(Received for publication, March 7, 1997, and in revised form, March 31, 1997)
From the Epidermal growth factor (EGF) receptor pathway
substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor
substrate. It has been shown to bind to the EGF receptor, adaptor
protein-2, and clathrin and is present at clathrin-coated pits and
vesicles. Upon stimulation of cells with EGF or transforming growth
factor Eps151 has been identified as a
142-kDa substrate of the EGF receptor (1). In quiescent cells Eps15 is
associated to the EGF receptor, and upon EGF stimulation this
association increases dramatically (2). In addition, Eps15 has been
shown to bind to both adaptor protein-2 and clathrin (2, 3).
Subcellular fractionation and immunolocalization studies have shown
that Eps15 is present in clathrin-coated pits and vesicles but not in
early endosomes (2, 4). Eps15 shares homology with the yeast proteins End3p and Pan1p. Both proteins contain multiple Eps15 homology domains,
a motif proposed to mediate protein-protein interaction, and have been
implicated in the endocytosis of the Tyrosine phosphorylation of Eps15 is transient and occurs within 2 min
of EGF stimulation (1). In addition, EGF stimulation results in the
appearance on SDS-polyacrylamide gels of a slowly migrating band of
Eps15 of approximately 150 kDa. Tyrosine kinase activity of the EGF
receptor was found to be required for this apparent increase in
molecular mass of Eps15 (7). Expression of Eps15 cDNA in bacteria
shows the presence of only the 142-kDa form, suggesting that Eps15 is
undergoing an EGF-induced post-translational modification (1).
In this paper we investigated the nature of this post-translational
modification of Eps15. We found that the appearance of the high
molecular mass form of Eps15 is not due to EGF-induced hyperphosphorylation. Instead, we found that the 8-kDa increase in
molecular mass was caused by monoubiquitination of Eps15. This indicates that EGF induces two different modes of post-translational modification of Eps15: tyrosine phosphorylation and ubiquitination.
HER14 fibroblasts (NIH3T3 fibroblasts stably
transfected with human EGF receptor cDNA) were cultured in
bicarbonate buffered Dulbecco's modified Eagle's medium (DMEM) (Life
Technologies, Inc., Paisely, UK) supplemented with 7.5% (v/v) fetal
calf serum (FCS) (Life Technologies, Inc.) in a humidified atmosphere
at 37 °C.
Cells were grown in 60-, 100-, or
175-mm dishes (Nunc Life Technologies, Gaithersburg, MD) till 80%
confluency. Cells were serum-starved in DMEM with 0% v/v FCS for
24 h before stimulation with 50 ng/ml EGF. Cells were lysed in
RIPA buffer (20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 0.5% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 100 mM NaF, and 1 mM
Na3VO4) at 4 °C for 5 min, scraped from the
dish, and centrifuged for 5 min at 12,000 × g in an
Eppendorf centrifuge. Total cell lysate samples were prepared by adding
Laemmli sample buffer to the RIPA lysates. For immunoprecipitations,
the RIPA lysates were incubated with 25 µl of a 1:1 suspension of
protein A-Sepharose for 1 h at 4 °C and centrifuged.
Supernatants were incubated with anti-Eps15 antibody (8) for 2 h
at 4 °C. Subsequently, protein A-Sepharose was added, and after a
further incubation of 2 h, the immunoprecipitates were washed
three times, once with RIPA-buffer, once with high salt buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, and 1% Triton
X-100), and finally once with low salt buffer (20 mM
Tris-HCl, pH 7.4, 0.15 M NaCl, and 1% Triton X-100). For
alkaline phosphatase treatment, immunoprecipitates were incubated in
phosphatase buffer (50 mM Tris, pH 8.5, and 1 mM EDTA) with 50 units of alkaline phosphatase (Boehringer
Mannheim, Mannheim, Germany) at 37 °C for 30 min. Heat-inactivated
alkaline phosphatase was prepared by incubating the alkaline
phosphatase at 95 °C for 15 min. The beads were boiled in 20 µl of
Laemmli sample buffer for 5 min, and proteins were separated by 8%
SDS-polyacrylamide gel electrophoresis, Western blotted onto PVDF
membrane (Immobilon-P, Millipore, Bedford MA, USA), and probed with
rabbit polyclonal antibodies against Eps15, mouse monoclonal
anti-phosphotyrosine PY20 (Transduction laboratories, Lexington KY), or
rabbit polyclonal anti-ubiquitin antibodies (antibody kindly provided
by Dr. A. Ciechanover). Protein bands were visualized by Enhanced
Chemiluminescence (Renaissance, DuPont NEN, Boston, MA) using
peroxidase-conjugated goat-anti-rabbit or rabbit-anti-mouse
immunoglobulins (Jackson ImmunoResearch, Pennsylvania, PA).
