml aprotinin, 2.5 µg/ml
leupeptin, 1 mM ß-glycerophosphate, 4-(2-aminoethyl)benzenesulfonyl
fluoride (AEBSF) and 60 µM sodium orthovanadate). Further sample
preparations were performed as described elsewhere
(Oksvold et al., 2000). The
nitrocellulose membranes were incubated overnight at 4°C with primary
antibodies. Peroxidase-conjugated donkey anti-sheep or mouse IgG and goat
anti-rabbit IgG were used for detection. All antibodies were diluted in 1%
(w/v) nonfat dry milk in TBS containing 0.01% thimerosal. The filters were
washed in TBS before detection by the enhanced chemiluminescence (ECL) method
with Hyperfilm (Amersham Pharmacia).
Immunoprecipitation
Whole cell lysates (2 mg protein/ml) from UV or EGF-exposed cells were
prepared in IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 50 mM NaF, 10 mM
ß-glycerophosphate, 0.5% (v/v) NP-40, 1 mM EDTA, 1 mM orthovanadate, 1 mM
DTT, 1 mM PMSF, 1 mM benzamidine, trypsin inhibitor (10 µg/ml), and
aprotinin and leupeptin (2.5 µg/ml)). The lysates were passed four times
through a 25G needle, and homogenised by 15 strokes in a dounce homogenizer.
Sheep anti-EGFR or rabbit anti-Cb1 (7 µg) were conjugated to 50 µl
protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) by incubation for 1
hour at room temperature. The anti-EGFR-protein G-Sepharose conjugate was
incubated with 100 µl cell lysates for 2 hours on ice. The pellets were
washed three times in IP-buffer, before resuspension in 100 µl Tris lysis
buffer containing 2% ß-mercaptoethanol and 0.002% bromophenol blue, and
heated at 100°C for 5 minutes. Immunoprecipitates were separated by
SDS-PAGE, and prepared for autoradiography or processed for immunoblotting as
described above.
In vivo labelling of EGFR
Cells were washed with phosphate-free MEM, and incubated in the same buffer
for 2 hours at 37°C, before incubation with [32P]orthophosphate
(0.2 mCi/ml) for 2 hours. Cells were exposed to UV (50 or 200 J/m2)
or EGF (5 nM) and lysated directly, or incubated at 37°C for 10 minutes.
The cells were rinsed in ice cold PBS, followed by lysation in Tris-lysis
buffer. EGFR was immunoprecipitated as described earlier. After separation of
the immunoprecipitated EGFR by SDS-PAGE, the gel was rinsed in TBS, and
incubated in 0.1% Coomassie blue R-250 in 40% methanol and 7% acetic acid for
15 minutes. The gel was washed in 40% methanol and 7% acetic acid, rinsed in
TBS and analysed for autoradiography using a Molecular Imager FX (Bio-Rad,
Hercules, CA).
Treatment with alkaline phosphatase
Immunoprecipitated EGFR from unstimulated, UV- or EGF-exposed cells were
incubated with 50 diethanolamine (DEA) units of alkaline phosphatase from
bovine intestinal mucosa in 150 µl DEA-buffer (1 M DEA, pH 9.8, containing
0.5 mM MgCl2) at 37°C for 30 minutes.
Crosslinking of the EGFR
Cells were either exposed to UV at 37°C and incubated for 2 minutes, or
rinsed in PBS and exposed to UV or EGF in HBSS on ice and further incubated
for 15 minutes on ice. Cells were rinsed in PBS, and treated with 3 mM of the
nonpermeable crosslinking agent bis(sulphosuccinimidyl) suberate
(BS3) (Pierce, Rockford, IL) in PBS for 20 minutes on ice. The
reaction was stopped by incubation in PBS with 250 mM glycine for 5 minutes on
ice. The cells were washed in PBS with 250 mM glycine, lysed in Tris lysis
buffer, and prepared for western immunoblotting as described elsewhere
(Oksvold et al., 2000). The
lysates were subject to SDS-PAGE and immunoblotting with an antibody to EGFR,
and further prepared as described above.
