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Suppression of EGFR endocytosis by dynamindepletion reveals that
EGFR signaling occursprimarily at the plasma membraneLeiliane P.
Sousaa, Irit Laxa, Hongying Shenb,c, Shawn M. Fergusonb, Pietro De
Camillib,c, and Joseph Schlessingera,1
aDepartments of Pharmacology; bCell Biology; and cHoward Hughes
Medical Institute, Yale University School of Medicine, New Haven,
CT 06520
Contributed by Joseph Schlessinger, January 5, 2012 (sent for
review December 13, 2011)
The role of endocytosis in the control of EGF receptor (EGFR)
acti-vation and cell signaling was explored by using mouse
fibroblastsin which dynaminwas conditionally depleted. Dynamin is a
GTPaseshown to play an important role in the control clathrin
mediatedendocytosis of EGFR and other cell surface receptors. In
this report,we demonstrate that EGF binding activity and the
display of highand low affinity EGFRs on the cell surface are not
affected by dy-namin depletion. By contrast, dynamin depletion
leads to a stronginhibition of EGFR endocytosis, robust enhancement
of EGFR au-tophosphorylation and ubiquitination, and slower
kinetics of EGFRdegradation. Surprisingly, MAPK stimulation induced
by either lowor high EGF concentrations is not affected by dynamin
depletion.While a similar initial Akt response is detected in
control or dyna-min depleted fibroblasts, a somewhat more sustained
Akt stimula-tion is detected in the dynamin depleted cells. These
experimentsdemonstrate that dynamin-mediated endocytosis leads to
attenua-tion of EGFR activation and degradation and that
stimulation ofthe MAPK response and Akt activation are primarily
mediated byactivated EGFR located in the plasma membrane.
membrane receptors ∣ tyrosine kinases
Epidermal growth factor receptor (EGFR) and other
receptortyrosine kinases (RTK) undergo rapid internalization and
de-gradation following ligand induced activation (1–3). At low
phy-siological concentrations, EGF induced EGFR internalization
isprimarily mediated by clathrin mediated endocytosis; a
processblocked by siRNA silencing of clathrin heavy chain
expressionor by overexpression of a dominant interfering dynamin
mutant(K44) (4–6). By contrast, when high EGF concentrations are
ap-plied, it is proposed that EGFR endocytosis is primarily
mediatedby clathrin-independent mechanisms (3, 7, 8).
It was initially thought that the function of EGFR
endocytosisand degradation was to terminate the signal initiated by
EGFbinding to EGFRs located at the plasma membrane (1, 2).
Sub-sequent studies demonstrated that EGFR and other RTKs, arealso
capable of recruiting signaling molecules and transmittingsignals
from endosomes (7, 9, 10). Moreover, it was proposed thatfollowing
EGFR activation clathrin mediated endocytosis playsan important
regulatory role in control of sustained activation ofthe MAPK/ERK
signaling pathway and in Akt stimulation (4,11). The current
prevailing view is that signals induced by acti-vated EGFR and
other RTKs can be transmitted from the plasmamembrane as well as
from endosomes and that the spatial loca-lizations of RTKs play an
important role in the control of signalspecificity, duration, and
robustness (4, 9–12).
Dynamin is large GTPase, that mediates the endocytic fissionof
coated pits (13, 14). In this report we describe the analysis ofthe
role played by dynamin in EGFR activation, endocytosis, andcell
signaling. Using dynamin conditional knockout mouse fibro-blasts we
demonstrate that the ligand binding characteristics andthe typical
display of high and low affinity EGFR binding sites onthe cell
surface are maintained and not affected by dynamin de-pletion.
However, dynamin depletion leads to a strong inhibition
of EGFR endocytosis that is accompanied by enhanced
tyrosineautophosphorylation, enhanced and prolonged EGFR
ubiquiti-nation, and reduced EGFR degradation. We also
demonstratethat depletion of dynamin expression results in
selective enhance-ment in tyrosine phosphorylation of the p66
isoform of the adap-tor protein Shc. Moreover, while MAPK
stimulation inducedby low or high concentrations of EGFare not
affected by dynamindepletion, a somewhat more sustained Akt
activation is observedin these cells. These experiments demonstrate
that dynamin-mediated endocytosis results in attenuation of EGFR
activation,autophosphorylation, and ubiquitination as well as in
enhancedEGFR degradation. Furthermore, stimulation of the MAPK/ERK
pathway and Akt activation can be effectively activated byEGFR
located in the plasma membrane.
ResultsThe role of dynamin in the control of EGFR activation,
endocy-tosis, and downstream signaling was explored by using
fibroblastsderived from Dnm1 flox∕flox; Dnm2 flox∕flox; Cre-Esr1þ ∕
−mice(15, 16) following tamoxifen-induced gene recombination
invitro. The Dnm1 and Dnm2 genes encode for dynamin 1 and
2,respectively, both of which are expressed in murine
fibroblastswhile the third dynamin isoform, dynamin 3, is largely
undetect-able in these cells. We therefore refer to these cells as
dynamindepleted cells, or dynamin knockout cells (DKO).