To determine the N-terminal amino acid
sequence of Eps15, proteins of immunoprecipitates were separated on a
8% SDS-polyacrylamide gel and transferred to a PVDF membrane. The
proteins were stained with Ponceau S (Sigma, St. Louis, MO), and the
Eps15 bands were cut out of the membrane, washed thoroughly with
distilled water, and subjected to Edman degradation (9, 10). High
pressure liquid chromatography was used for analysis of degradation
products.
Inhibition of endocytosis in
HER14 cells was performed by potassium depletion (11), by incubating
the cells in hypertonic medium (12), by acidification of the cytosol
(13), or by an incubation of the cells at 4 °C (14).
For potassium depletion, cells were washed twice with depletion buffer
(20 mM Hepes, pH 7.4, 0.14 M NaCl, 1 mM CaCl2, 1 mM MgCl2,
and 1 g/l D-glucose). Subsequently, cells were incubated for 5 min with a hypotonic buffer consisting of one part depletion buffer and one part H2O. Next, the cells were incubated for
a further 30 min in depletion buffer at 37 °C. Control cells were incubated with the same buffer supplemented with 10 mM KCl.
Inhibition of endocytosis by hypertonic shock of cells was performed
using hypertonic medium consisting of DMEM supplemented with 0.45 M sucrose. Cells were washed twice with hypertonic medium
before a 30-min incubation at 37 °C in this medium. For inhibition
of endocytosis by acidification of the cytosol, cells were incubated for 10 min in DMEM, pH 5.0, supplemented with 10 mM acetic
acid. Control cells were incubated in DMEM, pH 5.0, without acetic
acid. Inhibition of endocytosis by an incubation at 4 °C was
performed by placing the cells for 30 min at this temperature in
DMEM-Hepes 0% FCS, supplemented with 0.1% bovine serum albumin. For
all experiments, cells were serum-starved for 24 h prior to
treatment with EGF.
Internalization assays were
performed using a protocol modified from Haigler et al.
(15). HER14 cells were grown on 6-well dishes to 80% confluency and
incubated for 30 min with 1 ng/ml 125I-EGF in DMEM
containing 20 mM Hepes, pH 7.2, and 0.1% bovine serum
albumin. Nonspecific EGF binding was measured in the presence of
500-fold molar excess of unlabeled EGF. Following this incubation, the
medium was removed and the cells were washed twice with ice-cold phosphate-buffered saline. Surface-bound EGF was removed by successive incubation of the cells with 0.2 M sodium acetate, pH 3.5, containing 150 mM NaCl (acid wash) for 5 and 1 min,
respectively. The acid wash solution represented the surface-bound
radioactivity. Internalized ligand was determined by lysis of the cells
by incubation with 1 N NaOH for 5 min at 37 °C. The rate
of endocytosis was expressed as the ratio of internal and surface-bound
EGF.
To determine the nature of the EGF-induced
increase in the molecular mass of Eps15, we investigated whether this
increase is caused by tyrosine phosphorylation. HER14 fibroblasts
expressing the human EGF receptor were stimulated with EGF, and Eps15
was immunoprecipitated from the cell lysates. One Eps15
immunoprecipitate was treated with alkaline phosphatase, whereas two
controls were either left untreated or treated with heat-inactivated
phosphatase. The proteins were separated on 8% SDS-polyacrylamide gels
and blotted onto PVDF membrane. After detection of tyrosine
phosphorylated Eps15 by anti-phosphotyrosine antibodies, two Eps15
bands of 142 and 150 kDa were visible in untreated cells. This
indicates that both forms of Eps15 are phosphorylated (Fig.