Immunofluorescence staining and microscopy
Cells exposed to UV or EGF were fixed in 4% paraformaldehyde in PBS for 20
minutes at room temperature and prepared for immunocytochemistry as described
elsewhere (Oksvold et al.,
2000). Combinations of Cy2 and rhodamine Red-X-conjugated donkey
antibodies monospecific for IgG of the appropriate species were used. The
samples were mounted with coverslips using Dako fluorescent mounting kit
(Dako, Carpinteria, CA). The cells were examined with a Leica TCS SP confocal
microscope (Leica, Heidelberg, Germany) equipped with an Ar (488 nm) and two
He/Ne (543 and 633 nm) lasers. A Plan apochromat 100x/1.4 oil objective
was used. Images from multi-labeled cells were acquired sequentially.
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Results |
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To verify that UV-exposure did not induce an increased tyrosine phosphorylation of the EGFR, we examined the total phosphorylation of EGFR by in vivo labelling with 32P orthophosphate. Immunoprecipitated EGFR from UV-irradiated cells was analysed by SDS-PAGE and autoradiography. No increased phosphorylation was detected in the EGFR immunoprecipitates from cells exposed to 50-200 J/m2 UV and homogenised immediately, or after 10 minutes incubation (Fig. 2A). By contrast, EGFR immunoprecipitated from EGF-stimulated cells showed increased phosphorylation. Western immunoblotting analysis of the 32P-labelled EGFR immunoprecipitates from UV- and EGF-treated cells with anti-EGFR confirmed similar levels of EGFR in the different immunoprecipitates (Fig. 2B).
|
Also, two other stress factors and their effects on EGFR tyrosine phosphorylation were examined. Both hydrogen peroxide and heat shock induced tyrosine phosphorylation of the EGFR, as revealed by western immunoblotting of whole cell lysates (data not shown).
The results from HeLa cells were reproduced in A431 and PC12 cells, and primary cultures of hepatocytes from Sprague-Dawley rats (data not shown). In addition, EGF dose-response studies showed that 0.05 nM EGF induced EGFR tyrosine phosphorylation detectable by immunoblotting (data not shown).
EGFR from UV-exposed cells is not dimerised and displays an
electrophoretic mobility shift associated with receptor internalisation
without degradation
To examine whether UV-exposure induced EGFR dimerisation, we used the
chemical crosslinking agent BS3 to preserve dimerised receptors
(Sorkin and Carpenter, 1991).
UV- or EGF-treated cells were incubated with BS3 before lysis of
the cells. The cell lysates were analysed by SDS-PAGE and western
immunoblotting with antibody to EGFR. In cells stimulated with EGF (10 nM) for
15 minutes on ice, increased dimerisation of the EGFR was observed by a 340
kDa band (Fig. 3, arrow). By
contrast, cells exposed to UV (200 J/m2) and incubated for 2
minutes at 37°C, or exposed to UV on ice and further incubated on ice for
15 minutes, showed no increased dimerisation
(Fig. 3).
|
It has earlier been suggested that UV induces a conformational change of
the EGFR (Rosette and Karin,
1996; Huang, R. P. et al.,
1996
). As shown in Fig.
1A, we observed a shift in the electrophoretic mobility of EGFR
from cells exposed to UV compared with untreated cells. When irradiated with
10 J/m2, gel retardation of the receptor was not observed. With 50
J/m2, a mobility shift was seen 10 minutes, but not 20 minutes,
after UV-exposure. Irradiation with 200 J/m2 induced
electrophoretic retardation more rapidly and prolonged compared with lower
exposure levels (Fig. 1A,
EGFR). The UV-induced EGFR mobility shift was not an immediate event since it
was not observed less than 1 minute after UV-treatment. The EGFR gel mobility
shift was seen 5 minutes after irradiation, and was present even 2 hours after
exposure (Fig. 4A). In
addition, while degradation of the EGFR was observed in EGF-stimulated cells
incubated for 2 hours, EGFR-degradation was not detected in UV-exposed cells
(Fig. 4A).
|
To exclude the possibility that the UV-induced electrophoretic mobility shift of EGFR was due to phosphorylation not detectable by immunoblotting and 32P-labelling methods, we treated UV- or EGF-exposed EGFR with alkaline phosphatase. Incubations of EGFR-immunoprecipitates from EGF-stimulated cells with alkaline phosphatase effectively dephosphorylated the receptor (Fig. 4B, pY1173). However, the UV-induced EGFR mobility shift in SDS-PAGE was still present after treatment with alkaline phosphatase (Fig. 4B, EGFR).
