From the Howard Hughes Medical Institute and
Department of Cell Biology, Yale University School of Medicine, New
Haven Connecticut 06510, the § Department of Experimental
Oncology, European Institute of Oncology, Milan 20141, Italy, and the
¶ Istituto di Microbiologia, Universitá di Bari,
Bari 70124, Italy
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ABSTRACT |
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Clathrin-mediated endocytosis was shown to be
arrested in mitosis due to a block in the invagination of
clathrin-coated pits. A Xenopus mitotic phosphoprotein,
MP90, is very similar to an abundant mammalian nerve terminal protein,
epsin, which binds the Eps15 homology (EH) domain of Eps15 and the
Recent studies have implicated several cytosolic proteins besides
clathrin and the clathrin adaptor AP-2 in clathrin-mediated endocytosis, including the endocytosis of synaptic vesicles in nerve
terminals (1-5). Two such proteins are Eps15 and epsin (6, 7). Eps15
was first identified as an endogenous substrate for
EGF1 receptor kinase (8) and
was subsequently found to be an interacting partner for the
"appendage domain" of the AP-2 subunit Clathrin-mediated endocytosis is blocked during mitosis. In mitotic
cells clathrin coats assemble, but their invagination is impaired (19,
20). The identification of substrates of mitotic kinases responsible
for this effect may therefore shed new light on the still elusive
mechanisms underlying the invagination reaction. Epsin is highly
homologous to the Xenopus mitotic phosphoprotein MP90, which
was identified in a screen for substrates of mitotic kinases (21), and
contains a single putative consensus site for Cdc2 kinase, which is
conserved in mammalian epsin. These considerations prompted us to
investigate whether epsin undergoes mitotic phosphorylation. We report
here that both epsin and Eps15 are phosphorylated in mitosis and that
their phosphorylation inhibits binding to the clathrin adaptor AP-2. We
also report that both epsin and Eps15, like other accessory proteins of
clathrin-mediated endocytosis, undergo
stimulation-dependent dephosphorylation in nerve terminals
(22-25), with a resulting increase in their binding to each other and
to AP-2. Their dephosphorylation may facilitate endocytosis of synaptic
vesicle membranes following an exocytotic burst.
Cells and Reagents--
B82 mouse fibroblasts were cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 20% calf serum and 5 mM methotrexate.
Antibodies directed against epsin, Eps15, amphiphysin 1, and GST fusion
proteins of Analysis of Interphase and Mitotic Cells--
B82 mouse
fibroblasts were grown to 60-80% confluency and synchronized with 50 ng/ml nocodazole for 4 h. Mitotic cells were collected by
detaching rounded up cells. Interphase (G1 phase) cells
were obtained by washing away the nocodazole from the mitotic cells,
replating cells in fresh medium, and allowing cells to grow for 4 h before harvesting. Particulate and soluble fractions from these cells
were obtained as described (7). Cells extracts for affinity
purification were prepared by lysing cells in 50 mM Hepes
(pH 7.4), 150 mM NaCl, 1% Triton X-100, 1.5 mM
MgCl2, 5 mM EGTA, 10% glycerol, protease
inhibitors (100 µg/ml aprotinin, 100 µg/ml leupeptin, 100 µg/ml
pepstatin, 100 µg/ml antipain, and 1 mM PMSF), and
phosphatase inhibitors (1 mM sodium orthovanadate, 2 µM cyclosporin, and 100 nM okadaic acid) for
15 min on ice followed by centrifugation.
Generation of a Mutant DPW Domain--
The mutant DPW domain was
obtained by polymerase chain reaction-based site-directed mutagenesis.
A pair of primers harboring the serine 328 to aspartate mutation were
generated: 5'-GACCCTTGGGGAGGTGATCCT-3' and 5'-AGGATCACCTCCCCAAGGGTC-3'.