Ligand Binding Activity of EGFR Is Not Influenced by Dynamin
Deple-tion. In order to evaluate the effect of dynamin depletion on
theligand binding characteristics of cell surface EGFRs, murine
fi-broblasts were either pretreated with 4-hydroxy-tamoxifen or
buf-fer alone and then subjected to quantitative binding
experimentswith 125I-EGF. The effect of dynamin depletion on the
ligandbinding characteristics of EGFR were determined by
comparingdisplacement curves or Scatchard analyzes (17–19) of
quantita-tive 125I-EGF binding experiments to live cells (Fig. 1).
To con-firm that dynamin 1 and 2 were indeed depleted by
tamoxifentreatment, samples of tamoxifen treated or untreated cells
werelysed and subjected to immunoblotting with anti-dynamin
antibo-dies (Fig. 1B). Control or dynamin depleted fibroblasts were
in-cubated with 5 ng∕mL of 125I-EGF for 1 h at room temperaturein
the presence of increasing concentrations of unlabeled EGF.The
treated cells were lysed and the radioactive contents of thesamples
were determined using a scintillation counter. The ex-periment
presented in Fig. 1A depicts displacement curves of125I-EGF binding
to control fibroblasts or to dynamin depletedfibroblasts. This
experiment reveals similar 125I-EGF displace-
Author contributions: L.P.S., I.L., H.S., S.M.F., P.D.C., and
J.S. designed research; L.P.S. andI.L. performed research; H.S.,
S.M.F., and P.D.C. contributed new reagents/analytic tools;L.P.S.,
I.L., H.S., S.M.F., P.D.C., and J.S. analyzed data; and L.P.S.,
I.L., H.S., S.M.F., P.D.C.,and J.S. wrote the paper.
The authors declare no conflict of interest.1To whom
correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1200164109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1200164109 PNAS ∣ March 20,
2012 ∣ vol. 109 ∣ no. 12 ∣ 4419–4424
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ment curves for control and dynamin depleted fibroblasts(Fig.
1B) with concentrations required for 50% inhibition, IC50of 0.62
and 0.40 nM, respectively. This experiment demonstratesthat the
display of EGFR on the plasma membrane and the over-all binding
affinity of EGFR towards EGF was not affected bydynamin loss. A
similar conclusion was reached from Scatchardanalysis of
quantitative binding experiments with concentrationsranging from
0.1 to 100 ng∕mL 125I-EGF to control or dynamindepleted
fibroblasts. Similar numbers of EGFRs were present onthe cell
surfaces of control and dynamin depleted cells and bothcells
displayed a typical pattern of low and high affinity EGFRson their
cell surfaces (Fig. 1C). It is noteworthy that under theconditions
in which the 125I-EGF binding experiments were per-formed a
substantial fraction of bound 125I-EGF molecules be-come
internalized into dynamin depleted cells and even moreinto WTcells
indicating that the profile of EGF binding charac-teristic is not
sensitive to EGFR internalization. These experi-ments support the
conclusion that the display of EGFR on thecell surface and the
ligand binding characteristics of EGFRsare not affected by dynamin
loss. However, these conclusionscontradict an earlier study
demonstrating that overexpressionof a dominant interfering dynamin
mutant (K44A) prevent highaffinity EGF binding and reduces EGF
stimulation of EGFR au-tophosphorylation (20).
Endocytosis of EGFR Is Strongly Impaired in Dynamin Depleted
Cells. Itwas previously reported that clathrin-mediated endocytosis
isthe primary route for internalization of EGFR at low EGF
con-centrations (1.5 ng∕mL), while at higher ligand
concentrationsEGFR internalization may occur via both
clathrin-dependent andclathrin-independent mechanisms (4, 8). To
investigate whetherthe absence of dynamin affects EGFR
internalization, we next ana-lyzed the kinetics of internalization
of 125I-EGF using a previouslydescribed, well established
quantitative procedure (21, 22). To thisend, control or dynamin
depleted fibroblasts were incubated witheither low (1.5 ng∕mL) or
high (100 ng∕mL) 125I-EGF concentra-tions for 90 min at 4 °C. At
the end of the incubations, the cellswere washed and further
incubated at 37 °C for different times. Toquantitatively determine
the amount of EGFRs located at the cellmembrane, the cells were
subjected to a mild acid wash at differenttime points to
selectively release only cell surface bound 125I-EGF
molecules into the medium (21). The washed cells were then
so-lubilized and the radioactive contents of the cell surface bound
andthe internalized 125I-EGF molecules were separately
determinedusing a scintillation counter (21).
The experiments presented in Fig. 2 A–H show that endocyto-sis
of EGFR is strongly impaired in dynamin depleted fibro-blasts. The
results show that after 15 min most cell surface EGFRin control
fibroblasts became internalized in response to stimula-tion with
either low or high EGF concentrations (Fig. 2 A–C andFig. 2 E–G).
In contrast, a robust inhibition of EGFR internali-zation was
detected in dynamin depleted fibroblasts stimulatedwith 1.5 ng∕mL
(low dose) of EGF (Fig. 2B). Internalizationof EGFR was also
compromised in dynamin depleted cells stimu-lated with 100 ng∕mL
(high dose) of EGF (Fig. 2E). Under theseconditions approximately
70% inhibition of EGFR internaliza-tion was observed in dynamin
depleted cells (Fig. 2G). These ex-periments also show that EGFR
internalization is not completelyblocked in dynamin depleted cells.