1, Con). The two Eps15 bands of 142 and 150 kDa are each resolved into a tightly spaced doubled, and both forms of
the doubled are tyrosyl phosphorylated. The reason for the slight
difference in molecular mass is not known but could be due to
differential splicing. Treatment of Eps15 with alkaline phosphatase
resulted in the complete dephosphorylation of Eps15 (Fig. 1,
AP), whereas treatment with heat-inactivated alkaline
phosphatase did not change the phosphorylation state of Eps15 (Fig. 1,
HI-AP). Reprobing the same blot with anti-Eps15 antibodies
showed that irrespective of the phosphorylation state of Eps15, the
142- and 150-kDa forms were present (Fig. 1). These results demonstrate
that the appearance of high molecular mass form of Eps15 is not the
result of tyrosine phosphorylation.
The
approximate increase of 8 kDa in the modified form of Eps15 stimulated
us to investigate the possible monoubiquitination of Eps15. Ubiquitin
is a highly conserved protein of about 8 kDa, which is abundant in
eukaryotes. Ubiquitin is found free or covalently linked via its C
terminus to NH2 groups of one or more lysine residues of a
variety of cytoplasmic, nuclear, and integral membrane proteins
(16).
To investigate the monoubiquitination of Eps15, Eps15 was
immunoprecipitated from HER14 cells that were either left unstimulated or stimulated with 50 ng/ml EGF. The protein samples were separated on
8% SDS-polyacrylamide gels, and the Western blot was probed with
anti-Eps15 antibodies. A clear mobility shift was seen after EGF
stimulation but not in unstimulated cells (Fig. 2).
Subsequently, the Western blot was stripped and reprobed with
anti-ubiquitin antibodies. In this case only the 150-kDa form of Eps15
was detected, demonstrating that Eps15 becomes monoubiquitinated upon
EGF stimulation (Fig. 2). In addition to the appearance of the 150-kDa
band, a slight staining of higher molecular mass Eps15 was detected
upon EGF addition. This phenomenon was better visible upon longer
exposures (data not shown). Eps15 of higher molecular mass was
previously also found on Western blots containing immunoprecipitated
Eps15 that were stained for phosphotyrosine residues (2). These
observations suggest that Eps15 is not only monoubiquitinated but that
a minority of Eps15 may also be multiubiquitinated.
To obtain further proof for the ubiquitination of
Eps15, the N-terminal sequences of the 142- and 150-kDa Eps15 isoforms
were determined by Edman degradation. Because ubiquitin is conjugated via its C terminus to the target proteins, the N terminus of conjugated ubiquitin is still available for Edman degradation. Sequencing of the
142-kDa form of Eps15 did not result in any signal, most probably due
to N-terminal blocking. Sequencing of the 150-kDa form of Eps15
resulted in a single protein sequence (Fig. 3). Comparison of these 10 amino acids with the published sequence of
bovine ubiquitin revealed that the obtained amino acids are identical
to the first amino acids of ubiquitin. Comparison of this sequence with
sequences in the SWISS-PROT protein data base did not reveal a relevant
match with any other protein than ubiquitin.
Based on both the Western blotting results and the N-terminal amino
acid sequence, we conclude that Eps15 becomes ubiquitinated after
stimulation of the cell with EGF. Because the increase in molecular
mass of Eps15 is similar to the molecular mass of ubiquitin (8 kDa), we
conclude that Eps15 becomes predominantly monoubiquitinated. Because
the approximate ratio of the two Eps15 forms in EGF-stimulated cells
was previously determined as 1:1, we estimate that about 50% of Eps15
becomes monoubiquitinated after stimulation of cells with EGF (2). Both
forms of Eps15 become phosphorylated on tyrosine residues (Fig. 1),
which indicates that ubiquitination of Eps15 is not required for its
phosphorylation.
Monoubiquitination of proteins has not frequently been reported.
Examples of monoubiquitination are the T cell antigen receptor (17),
histone H2A (18), and cytochrome c (19). The yeast The binding of Eps15 to both adaptor protein complex-2 and clathrin and
the presence of Eps15 in clathrin-coated pits and vesicles of mammalian
cells suggest a role for Eps15 in the endocytosis of the EGF receptor.
To investigate the possible relationship between Eps15 ubiquitination
and EGF receptor internalization, we examined the effect of blocking
EGF receptor internalization on Eps15 ubiquitination. Internalization
of EGF receptors was inhibited in four different ways: by incubation at
low temperature, by depleting potassium from the cytosol, by a
hypertonic shock of the cells, or by acidification of the cytosol.