Using 5'-GACCCTTGGGGAGGTGATCCT-3' together with
5'-AAACGCGTCGACGTCGAAGTCTGAGAACTCATC-3' and
5'-AAACCGGAATTCCGGATCCGTCGTGGGGAT-3' together with
5'-AGGATCACCTCCCCAAGGGTC-3', two cDNA fragments were obtained and
purified by a QIAGEN kit (QIAGEN). These two DNA fragments were used as
both primers and templates in a standard polymerase chain reaction to
generate a full-length mutant DPW domain, which was subsequently cloned
into the PGEX6-1 vector (Amersham Pharmacia Biotech). The sequence of
the mutant DPW domain was confirmed by standard double-strand sequencing.
p34cdc2-Cyclin B Kinase Reaction--
10 µg of
recombinant wild type or mutant DPW domain were obtained from a GST-DPW
fusion protein by thrombin (Novagen) cleavage and incubated at 30 °C
for 30 min with 3 nM Xenopus
p34cdc2-cyclin B kinase (26) (a kind gift of M. Solomon, Yale
University) in the presence of 0.25 µCi/µl
[ Phosphorylation of Brain Cytosol--
Brain cytosol was prepared
by homogenizing rat brains in 2 volumes of 10 mM Hepes (pH
7.4), 1 mM EDTA, and a protease inhibitor mixture (3 µg/ml each of aprotinin, antipain, leupeptin, and pepstatin). The
lysate was centrifuged at 100,000 × g for 2 h, and the
resulting supernatant (cytosol) was desalted on Sephadex G-25 (Amersham Pharmacia Biotech) into 20 mM Hepes (pH 7.4), 120 mM KCl, and 1 mM MgCl2 at room
temperature. The desalted cytosol was incubated at 30 °C for 30 min
either with a general protein kinase inhibitor (K252a)
(dephospho-cytosol) or with 5 mM ATP, 5 mM
MgCl2, 1 mM CaCl2, 1 mM
sodium orthovanadate (Sigma), 2 µM cyclosporin
(Calbiochem), and 100 nM okadaic acid (Calbiochem)
(phospho-cytosol). The incubation was terminated by the addition of 1%
SDS, and SDS was then "neutralized" by the addition of 2% Triton
X-100 in 10 mM Hepes (pH 7.4), 150 mM NaCl to
achieve a Triton X-100:SDS ratio > 4 (w/w).
Immunoprecipitations--
Immunoprecipitations from B82 cells
and brain extracts were performed as described (7, 27). For calf
intestinal phosphatase treatment of the immunoprecipitates,
immunocomplexes were treated with 100 units of calf intestinal
phosphatase (Boheringer Mannheim) for 1 h at 37 °C in a buffer
containing 10 mM Tris-HCl (pH 8), 10 mM
MgCl2, 50 mM NaCl, 0.1% Nonidet P-40, 100 µg/ml aprotinin, 100 µg/ml leupeptin, and 1 mM PMSF.
Treatment with phosphatase inhibitors was performed with 10 mM sodium orthovanadate, 50 mM sodium fluoride,
and 20 mM sodium pyrophosphate.
Miscellaneous Procedures--
Affinity purification of
brain extracts and experiments on intact synaptosomes were performed as
described previously (27).
We investigated whether mammalian epsin undergoes phosphorylation
in mitotic cells as predicted by the property of Xenopus MP90 to act as a substrate for mitotic kinases (21). The mitotic phosphorylation of MP90 was shown to lower its mobility in SDS-PAGE (21). Cell extracts from mitotic and interphase (G1 phase)
B82 cells (a mouse fibroblastic cell line) were separated by SDS-PAGE, and the mobility of epsin was analyzed by Western blotting. As shown by
Fig. 1A, epsin from mitotic
extracts had a slower mobility than epsin from interphase cells. A
similar shift was observed for Eps15. A putative Cdc2 phosphorylation
site (28) is present in the COOH-terminal region of Eps15 (threonine
779 of mouse Eps15 (8)), which is the -adaptin subunit of the clathrin adaptor AP-2. We show here that
both rat epsin and Eps15 are mitotic phosphoproteins and that their
mitotic phosphorylation inhibits binding to the appendage domain of
-adaptin. Both epsin and Eps15, like other cytosolic components of
the synaptic vesicle endocytic machinery, undergo constitutive
phosphorylation and depolarization-dependent dephosphorylation
in nerve terminals. Furthermore, their binding to AP-2 in brain
extracts is enhanced by dephosphorylation. Epsin together with Eps15
was proposed to assist the clathrin coat in its dynamic
rearrangements during the invagination/fission reactions.
Their mitotic phosphorylation may be one of the mechanisms by which the
invagination of clathrin-coated pits is blocked in mitosis and their
stimulation-dependent dephosphorylation at synapses may
contribute to the compensatory burst of endocytosis after a secretory stimulus.