The partial internalizationthat takes place in dynamin depleted
cells is probably not causedby the residual expression of dynamins
in these cells, because re-
A
C
B
Fig. 1. Ligand binding characteristics of EGFR are not
influenced by dynamindepletion. (A). Binding experiments were
performed using a single concen-tration of 125I-labeled EGF in the
presence of increasing concentration on un-labeled native EGF. The
graphs depict competition experiments of 125I-labeled EGF binding
to control (blue squares) or DKO (red circles) fibroblastsin the
presence of increasing concentration of native EGF. Curve fitting
tobinding data shown by lines and bars indicate standard deviation
values.(B). Immunoblotting analysis of total cell lysates of
control or DKO fibroblastswith anti-dynamin antibodies.
Immunobloting with anti-AKT antibodieswas used as loading control.
(C). Dissociation constants (Kd) and numbersof low and high
affinity EGFR binding sites on control and DKO fibroblastswere
determined using Scatchard analysis of quantitative 125I-EGF
bindingexperiments to these cells. Similar results were obtained in
three separate125I-EGF binding experiments.
A B
C D
E F
G H
Fig. 2. Endocytosis of EGFR is strongly impaired in dynamin
depleted cells.Quantitative ligand internalization experiments
using 1.5 ng∕mL (A–D) or20 ng∕mL (E–H) of 125I-EGF to control (A,
E) and DKO (B, F) fibroblastsare shown. Surface bound 125I-EGF
(blue), internalized 125I-EGF (red) anddegraded 125I-EGF (green)
are shown. The TCA precipitatable radioactivity re-presenting
intact released or recycled 125I-EGF molecules are not included
inthe figures. The ratio of the amount of 125I EGF internalized vs.
surface bound125I-EGF molecules are shown in (C) and (G) for
control (solid line) and DKO(dashed line) fibroblasts. Each data
point is the average value of duplicate re-sults. Data are
presented as mean� SD, as indicated by the bars. Also
shownimmunobloting analyzes with anti-dynamin antibodies or
anti-EGFR antibo-dies to reveal dynamin or EGFR expression
respectively, in control or DKO fi-broblasts. Similar results were
obtained in three different experiments.
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sidual dynamin expression is extremely low (Fig. 2H). For
thesame reason, it is also unlikely that the incomplete block
ofEGFR endocytosis may reflect the presence of few cells
whererecombination did not occur. A plausible explanation is that
EGFinduced internalization of EGFR can follow both
dynamin-dependent and dynamin-independent routes of
internalization,especially when the cells are stimulated with high
EGF concen-trations.
Ligand Induced Degradation of EGFR Is Compromised in
DynaminDepleted Cells. Following EGF induced receptor activation
andendocytosis, a fraction of the internalized EGFRs are
degraded(3, 23, 24). The process of ligand induced EGFR
eliminationfrom the cell surface and ensuing degradation that
terminates sig-naling is known as “receptor down regulation” (25,
26). To ex-plore the effect of dynamin depletion on EGFR
degradation,control or dynamin depleted fibroblasts were first
incubated withcycloheximide for an hour to block protein synthesis
and blockthe formation of new EGFRs. The cells were then treated
with1, 10, or 100 ng∕mL of EGF, solubilized at different time
pointsand subjected to immunoblotting analyses with anti-EGFR
anti-bodies to reveal the amount of EGFR. Because minimal
EGFRdegradation was detected in cells stimulated with 1 ng∕mL
ofEGFafter 4 h, the experiment presented in Fig. 3 shows the
effectof stimulation with 10 or 100 ng∕mL of EGF. As previously
de-scribed (27), stimulation of control fibroblasts with 100 ng∕mL
ofEGF led to robust degradation of EGFR with a half-time of
ap-proximately 45 min (Fig. 3B). Slower kinetics of EGFR
degrada-tion were detected in control fibroblasts treated with 10
ng∕mLof EGF with a half-time of 2–3 h (Fig. 3A). Previously
describeddegradation products of EGFR were clearly detected after30
min of ligand stimulations in both experiments. The experi-ments
presented in Fig. 3 A and B demonstrate that the stabilityof EGFR
is increased in dynamin depleted fibroblasts that werestimulated
with either 10 or 100 ng∕mL EGF. In dynamin de-pleted fibroblasts
that were stimulated with 100 ng∕mL the half-time of EGFR
degradation was extended to approximately 2 hwithout the appearance
of EGFR degradation products seenin control stimulated cells. It is
noteworthy that the typical EGFRdegradation products detected in
control fibroblasts stimulated
with 100 ng∕mL EGF (21, 27) were not detected in dynamin
de-pleted fibroblasts stimulated with the same EGF
concentrationsuggesting that EGF induced degradation of EGFR may
proceedvia a different mechanism in dynamin deficient cells.