These methods inhibit different steps in endocytosis: hypertonic shock
and incubation at low temperature prevent clustering of receptors (12),
potassium depletion inhibits the assembly of coated pits (11), whereas acidification of the cytosol is suggested to inhibit pinching off of
clathrin-coated pits from the plasma membrane (13). The effect of these
conditions on EGF endocytosis was measured using 125I-labeled EGF. In control cells EGF was rapidly
internalized, whereas under all four endocytosis inhibiting conditions
EGF internalization was inhibited for more than 80% (Fig.
4). Analysis of Eps15 phosphorylation revealed that in
all cases Eps15 became phosphorylated on tyrosine residues (Fig.
5B). These results demonstrate that EGF
receptor activity has not been affected by either of these treatments. Interestingly, it was recently reported by Vieira and co-workers (28)
that inhibition of endocytosis using a dynamin mutant resulted in
differences in EGF receptor substrate phosphorylation. This indicates
that maximum Eps15 phosphorylation is already achieved before the EGF
receptor internalization process is initiated. Detection of Eps15 by
Western blotting showed that in control cells Eps15 became
ubiquitinated after 10 min of EGF stimulation (Fig. 5A).
However, when cells were incubated and stimulated at 4 °C,
ubiquitination of Eps15 was completely abolished (Fig. 5A, row 1). The same results were obtained when endocytosis was
inhibited by alternative methods such as potassium depletion (Fig.
5A, row 2), acidification of the cytosol (Fig.
5A, row 3), and hypertonic shock (Fig.
5A, row 4). Together these data show that when
endocytosis is inhibited Eps15 monoubiquitination is abolished, but
tyrosine phosphorylation remains undisturbed.
An interesting question is the possible function of the
monoubiquitination of Eps15. The absence of Eps15 ubiquitination under conditions that inhibit EGF receptor internalization indicates that
either Eps15 ubiquitination is required for Eps15 endocytosis or EGF
receptor endocytosis is a prerequisite for Eps15 ubiquitination. The
first possibility is an analogy to what has been reported for the
growth hormone receptor in mammalian cells (23) and the We thank Bram Dijker for practical
assistance, Fridolin van der Lecq and Ton Aarsman (Sequence Center,
Institute of Biomembranes, University Utrecht, The Netherlands) for
performing and interpreting the Edman Degradation experiments, and
Lisette Verspui and Theo van der Krift for photographic
reproductions.
Department of Molecular Cell Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, Eps15 becomes rapidly and transiently phosphorylated on
tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by
Western blotting and protein sequencing that this EGF-induced increase
in molecular mass is the result of monoubiquitination. Eps15
ubiquitination but not tyrosine phosphorylation was inhibited under
conditions that blocked EGF-induced internalization of the EGF
receptor. Our results establish ubiquitination as a second form of
EGF-stimulated covalent modification of Eps15.
-factor and lipids,
respectively (5, 6).
Tissue Culture
Eps15 Mobility Shift Is Not Due to Tyrosine
Phosphorylation
Fig. 1.
Alkaline phosphatase treatment of Eps15
immunoprecipitates. HER14 cells were stimulated for 10 min with 50 ng/ml EGF and lysed in RIPA buffer. Eps15 was immunoprecipitated from
the cell lysates, and the immunoprecipitates were either left untreated (Con) or treated with alkaline phosphatase (AP)
or heat-inactivated alkaline phosphatase (HI-AP). Proteins
were separated on an 8% SDS-polyacrylamide gel, and the Western blot
was incubated with anti-phosphotyrosine antibodies (WB
P-Tyr). Subsequently, the blot was stripped and incubated with
anti-Eps15 antibodies (WB Eps15).
[View Larger Version of this Image (25K GIF file)]
Fig. 2.
Stimulation of HER14 cells with EGF induces
monoubiquitination of Eps15. HER14 cells were left unstimulated or
stimulated for 10 min with 50 ng/ml EGF and lysed in RIPA buffer. Eps15
was immunoprecipitated from the cell lysates, and the proteins were separated on an 8% SDS-polyacrylamide gel. The Western blot was incubated with anti-Eps15 antibodies (WB Eps15).
Subsequently, the blot was stripped and incubated with anti-ubiquitin
antibodies (WB Ubiquitin).
[View Larger Version of this Image (47K GIF file)]
Fig. 3.
Obtained N-terminal amino acid sequence of
the 150-kDa form of Eps15 as determined by Edman degradation.