INTRODUCTION
Top
Abstract
Introduction
References
-adaptin. Binding of
Eps15 to AP-2 is mediated by its COOH-terminal region (9, 10), whereas
the NH2-terminal region of Eps15 includes three Eps15
homology (EH) domains (11). Via these modules, Eps15 binds proteins
with the consensus amino sequence NPF (12). Epsin, which contains three
NPF motifs in its COOH-terminal region (NPF domain), is a major binding
partner for Eps15 (7). Its NH2-terminal portion comprises
an evolutionary conserved domain of unknown function, the ENTH domain
(epsin NH2 terminal
homology domain), whereas its central region, which
contains eight DPW repeats (DPW domain), binds the appendage domain of
-adaptin at a site that overlaps with the Eps15-binding site (7).
Perturbation of the interactions of both Eps15 and epsin with AP-2, as
well as disruption of the function of both proteins by antibody
injection, block clathrin-mediated endocytosis (7, 13-16). It was
proposed that Eps15 and epsin play an important role in
clathrin-mediated endocytosis, possibly by participating in dynamic
rearrangements of the clathrin coat during bud invagination and fission
(7, 17, 18).
MATERIALS AND METHODS
-adaptin and the DPW domain of epsin were previously
described (7). Antibody against
-adaptin was purchased from Sigma.
-32P]ATP, 0.4 mM ATP, 15 mM
MgCl2, 20 mM EGTA, 10 mM
dithiothreitol, 80 mM potassium
-glycerophosphate (pH
7.3), and 1 mg/ml ovalbumin. The reaction was stopped by addition of a
20-fold excess of 10 mM Hepes (pH 7.4), 150 mM
NaCl, and 5 mM EDTA.
RESULTS
-adaptin-binding region.
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Fig. 1.
Mitotic phosphorylation of epsin and Eps15 in
fibroblastic B82 cells. A, epsin and Eps15 Western
blots of total extracts of interphase (G1) and mitotic (M)
cells demonstrating the upper mobility shift of the two proteins.
B, epsin and Eps15 immunoprecipitates generated from Triton
X-100 extracts from interphase (G1) and mitotic
(M) B82 cells were processed by Western blotting after
incubation in the absence or in the presence of alkaline phosphatase
(Alk. P.) or alkaline phosphatase and protein phosphatase
inhibitors (P. I.) as indicated.
To confirm that the electrophoretic shifts were due to phosphorylation, epsin and Eps15 were immunoprecipitated from interphase and mitotic cell extracts. The immunoprecipitates obtained from mitotic extracts were then incubated with or without alkaline phosphatase or with both alkaline phosphatase and a phosphatase inhibitor mixture. As shown by Fig. 1B, incubation with alkaline phosphatase completely reversed the mobility shift due to mitosis, and this effect was blocked by the presence of phosphatase inhibitors.
Clathrin-coated pits are present in mitotic cells, and the main
difference from interphase cells is the predominance of pits with
shallow curvature (19, 20). A block of the invagination reaction may
result from a dissociation from the pits of factors that normally
assist the coat during its progressive rearrangement. We therefore
compared the partitioning of clathrin, epsin, and Eps15 between the
soluble and particulate fraction of interphase (G1) and
mitotic (M) cells. As shown by Fig.
2A, the fraction of clathrin
recovered in the two fractions was similar in both conditions. In
contrast, the mitotic phosphorylation of epsin and Eps15 (revealed by
their upper mobility shift), correlated with a drastic decrease of
their recovery in the particulate fraction. One of the factors
responsible for this effect may be a decreased affinity of mitotic
epsin and Eps15 for the clathrin adaptor AP-2. To address this
question, Triton X-100 extracts of interphase and mitotic B82 cells
were affinity-purified onto a GST fusion protein comprising the
appendage domain of -adaptin (Fig. 2B). Analysis of the
bound fraction by Western blotting revealed a major decrease in binding
of the mitotic forms of both epsin and Eps15 compared with the binding
of the corresponding interphase proteins (Fig. 2B).
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Epsin and MP90 contain a single putative phosphorylation site for Cdc2
kinase in the DPW domain (serine 328 of rat epsin) (7, 21). To
determine whether this site acts as a substrate for the Cdc2 kinase and
mediates the inhibition of AP-2 binding in mitosis, a mutant DPW domain
of rat epsin was generated harboring a S328D mutation. Purified wild
type and mutant DPW domains were incubated with
[-32P]ATP in the presence of the purified
Xenopus p34cdc2-cyclin B kinase complex (26). As
shown in Fig. 3A, a very
strong difference was observed in the 32P incorporation of
the two proteins, indicating that serine 328 is a key substrate site
for the kinase in vitro. Furthermore, affinity purification
on the immobilized
-adaptin appendage domain of the
32P-labeled wild type DPW domain revealed no binding of the
32P-labeled protein, proving that its phosphorylation by
Cdc2 kinase blocks the interaction (Fig. 3B). Finally, as
shown by Fig. 3C, affinity purification of a brain cytosolic
extract on wild type and mutant DPW domains demonstrated a significant
decrease in the binding of
-adaptin to the mutant domain, confirming
that the introduction of an acidic charge at position 328 affects the epsin-AP-2 interaction.