Enhanced Ligand Induced Autophosphorylation and Ubiquitination
ofEGFR in Dynamin Deficient Cells. We next examined the effect
ofdynamin depletion on EGF stimulated EGFR tyrosine
phosphor-ylation and ubiqutination (Fig. 4 A–C). Control or dynamin
de-pleted fibroblasts were stimulated with 1.5, 5, or 100 ng∕mL
ofEGF. Cell lysates of EGF stimulated or unstimulated cells
weresubjected to immunoprecipitation with anti-EGFR
antibodiesfollowed by immunoblotting with either anti-pTyr or
anti-ubiqui-tin antibodies. The experiments presented in Fig. 4 A–C
show anoverall strong increase in tyrosine phosphorylation and
ubiquiti-nation of EGFR in dynamin depleted cells stimulated
with1.5 ng∕mL of EGF. Interestingly bimodal activation of
EGFRautophosphorylation was reproducibly observed in cells
stimu-lated with 1.5 or 5 ng∕mL of EGF. The experiment presentedin
Fig. 4A shows that in WTcells enhanced EGFR autophosphor-ylation
occurs after 5 min of stimulation with 1.5 ng∕mL of EGFfollowed by
reduced autophosphorylation at the 15 and 30 mintime points that is
followed after 1 and 3 h by a second peak. Indynamin depleted
fibroblasts stimulated with 1.5 ng∕mL EGF, astronger and earlier
onset of the first peak was seen after 2 minfollowed by reduced
autophosphorylation at the 5, 15, and 30 mintime points that is
followed after 1 and 3 h by a strong secondpeak of
autophosphorylation. These experiments also show thatthe levels of
EGFR ubiquitination, which is weakly detected incontrol
fibroblasts, is strongly stimulated in dynamin depleted
fi-broblasts stimulated with 1.5 ng∕mL of EGF (Fig. 4A).
Upon stimulation with 5 ng∕mL EGF stronger enhancementof
autophosphorylation of EGFR was detected in control anddynamin
depleted fibroblasts (Fig. 4B) with a robust early onsetafter 2 min
stimulation of the dynamin depleted cells. Similarly,enhanced
ubiquitination of EGFR was also detected in cells sti-mulated with
5 ng∕mL of EGF (Fig. 4B).
While autophosphorylation and ubiquitination of EGFR weremuch
more robust in both control and dynamin depleted fibro-blasts
during the first thirty minutes of stimulation with 100 ng∕mL EGF
(Fig. 4C) the initial decline and the second elevatedphase of
autophosphorylation that was seen in 1.5 or 5 ng∕mLEGF stimulated
cells at the 1 and 3 h time points were not detectedat this higher
EGF concentration.
We also examined the effect of pretreatment with cycloheximideon
ligand induced EGFR autophosphorylation and ubiquitination.These
experiments showed minor effects of cycloheximide treat-ment on
these posttranslational modifications (Fig. S1), indicatingthat
preexisting pools of EGFR play a major role in the controlof EGFR
autophosphorylation and ubiquitination during the first3 h of
ligand stimulation.
Altered Pattern of Tyrosine Phosphorylation of Shc Isoforms in
Dyna-min Depleted Fibroblasts. Shc is an adapter protein
containingan SH2 and PTB domain that functions upon tyrosine
phosphor-ylation by EGFR and other RTKs as an important link
betweenEGFR and the RAS/MAP kinase signaling pathway by
recruit-ment of Grb2/Sos complexes (28). Grb2 is an adaptor
proteincomposed of one SH2 domain flanked by two SH3 domains
whichplays an important role via direct or indirect interactions
withEGFR and other RTKs to link between RTK stimulation andthe
Ras/MAP kinase signaling pathway (2, 28). Most cells expressthree
known Shc isoforms designated p42, p52, and p66 (29,30). The
experiments presented in Fig. 4 D–F show that boththe p42 and p52
isoforms of Shc are similarly tyrosine phosphory-lated in control
or dynamin depleted fibroblasts in response tolow (1.5 ng∕mL) or
high (100 ng∕mL) EGF concentrations(Fig. 4 D–F, lower boxes).
Interestingly, a more robust and sus-
A
B
Fig. 3. Ligand induced degradation of EGFR is compromised in
dynamin de-pleted cells. Serum starved control or DKO fibroblasts
were pretreated with10 μM of cycloheximide for 1 h followed by
stimulation with 10 ng∕mL (A) or100 ng∕mL (B) of EGF for indicated
times. Equal amounts of cell lysates weresubjected to immunobloting
with anti-EGFR antibodies and as controls withanti-dynamin or
anti-AKT antibodies. Arrow points to an EGFR degradationproduct
that is detected in EGF stimulated WT cells and not in dynamin
de-pleted cells. Similar results were obtained in three different
experiments.
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tained tyrosine phosphorylation of the p66 isoform of Shc
wasdetected in dynamin depleted fibroblasts stimulated by eitherlow
or high EGF concentrations (Fig. 4 D–F, lower boxes).
Theseexperiments showed similar Grb2 recruitment in Shc
immunopre-cipitates in lysates from stimulated control or dynamin
depletedfibroblasts suggesting that Grb2 becomes associated
primarilywith the tyrosine phosphorylated p42 and/or p52 Shc
isoformsand that the p66 isoform may be involved in mediating a
Grb2independent process (31).