Polyubiquitination, in addition to other functions, is a signal for
degradation by the 26S proteasome (Dubiel
and Gordon, 1999). Since polyubiquitination leads to
electrophoretic mobility shift of targeted proteins, we compared EGFR
polyubiquitination in UV- and EGF-exposed cells. Whole cell lysates from EGF-
and UV-treated cells incubated for the indicated time intervals were analysed
by western immunoblotting with anti-EGFR. In western immunoblots,
polyubiquitination can be detected by gel retardation observed as smears, as
previously shown in HeLa cells (Stang et
al., 2000
). EGF-stimulated EGFR appeared polyubiquitinated from
1-10 minutes after incubation at 37°C
(Fig. 5A). In comparison, no
ubiquitination was observed in cells exposed to 200 J/m2 UV
(Fig. 5A). It has previously
been shown that EGFR polyubiquitination is dependent of tyrosine
phosphorylation (Levkowitz et al.,
1998
).
|
To further compare EGFR ubiquitination in UV- and EGF-exposed cells, we
studied the product of the proto-oncogene c-Cbl, which binds activated protein
tyrosine kinases such as EGFR, and regulates their polyubiquitination
(Waterman et al., 1999).
Increased tyrosine phosphorylation of Cbl was not observed in
Cbl-immunoprecipitates from cells exposed to UV
(Fig. 5B). In addition, complex
formation between EGFR and Cbl was not observed
(Fig. 5B).
Cbl-immunoprecipitates from EGF-treated cells showed increased Cbl tyrosine
phosphorylation, and complex formation between tyrosine phosphorylated EGFR
and Cbl (Fig. 5B).
It has previously been described that, similar to EGF, UV induces
internalisation of the EGFR (Rosette and
Karin, 1996). We therefore studied the effects of UV on EGFR
distribution. Whereas EGFR in unstimulated cells are located in the plasma
membrane, we have previously described that the activated receptor was located
in EEA1-positive early endosomes 10 minutes after EGF stimulation
(Oksvold et al., 2000
). In
unstimulated cells EGFR was located in the plasma membrane, as seen with
immunofluorescence confocal microscopy
(Fig. 6A). We did not observe
internalisation of EGFR in cells exposed to 10 J/m2 UV and
incubated for 10 minutes (data not shown). When irradiated with 50
J/m2, some EGFR internalisation was seen (data not shown). After
exposure to 200 J/m2, an extensive receptor internalisation
comparable with that seen after EGF-stimulation was observed
(Fig. 6B). The internalised
EGFR was located to early endosomes, as shown by co-localisation with EEA1
(Fig. 6B-D).
|
To exclude the possibility that the observed UV-induced internalisation of EGFR was due to a general increased inward membrane flow, we compared the liquid phase endocytosis of horseradish peroxidase (HRP) in unstimulated and UV-exposed cells. We observed a decrease in the internalisation of HRP in UV-exposed cells compared with that in unstimulated cells (data not shown).
We have earlier reported that EGFR was relocated to CD63 and
LAMP-1-positive late endosomes in EGF-stimulated HeLa cells incubated for 20
minutes (Oksvold et al.,
2001). EGFR was not detected in CD63-positive compartments of
UV-exposed cells incubated for the same time interval
(Fig. 7A-C). UV-exposed cells
incubated for 20-120 minutes showed no colocalisation of EGFR and the early
(EEA1) and late (CD63 and LAMP-1) endosome markers (data not shown). To
determine whether the compartments containing internalised EGFR 60-120 minutes
after UV-exposure were part of the endocytic pathway, we studied the
accessibility of these compartments for transferrin. Transferrin bound to the
transferrin receptor is transported from the cell surface to early endosomes,
from where it recycles back to the cell surface
(Lok and Loh, 1998
). In
UV-irradiated cells chased for 60 minutes and co-incubated with FITC-labeled
transferrin (50 µg/ml) the last 20 minutes, EGFR was located in
transferrin-positive vesicles (Fig.
7D-F). Aggregates of compartments containing EGFR and transferrin
were observed close to the cell surface
(Fig. 7D-F, arrow). These
aggregates were seen in the majority of the cells studied (data not shown). In
EGF-stimulated cells incubated for 60-120 minutes, little or no EGFR was
located in vesicles (data not shown). This finding was in agreement with our
previous result, which showed EGF-induced degradation of EGFR, but little or
no degradation in UV-exposed cells (Fig.