HER14 cells were stimulated with EGF, and Eps15 was immunoprecipitated
from all the lysates. Proteins were separated on an 8%
SDS-polyacrylamide gel. The 150-kDa band of Eps15 was cut out of the
PVDF membrane and subjected to Edman degradation. The obtained
N-terminal sequence of the 150-kDa mouse Eps15 and the bovine ubiquitin
sequence are shown.
[View Larger Version of this Image (6K GIF file)]
-factor receptor has recently been shown to become either mono- or
diubiquitinated (20). Multiubiquitination of proteins usually starts on
one lysine residue (16). Subsequently, this ubiquitin becomes
ubiquitinated, resulting in the formation of multiubiquitin chains.
Examples of multiubiquitination include cytoplasmic and nuclear
proteins but also integral membrane proteins such as receptors for EGF
(21), growth hormone (22, 23), platelet-derived growth factor (24), and
the tumor necrosis factor (25). Protein ubiquitination has been
implicated in many cellular processes (16). The most widely studied
function of ubiquitination lies in the targeting of
(multiubiquitinated) proteins for degradation to the 26 S proteasome.
However, not all ubiquitinated proteins are degraded, suggesting
additional functions for ubiquitination besides proteolysis. Treatment
of cells with transcription inhibitors resulted in a reduced level of
ubiquitinated histone H2B, suggesting a role for ubiquitination in
chromatin organization (18). Recently, it has been suggested that
ubiquitination plays a role in the activation of I
B
, a regulator
of the transcription factor NF
B (26). Interestingly, a new function
for ubiquitination has been recently described for the ubiquitination
of plasma membrane receptors. Ubiquitination of both the growth hormone
receptor and the
-factor receptor in S. cerevisiae have
been implicated in the endocytosis of these receptors (20, 23, 27).
Fig. 4.
Inhibition of EGF receptor endocytosis.
Inhibition of EGF receptor endocytosis in HER14 cells was done by four
different methods as described under "Experimental Procedures." The
rate of endocytosis in control cells (C) was set at 100%,
and the endocytosis after treatment with low temperature, hypertonic
shock, potassium depletion, or cytosol acidification (E) is
presented as a percentage of the control.
[View Larger Version of this Image (38K GIF file)]
Fig. 5.
Inhibition of endocytosis prevents Eps15
monoubiquitination. HER14 cells were subjected to four different
methods to inhibit endocytosis: low temperature, hypertonic shock,
potassium depletion, or cytosol acidification. Subsequently the cells
were left unstimulated or stimulated with 50 ng/ml EGF for 10 min. Proteins from the cell lysates were separated on 8% SDS-polyacrylamide gels, and the Western blots were incubated with anti-Eps15 antibodies (A). Immunoprecipitated (IP) Eps15 was separated
in a similar manner, and the Western blots (WB) were
incubated with anti-phosphotyrosine (-p-tyr) antibodies
(B). The 142-kDa band of Eps15 and the ubiquitinated 150-kDa
band of Eps15 (Eps15-Ub) are indicated.
[View Larger Version of this Image (55K GIF file)]
-factor
receptor in yeast (20). In this case Eps15 ubiquitination could be
involved in the early steps of endocytosis of the EGF receptor.
However, inhibition of endocytosis of the
-factor receptor in yeast
resulted in an increased ubiquitination of the receptor, which is in
contrast to the results presented in this paper (20). Alternatively,
our results may indicate that endocytosis is required for Eps15
ubiquitination. This would imply that Eps15 ubiquitination occurs
exclusively at a post-surface endocytic transport step. We have shown
previously that Eps15 localization is restricted to coated pits and
coated vesicles and absent from early endosomes (2). The
monoubiquitination of Eps15 could thus be involved in the targeting of
coated pits to the early endosome or in the uncoating of the coated
vesicle. Another possibility is that Eps15 ubiquitination could be
involved in the targeting of Eps15 to the 26 S proteasome for its
degradation, which is in fact the first described function of protein
ubiquitination.
*
This work was supported by Grant 17.182 (to S. v. D.) from
the Life Sciences Foundation, which is subsidized by the Netherlands Organization for Scientific Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 31-30-253-3349;
Fax: 31-30-251-3655; E-mail: sanne{at}emsaserv.biol.ruu.nl.
1
The abbreviations used are: Eps15, epidermal
growth factor receptor pathway substrate clone 15; EGF, epidermal
growth factor; FCS, fetal calf serum; DMEM, Dulbecco's modified
Eagle's medium; PVDF, polyvinylidene difluoride.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.