Similar Stimulation of MAP Kinase and Akt Responses in
DynaminDepleted Cells.We next examined the effect of dynamin
depletionon EGF stimulation of the MAP kinase (ERK) and Akt
signalingpathways in response to either low (1.5 ng∕mL) or high(100
ng∕mL) EGF concentrations (Fig. 5). Control or dynamindepleted
fibroblasts were stimulated with EGF. At different timepoints, the
cells were solubilized and subjected to immunoblot-ting with
antibodies that recognized either MAPK (anti-ERK) oractivated MAPK
(anti-pERK). The samples were also subjectedto immunoblotting with
antibodies that selectively recognize totalAkt and the activated
form of Akt (anti-pAkt). The experimentspresented in Fig. 5 show a
very similar profile of MAPK stimula-tion in control and dynamin
depleted fibroblasts over a 3 h periodin response to 1.5, 5, or 100
ng∕mL of EGF stimulation. A similarAkt response was also observed
in control or dynamin depletedfibroblasts stimulated with either
low or high EGF concentra-tions. However, a somewhat more sustained
Akt activation wasreproducibly detected in dynamin depleted cells
stimulated withany of these EGF concentrations (Fig. 5).
DiscussionIt is well established that clathrin-mediated
endocytosis of EGFRor other RTKs plays an important role in the
control of receptordown regulation; a process mediated by
intracellular degradationof both EGF and EGFR which results in
signal termination (1, 2,21, 25, 26). Subsequent studies reporting
experiments in whichclathrin mediated endocytosis of EGFR was
blocked by eitherectopic overexpression of a dominant interfering
dynamin mutant(5, 32) or by silencing the expression of clathrin
heavy chain usingspecific siRNAs (4, 6) concluded that EGFR
molecules interna-lized by means of clathrin mediated endocytosis
are capable ofrecruitment and activation of critical intracellular
signaling path-ways from endosomal compartments (5, 12, 33).
In this report we use dynamin depleted murine fibroblasts
toexplore the role played by endocytosis in the control of EGFR
display on the cell surface, in regulation of EGFR
activation,EGFR degradation, and in cell signaling via EGFR. Our
experi-ments demonstrate that the expression and display of high
andlow affinity EGFRs on the cell surface are not affected by
dyna-min loss. These experiments contradict earlier studies
demon-strating that overexpression of a dominant interfering
dynaminmutant prevent high affinity EGF binding and reduces
EGFRautophosphorylation (20). Ligand induced endocytosis of EGFRis
strongly impaired in dynamin depleted fibroblasts stimulatedwith
either low EGF concentrations [1-1.5 ng∕mL, conditionsunder which
internalization of EGFR is primarily driven by cla-thrin mediated
endocytosis (4, 8)], or high EGF concentrations(100 ng∕mL, a
condition under which EGFR endocytosis isthought to be mediated by
both clathrin-dependent and clathrin-independent mechanisms). The
experiments performed withdynamin depleted fibroblasts stimulated
with low EGF concen-tration provide an opportunity to address the
role of clathrin-mediated endocytosis in the control of EGFR
activation andsignaling via EGFR. These experiments clearly
demonstrate thatduring the early phase of EGF stimulation under
conditions inwhich the majority of EGFR are still located at the
cell surface,autophosphorylation and ubiquitination of EGFR are
stronglyenhanced indicating that autophosphorylation and
ubiquitinationof EGFR are taking place primarily by activated EGFR
locatedat the cell membrane. Autophosphorylation of EGFR leads
torecruitment of Cbl which is responsible for the ubiquitinationand
subsequent degradation of internalized EGFR molecules(34–36). The
enhanced tyrosine autophosphorylation and ubi-quitination of EGFR
observed under conditions in which EGFRendocytosis is strongly
compromised suggests that tyrosine phos-phatases and
deubiquitinating enzymes (DUB) may start to actshortly after
activated EGFR become internalized and that Cblactivity is reduced
prior to the onset of EGFR degradation bylysosomal enzymes. It is
noteworthy that similar results wereobtained using HeLa cells in
which endocytosis of EGFR wasimpaired by siRNA silencing of
clathrin heavy chain. Namely,cells in which endocytosis is impaired
by depletion of clathrinheavy chain expression also exhibit
enhanced EGFR autopho-sphorylation and ubiquitination (Fig. S2).
Moreover, similarMAPK stimulation was observed in control cells or
in cells inwhich EGFR endocytosis was impaired by clathrin heavy
chaindepletion (Fig. S3). Similar EGF stimulation of Map Kinaseof
ERK Kinase (MEK) and ERK activation was previously de-scribed in
HeLa cells in which clatharin expression was silenced
A B C
D E F
Fig. 4. Enhanced EGF induced autophosphorylation and
ubiquitination of EGFR and altered pattern of phosphorylation of
Shc isoforms in dynamin depletedcells. Serum starved control or DKO
fibroblasts were stimulated with 1.5 ng∕mL (A, D), 5 ng∕mL (B, E)
or 100 ng∕mL (C, F) of EGF for different times. Equalamounts of
cell lysates were subjected to immunoprecipitation with either
anti-EGFR or anti-Shc antibodies. (A–C) The anti-EGFR
immunoprecipitates wereimmunobloted with anti-phosphotyrosine (pY)
and reprobed with anti-ubiquitin (UB) antibodies. Enhanced tyrosine
phosphorylation of EGFR is marked withasterisks. (D–E) Anti-Shc
immunoprecipitates were subjected to immunobloting with anti-pY
antibodies (asterisk marks the presence of an nonspecific
proteinband observed in control cells) and reprobed using anti-Shc
or anti-Grb2 antibodies. The efficiency of tamoxifen induced
dynamin depletion as well as the levelsof EGFR expression in
control and DKO fibroblasts were determined by immunobloting with
anti-dynamin or anti-EGFR antibodies, respectively (not shown
forclarity). Similar results were obtained in three different
experiments.