4A).
|
It has earlier been suggested that the reported UV-induced activation of
EGFR is due to production of ROS (Huang,
R. P. et al., 1996). We therefore studied the effects of ROS
scavengers on the UV-induced EGFR gel mobility shift and internalisation.
Treatment with the ROS scavenger NAC (10 mM) inhibited
H2O2-induced tyrosine phosphorylation of the receptor,
observed by western blotting with anti-pY1173 (data not shown). However,
incubation with NAC for 15 minutes to 12 hours before exposure to UV showed
minor effects on both the EGFR gel mobility shift and receptor internalisation
(data not shown). These results were confirmed by use of two other known ROS
scavengers,
-tocopherol and L-ascorbic acid (data not shown).
UV-induced EGFR internalisation is independent of receptor tyrosine
kinase activity
It has previously been reported that EGFR kinase activity is required for
ligand-induced sequestration of receptors into coated pits
(Lamaze and Schmid, 1995) and
for EGFR internalisation (Wiley et al.,
1991
). In order to test whether UV-induced EGFR internalisation
was dependent on receptor tyrosine kinase activity, we used the specific EGFR
tyrosine kinase inhibitor PD153035 (Kunkel
et al., 1996
) prior to UV exposure. Incubation with 100 nM
PD153035 for 2 hours effectively inhibited EGF-induced EGFR tyrosine
phosphorylation (Fig. 8, upper
panel). The EGF-induced internalisation of the receptor was also inhibited in
cells incubated with PD153035, as shown by immunofluorescence confocal
microscopy (Fig. 8A-C). In
UV-exposed cells, inhibition of the EGFR tyrosine kinase with PD153035 had no
effect on the internalisation of EGFR (Fig.
8D). Our result showed that UV-induced EGFR internalisation was
independent of the receptor tyrosine kinase activity.
|
EGF-EGFR complexes are internalised by clathrin-coated pit-mediated
endocytosis. To examine whether UV-irradiated EGFR follows the same pathway,
we used the cholesterol-extracting drug ß-cyclodextrin to inhibit
clathrin-coated pitmediated endocytosis. Recently it was reported that
ß-cyclodextrin treatment strongly inhibited endocytosis of transferrin
and EGF in HEp-2 and other cell lines
(Rodal et al., 1999).
Incubation with 15 mM ß-cyclodextrin revealed strong inhibition of both
EGF-mediated EGFR internalisation, and UV-induced EGFR internalisation (data
not shown). We also studied the effects of ß-cyclodextrin on the
UV-induced EGFR mobility-shift in SDS-PAGE. The UV-induced electrophoretic
retardation of EGFR was similar with and without ß-cyclodextrin,
suggesting that the receptor mobility shift was not dependent on
internalisation (data not shown).
Effects of UV on signal proteins downstream of EGFR
To examine the effects of UV-exposure on intracellular signal transduction
downstream of EGFR, we studied the effects of UV on the activation of the
signalling proteins Shc, Grb2, Raf, MEK and MAPK. The adaptor proteins Shc and
Grb2 bind to the activated tyrosine autophosphorylation sites on the EGFR
after growth factor activation, and initiates the Ras-Raf-MEK-MAPK signalling
cascade. In unstimulated cells, Shc and Grb2 showed a diffuse distribution
throughout the cytosol (Fig. 9A and
B, respectively). Their association with centrosomes was seen as
co-localisation with the centrosome marker CRT453 (data not shown). In
response to EGF for 10 minutes, a redistribution of Shc and Grb2 to vesicles
was observed (Fig. 9C and D,
respectively). These vesicles contained both EGFR and EEA1 (data not shown).
In cells exposed to UV, no redistribution of Shc and Grb2 was found
(Fig. 9E and F, respectively).
Next, we analysed the activation of Raf and MEK in UV-irradiated cells. To
sort out whether inactivation of the EGFR tyrosine kinase had any effect on
this activation, we studied UV-induced activation of Raf and MEK with and
without PD153035. The MEK effector Raf is translocated by Ras to the plasma
membrane, where it is activated (Morrison
and Cutler, 1997). Immunoblotting of lysates from EGF-stimulated
cells with anti-Raf-1 showed a Raf gel mobility shift in SDS-PAGE, indicating
an EGF-induced phosphorylation of Raf
(Fig. 10A, Raf). The induced
Raf phosphorylation was inhibited by pretreatment with the EGFR tyrosine
kinase inhibitor PD153035 (Fig.