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by specific siRNA (37). These experiments provide further
sup-port to the conclusion that MAP kinase stimulation is
primarilymediated by activated EGFR located at the plasma
membrane.
We have also shown that unaltered tyrosine phosphorylation ofthe
p42 and p52 isoforms of the adapter protein Shc and
complexformation between Shc and Grb2 are detected both in
controland in dynamin depleted fibroblasts. By contrast, selective
tyro-sine phosphorylation of the p66 isoform of Shc is detected
indynamin depleted cells in which the majority of activated EGFRare
located on the cell membrane.
Surprisingly and in contrast to earlier publications
demonstrat-ing that MAPK signaling is compromised when endocytosis
wasprevented (4, 5), MAP kinase stimulation by EGF was compar-able
in control and in dynamin depleted cells. Thus, the experi-ments
presented in this report demonstrate that MAP kinasestimulation is
driven primarily by activated EGFR located atthe plasma membrane
rather then by activated EGFR locatedinside the cell. Although MAP
kinase activation is primarily
mediated by activated EGFR located on the cell surface, the
factthat a similar MAP kinase response is observed in cells in
whichthe majority of EGFR are internalized suggests that the
fractionof activated EGFR that are located on the cell surface are
cap-able stimulating the entire MAP kinase response.
Interestingly, EGF stimulation of Akt differs from the MAPkinase
response, as we observed a somewhat more sustainedAkt stimulation
in dynamin depleted fibroblasts stimulated witheither low or high
EGF concentrations. This experiment suggeststhat Akt stimulation
may be driven both by activated EGFRlocated at the cell membrane
and by activated EGFR locatedin the membranes of intracellular
organelles such as endosomes.These results also suggest, however,
that termination of Akt sig-naling is enhanced by endocytosis of
the EGFR.
All in all, while the experiments presented in this report donot
rule out the possibility that activated EGFRs are capableof
stimulating signaling pathways from within the cells; i.e.,
fromendosomes, it is clear that the MAP kinase response is
primarilymediated by activated EGFR located at the cell
membrane.Moreover, one of the functions of endocytosis is to
suppressEGFR autophosphorylation and ubiquitination which are
pri-marily taking place at the cell membrane likely by reduced
actionof Cbl and possibly by the action of tyrosine phosphatases
andDUB that operate during endocytosis.
Material and MethodsCells and Culture. Tamoxifen-inducible
dynamin conditional knock-out mouse fibroblasts (dnm1 flox∕flox;
dnm2 flox∕flox; Cre-Esr1þ∕0)were previously described (38). Mouse
fibroblasts were grownin Dulbecco modified Eagle medium (DMEM)
containing 10%serum and 1% streptomycin and penicillin mixture.
Dynamin de-pleted fibroblasts were generated by treating the cells
twice with2 μM 4-hydroxy-tamoxifen (Sigma) on sequential days. Six
daysafter initiating tamoxifen treatment, cells were either plated
intosix-well plates coated with collagen (BD Biosciences) or
serumstarved overnight prior to EGF stimulation.
125I-Labeled EGF Binding and Internalization Experiments.
MurineEGF (Biomedical Technologies) was labeled with 125I usingthe
Iodogen method (Pierce) according to the manufacturer’s
in-structions. Tamoxifen treated (DKO) and nontreated
(control)cells were plated into six-well plates and allowed to grow
over-night. 125I-EGF binding and internalization experiments
wereperformed as previously described (19, 39, 40). For binding
ex-periments, cells were incubated with the indicated
concentrationof 125I-EGF at room temperature for 1 h in the
presence of in-creasing concentration of nonlabeled EGF; conditions
permittinginternalization of bound 125I-EGF molecules. Cells were
lysed in0.5 M of NaOH overnight and their radioactive content
quanti-fied in 10 mL Optifluor (Perkin Elmer) with a scintillation
coun-ter (LS6500, Beckman Coulter). The half time of
displacementcurves of 125I-EGF binding to control and DKO
fibroblasts werecalculated by curve fitting using PRISM3 software
(GraphPad).
Scatchard analysis of 125I-EGF binding experiments was car-ried
out in triplicate using concentrations of 125I-EGF rangingfrom 0.1
to 100 ng∕mL. A 100-fold excess of nonlabeled EGFwas added for each
assayed concentration to measure nonspecificbinding. The bound
radioactivity was quantified as describedabove. The average values
per well for control and DKO cellswere determined from two
independent counts and used to cal-culate values of Bmax (number of
receptors per cell) using non-linear curve fitting to saturation
binding according to Scatchardanalysis as previously described (15,
17).
Quantitative analyzes of EGF internalization were performedby
incubating cells with 1.5 or 20 ng∕mL of 125I-EGF for 90 minat 4
°C. The labeled cells were then washed twice with cold PBS toremove
ligand excess followed by addition of prewarmed mediumand
incubation at 37 °C for the indicated times. After an acid
A
B
C
Fig. 5. Similar stimulation of MAP kinase and AKT responses in
dynamin de-pleted cells. Serum starved control or DKO fibroblasts
were stimulated with1.5 ng∕mL (A), 5 ng∕mL (B), or 100 ng∕mL (C) of
EGF. Cells were collected atthe indicated time points and equal
amounts of lysates were subjected toimmunobloting with anti-AKT or
anti-ERK antibodies. Membranes were re-probed with anti-pAKT or
anti-pERK antibodies to monitor their enzymaticactivities. EGFR and
dynamin levels were monitored by inmunoblotting withanti-EGFR or
anti-dynamin antibodies respectively. Similar results were
ob-tained in three different experiments.
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wash to remove cell surface bound ligand molecules, the cell
sur-face bound and intracellular 125I-EGF molecules were
collectedfor each time point and quantified using a scintillation
counter.The incubation medium was precipitated with 10% TCA
(tri-chloroacetic acid) in order to quantify the amount of
degraded125I-EGF molecules in the medium. All the results are
presentedas a percentage of total cell associated 125I-EGF
radioactivityafter 90 min incubation (time 0) at 4 °C (mean� SD).
To revealthe efficiency of dynamin depletions, cell lysates were
collectedfrom control and DKO cells plated in extra wells from each
bind-ing and internalization experiment followed by
immunoblottingwith anti-dynamin or anti-EGFR antibodies.
Immunoblotting and Immunoprecipitation Analysis.After serum
star-vation, control or DKO fibroblasts were stimulated with 1.5,
5, 10,or 100 ng∕mL of EGF at 37 °C for the indicated time points
andcollected in lysis buffer (50 mM Hepes, 150 mM NaCL, 1 mMEDTA, 1
mMEGTA, 10% glycerol, 1% TritonX-100, 25 mM NaF,10 μM ZnCl2, 1 mM
NaVO4) that includes a protease inhibitor
cocktail (Roche). To carry out the degradation
experiments,control and DKO fibroblasts were preincubated prior to
ligandstimulation with 10 μMof cycloheximide for 1 h. Identical
amountsof total cell lysates were subjected to inmunoprecipitation
withanti-EGFR antibodies. The samples were also subjected
toimmunoblotting analysis with anti-AKT, anti-pAKT (Cell
SignalingTechnology), anti-ERK, anti-pERK, anti-ubiquitin (Santa
CruzBiotechnology), anti-dynamin (clone 41, BD Biosciences),
andanti-4G10, anti-phosphotyrosine antibodies. The anti-EGFR(ab328)
antibodies used in the study were generated in our labora-tory.
Primary antibodies were detected using anti-mouse HRP andProtein
A-HRP (Santa Cruz Biotechnology), and visualized by
achemiluminescence kit (Denville Scientific Inc.). Equal loadingof
proteins analyzed by immunoblotting or immunoprecipitationanalyzes
were confirmed by reprobing the stripped membranes(0.2 M NaOH, 5
min) with anti-AKT antibodies.
ACKNOWLEDGMENTS. The authors thank members of the Schlessinger
groupfor helpful discussion.
1. Carpenter G, Cohen S (1979) Epidermal growth factor. Annu Rev
Biochem 48:193–216.2. Lemmon MA, Schlessinger J (2010) Cell
signaling by receptor tyrosine kinases. Cell
141:1117–1134.3. Sorkin A, Goh LK (2009) Endocytosis and
intracellular trafficking of ErbBs. Exp Cell Res
315:683–696.4. Sigismund S, et al. (2008) Clathrin-mediated
internalization is essential for sustained
EGFR signaling but dispensable for degradation. Dev Cell
15:209–219.5. Vieira AV, Lamaze C, Schmid SL (1996) Control of EGF
receptor signaling by clathrin-
mediated endocytosis. Science 274:2086–2089.6. Huang F, Khvorova
A, Marshall W, Sorkin A (2004) Analysis of clathrin-mediated
endocytosis of epidermal growth factor receptor by RNA
interference. J Biol Chem279:16657–16661.
7. von Zastrow M, Sorkin A (2007) Signaling on the endocytic
pathway. Curr Opin CellBiol 19:436–445.
8. Sigismund S, et al. (2005) Clathrin-independent endocytosis
of ubiquitinated cargos.Proc Natl Acad Sci USA 102:2760–2765.
9. Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett NW
(2009) Endosomes: alegitimate platform for the signaling train.
Proc Natl Acad Sci USA 106:17615–17622.
10. Miaczynska M, Pelkmans L, Zerial M (2004) Not just a sink:
endosomes in control ofsignal transduction. Curr Opin Cell Biol
16:400–406.
11. Taub N, Teis D, Ebner HL, Hess MW, Huber LA (2007) Late
endosomal traffic ofthe epidermal growth factor receptor ensures
spatial and temporal fidelity of mito-gen-activated protein kinase
signaling. Mol Biol Cell 18:4698–4710.
12. Hupalowska A, Miaczynska M (2011) The new faces of
endocytosis in signaling. Traffic3:9–18.
13. Ferguson S, De Camilli P (2012) Dynamin, a membrane
remodeling GTPase. Nat RevMol Cell Biol , 13 pp:75–88.
14. Schmid SL, Frolov VA (2011) Dynamin: functional design of a
membrane fissioncatalyst. Annu Rev Cell Dev Biol 27:79–105.
15. Ferguson SM, et al. (2009) Coordinated actions of actin and
BAR proteins upstream ofdynamin at endocytic clathrin-coated pits.
Dev Cell 17:811–822.