10A, Raf). In UV-exposed cells no gel mobility shift for Raf was
found, indicating that UV does not induce the Raf phosphorylation
(Fig. 10A, Raf).
Immunoblotting with anti-phosphorylated MEK revealed activation of MEK in
response to both UV and EGF (Fig.
10A, pMEK). The EGF-induced activation of MEK was effectively
inhibited in cells preincubated with PD153035
(Fig. 10A, pMEK). By contrast,
UV-induced activation of MEK was similar with and without PD153035. Whereas 10
and 50 J/m2 UV did not induce MEK-activation, high doses (200
J/m2) induced strong activation 20 minutes after irradiation
(Fig. 10B, pMEK). Activated
ERK 1/2 was observed in cells exposed to 50 J/m2 UV and incubated
for 5-10 minutes, but was no longer phosphorylated 20 minutes after
irradiation (Fig. 10B, pERK).
However, in cells exposed to 200 J/m2, ERK 1/2 activation was found
after incubation for 20 minutes (Fig.
10B, pERK). To find out whether activation of ERK was dependent of
MEK activation, we studied ERK phosphorylation in UV- and EGF-exposed cells
with and without the specific MEK inhibitor PD98059. UV- and EGF-induced
activation of ERK was totally inhibited in cells treated with PD98059 (data
not shown).
|
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![]() |
Discussion |
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We applied 10-200 J/m2 UVC 254 nm, similar to that used in previous studies reporting UV-induced EGFR tyrosine phosphorylation. Our finding that UV-exposure did not induce EGFR tyrosine phosphorylation was supported by a set of further observations: (1) UV did not induce increased incorporation of [32P]; (2) treatment of UV-exposed EGFR immunoprecipitates with alkaline phosphatase did not eliminate the UV-induced mobility shift in SDS-PAGE; (3) UV-exposure did not induce EGFR tyrosine kinase-dependent polyubiquitination of EGFR; (4) Inhibition of the EGFR tyrosine kinase revealed no effect on the UV-induced receptor gel mobility shift and internalisation of EGFR; (5) UV-exposure did not induce redistribution of Shc and Grb2 to vesicles containing EGFR, indicating lack of specific receptor phosphotyrosine-containing Shc and Grb2 binding sites.
It has previously been suggested that direct absorption of UV energy
induces an EGFR conformational change
(Rosette and Karin, 1996;
Huang, R. P. et al., 1996
). We
found by dose-response studies that there was a correlation between increasing
UV-doses and induced EGFR gel mobility shift. This observation suggested
either a UV-induced receptor conformational change, or a post-translational
modification of EGFR, directly or via other signal proteins. Although a
conformation alteration is not necessarily detectable by SDS electrophoresis,
the fact that the UV-induced mobility shift for EGFR was intact under reduced
conditions by western immunoblotting analysis indicated the involvement of an
unknown post-translational modification. Huang and co-workers have recently
suggested that ROS are involved in the UV-mediated activation of EGFR
(Huang, R. P. et al., 1996
).
We were not able to inhibit the UV-induced EGFR gel mobility-shift and
receptor internalisation by treatment with ROS scavengers. We therefore find
it unlikely that ROS have an important role in the UV-induced modification and
internalisation of EGFR.
Our finding that EGFR was internalised without phoshorylation and
independent of receptor tyrosine kinase activity was surprising. The
UV-induced EGFR gel mobility shift may serve as a signal for internalisation,
directly or indirectly through other signal mediators. It has been reported
earlier that EGF binding induces a conformational change in the external
domain of its receptor (Greenfield et al.,
1989). Opresko and co-workers have suggested that a conformational
change in the EGFR is important for ligand-induced internalisation of the
receptor (Opresko et al.,
1995
). It is possible that an EGFR modification induced by UV
resembles the receptor modification seen after ligand binding. This was
supported by our finding that treatment of EGFR immunoprecipitates from
EGF-stimulated cells with alkaline phosphatase eliminated tyrosine
phosphorylation, but not the EGFR mobility shift. In addition, inhibition of
clathrin-coated pit-mediated endocytosis with ß-cyclodextrin showed no
inhibitory effect on the EGFR mobility shift in SDS-PAGE. This supports our
hypothesis that the UV-induced EGFR modification observed as a receptor gel
mobility shift, in some way mimics an EGF-mediated internalisation signal.
Whereas polyubiqutination did not seem to be involved in UV-induced EGFR
internalisation, there is a possibility that monoubiqutination played a role.
We failed to detect monoubiqutination by immuno-coprecipitation studies.
However, it is generally difficult to detect monoubiquitin, and for that
reason we cannot exclude the possible involvement of monoubiqutination. The
findings that a single ubiquitin can serve as a signal for sorting to the
degradative pathway (Urbanowski and Piper,
2001), do not support a role for monoubiqutination in UV-induced
internalisation of EGFR.
We have shown that UV-exposed EGFR was internalised independently of
receptor dimerisation and its receptor tyrosine kinase activity. The
involvement of the EGFR tyrosine kinase activity. The involvement of the EGFR
tyrosine kinase for receptor internalisation and intracellular sorting has
been intensively investigated in the past years. Whereas receptor kinase
activity has been shown to be required for ligand-activated internalisation of
EGFR (Chen et al., 1989;
Lund et al., 1990
;
Wiley et al., 1991
;
Lamaze and Schmid, 1995
), its
role in intracellular receptor trafficking is still unclear. One hypothesis is
that endosomal sorting is controlled by tyrosine kinase activity, and that the
EGFR tyrosine kinase is necessary for trafficking to lysosomes for degradation
(Honegger et al., 1987
;
Honegger et al., 1990
;
Felder et al., 1990
;
Felder et al., 1992
;
Futter et al., 1993
). Felder
and co-workers found that the distribution to internal vesicles of
multivesicular bodies is inhibited in EGFR with a mutated tyrosine kinase,
suggesting that the receptor tyrosine kinase is important for trafficking to
lysosomes for degradation (Felder et al.,
1990
). Others have found that tyrosine kinase activity is not
crucial for lysosomal targeting (Sorkin et
al., 1991
; Helin and Beguinot,
1991
; Sorkin and Waters,
1993
; Wiley et al.,
1991
; Lamaze et al.,
1993
; French et al.,
1994
; Herbst et al.,
1994
; Opresko et al.,
1995
). These studies support the model originally introduced by
Linderman and Lauffenburger in which selective sorting occurs by endosomal
retention (Linderman and Lauffenburger,
1988
). Recently, a leucinebased lysosomal sorting signal was found
in the EGFR (Kil et al.,
1999
).
Whereas UV-exposed EGFR internalisation was similar to ligand-mediated internalisation, the receptor trafficking in UV-irradiated cells was altered. We did not observe degradation of EGFR in UV-exposed cells. The receptor was not transported from early endosomes to late endosomes and lysosomes, but instead redistributed to compartments accessible to transferrin, where it was arrested. We presume that these compartments were recycling vesicles. We have two possible explanations for the altered receptor trafficking in UV-exposed cells: (1) lysosomal targeting of EGFR is dependent on an active receptor tyrosine kinase, or intact tyrosine phosphorylated autophosphorylations sites; (2) the endocytic transport machinery is in some way abrogated by UV-irradiation.
It has been suggested that UV-induced activation of MEK and MAPK mimics the
ligand-induced signalling pathway that initiates from EGFR
(Huang, R. P. et al., 1996).
Our results reveal that the UV-mediated activation of MEK and MAPK occurs
independently of tyrosine phosphorylated EGFR and receptor binding of Shc and
Grb2. Other studies yield support to our findings. Migliaccio and co-workers
reported UV-induced gel mobility shift for p66 Shc, without tyrosine
phosphorylation (Migliaccio et al.,
1999
). Furthermore, it has been shown that UV-induced AP-1
activation does not require EGFR (Huang,
C. et al., 1996
).
In conclusion, our work demonstrated that the UV-induced internalisation of EGFR was independent of its receptor tyrosine kinase. The trafficking of UV-exposed EGFR was altered compared with ligand-activated receptor, and the internalised receptor was not directed to lysosomes for degradation. In addition, the UV-induced activation of MEK and MAPK was independent of an active EGFR tyrosine kinase.
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Acknowledgments |
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