16. Shen H, et al. (2011) Constitutive activated
Cdc42-associated kinase (Ack) phosphor-ylation at arrested
endocytic clathrin-coated pits of cells that lack dynamin. Mol
BiolCell 22:493–502.
17. Schlessinger J (1986) Allosteric regulation of the epidermal
growth factor receptorkinase. J Cell Biol 103:2067–2072.
18. Mattoon D, Klein P, Lemmon MA, Lax I, Schlessinger J (2004)
The tethered configura-tion of the EGF receptor extracellular
domain exerts only a limited control of receptorfunction. Proc Natl
Acad Sci USA 101:923–928.
19. Ozcan F, Klein P, Lemmon MA, Lax I, Schlessinger J (2006) On
the nature of low- andhigh-affinity EGF receptors on living cells.
Proc Natl Acad Sci USA 103:5735–5740.
20. Ringerike T, et al. (1998) High-affinity binding of
epidermal growth factor (EGF) to EGFreceptor is disrupted by
overexpression of mutant dynamin (K44A). J Biol
Chem273:16639–16642.
21. Honegger AM, et al. (1987) Point mutation at the ATP binding
site of EGF receptorabolishes protein-tyrosine kinase activity and
alters cellular routing. Cell 51:199–209.
22. Sorkin A, Duex JE (2010) Quantitative analysis of
endocytosis and turnover of epider-mal growth factor (EGF) and EGF
receptor. Current Protocols in Cell Biology46:15.14.1–15.14.20
Chapter 15:Unit 15.14.
23. Kirisits A, Pils D, Krainer M (2007) Epidermal growth factor
receptor degradation: analternative view of oncogenic pathways. Int
J Biochem Cell Biol 39:2173–2182.
24. Madshus IH, Stang E (2009) Internalization and intracellular
sorting of the EGF recep-tor: a model for understanding the
mechanisms of receptor trafficking. J Cell Sci122:3433–3439.
25. Beguinot L, Lyall RM, Willingham MC, Pastan I (1984)
Down-regulation of the epider-mal growth factor receptor in KB
cells is due to receptor internalization and subse-quent
degradation in lysosomes. Proc Natl Acad Sci USA 81:2384–2388.
26. Stoscheck CM, Carpenter G (1984) Down regulation of
epidermal growth factor recep-tors: direct demonstration of
receptor degradation in human fibroblasts. J Cell
Biol98:1048–1053.
27. Honegger AM, Schmidt A, Ullrich A, Schlessinger J (1990)
Separate endocytic pathwaysof kinase-defective and -active EGF
receptor mutants expressed in same cells. J Cell
Biol110:1541–1548.
28. Ravichandran KS (2001) Signaling via Shc family adapter
proteins. Oncogene20:6322–6330.
29. Pelicci G, et al. (1996) A family of Shc related proteins
with conserved PTB, CH1 and SH2regions. Oncogene 13:633–641.
30. Migliaccio E, et al. (1997) Opposite effects of the
p52shc/p46shc and p66shc splicingisoforms on the EGF receptor-MAP
kinase-fos signalling pathway. EMBO J 16:706–716.
31. Xi G, Shen X, Clemmons DR (2010) p66shc inhibits
insulin-like growth factor-I signalingvia direct binding to Src
through its polyproline and Src homology 2 domains, resultingin
impairment of Src kinase activation. J Biol Chem 285:6937–6951.
32. Johannessen LE, Ringerike T, Molnes J, Madshus IH (2000)
Epidermal growth factorreceptor efficiently activates
mitogen-activated protein kinase in HeLa cells andHep2 cells
conditionally defective in clathrin-dependent endocytosis. Exp Cell
Res260:136–145.
33. Roepstorff K, Grøvdal L, Grandal M, Lerdrup M, van Deurs B
(2008) Endocytic down-regulation of ErbB receptors: mechanisms and
relevance in cancer. Histochem Cell Biol129:563–578.
34. Bertelsen V, Breen K, Sandvig K, Stang E, Madshus IH (2007)
The Cbl-interactingprotein TULA inhibits dynamin-dependent
endocytosis. Exp Cell Res 313:1696–1709.
35. Argenzio E, et al. (2011) Proteomic snapshot of the
EGF-induced ubiquitin network.Mol Syst Biol 7:462.
36. Visser Smit GD, et al. (2009) Cbl controls EGFR fate by
regulating early endosomefusion. Science Signaling 2:ra86.
37. Galperin E, Sorkin A (2008) Endosomal targeting of MEK2
requires RAF, MEK kinaseactivity and clatharin-dependent
endocytosis. Traffic 9:1776–1790.
38. Ferguson SM, et al. (2007) A selective activity-dependent
requirement for dynamin 1 insynaptic vesicle endocytosis. Science
316:570–574.
39. Yang Y, Yuzawa S, Schlessinger J (2008) Contacts
betweenmembrane proximal regionsof the PDGF receptor ectodomain are
required for receptor activation but not for re-ceptor
dimerization. Proc Natl Acad Sci USA 105:7681–7686.
40. Mattoon DR, Lamothe B, Lax I, Schlessinger J (2004) The
docking protein Gab1 is theprimary mediator of EGF-stimulated
activation of the PI-3K/Akt cell survival pathway.BMC Biol
2:24.
4424 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1200164109 Sousa et
al.
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