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Stimulation of neurotransmitter release from nerve terminals correlates with an increased state of phosphorylation of several proteins with a putative role in synaptic vesicles exocytosis (29) and a decreased state of phosphorylation of several proteins with a putative role in their endocytosis including dynamin 1, synaptojanin 1, and amphiphysin (22, 23, 25, 30, 31). We investigated whether epsin and Eps15 as well undergo dephosphorylation. Fig. 4A shows immunoblots of rat synaptosomes incubated for 1 min in either control buffer or high K+ buffer. A downward shift of epsin in depolarized synaptosomes can be seen. This shift correlates with the concomitant downwards shift of amphiphysin 1 previously shown to reflect its Ca2+-dependent dephosphorylation (23). In control synaptosomes, Eps15 migrated as a doublet. Depolarization resulted in a decreased immunoreactivity on the upper bands with a corresponding increase of the immunoreactivity in the lower band, suggesting that Eps15 as well undergoes depolarization-dependent dephosphorylation.
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The phosphorylation of amphiphysin, dynamin 1, and synaptojanin 1 in
brain extracts inhibits their property to interact with partners
proteins implicated in endocytosis (25). The effect of epsin and Eps15
phosphorylation in brain extracts on their interaction with the AP-2
subunit -adaptin was therefore investigated. Brain cytosolic
extracts were incubated in either phosphorylating or dephosphorylating
conditions as described (25). Western blots of anti-epsin
immunoprecipitates generated at the end of these incubations
demonstrated an upper mobility shift of epsin in the phosphorylating
conditions that could be reversed by treatment of the
immunoprecipitates with alkaline phosphatase (Fig. 4B) consistent with the interpretation that the mobility shift of epsin and
Eps15 observed in Fig. 4A is due to dephosphorylation. Aliquots of the phospho- and dephospho-extracts were then incubated with the appendage domain of
-adaptin, and the bound material was
analyzed by Western blotting. Quantitation of the blots demonstrated that binding to
-adaptin is inhibited by phosphorylation (Fig. 4C).
We next performed immunoprecipitation experiments from these extracts
to test the effect of phosphorylation on the endogenous interactions
within the cytosolic extract of epsin and Eps15 with AP-2. In both
anti-Eps15 and anti-epsin immunoprecipitates coprecipitation of AP-2
was clearly decreased by the previous phosphorylation of the extracts
(Fig. 4D). This result is consistent with an inhibitory effect of the phosphorylation of epsin and Eps15 on their interaction with AP-2, although they do not rule out a contribution of
phosphorylation of AP-2 itself to their reduced interaction.
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DISCUSSION |
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The results of this study demonstrate that the interaction of
Eps15 and epsin with the -adaptin subunit of AP-2 is modulated by phosphorylation.
Both Eps15 and epsin undergo phosphorylation in mitosis, a stage of the cell cycle where clathrin-mediated endocytosis is blocked (19, 20). In mitosis, clathrin-coated pits are still present, but early stages of clathrin-coated pits (shallow domes and wide necks) predominate over late stages (narrow neck). This observation led to the speculation that invagination of clathrin-coated pits is a processive reaction and that some proteins that play a key role in this process may be the target of mitotic kinases (19, 20). Epsin/MP90 and Eps15 may be two such proteins. Both Eps15 and epsin were detected at clathrin-coated pits but were found not to be co-enriched, together with clathrin, in clathrin-coated vesicles (7, 18), suggesting that they are accessory and not key intrinsic components of the clathrin coat. The phosphorylation-dependent block of the interaction of epsin and Eps15 with AP-2 may dissociate them from the clathrin coat and play a role in the inhibition of the invagination process. If further studies confirm a role of epsin and Eps15 in the mitotic block of endocytosis, the elucidation of the function of these proteins may be crucial to understand the still elusive mechanism of clathrin-coated pit invagination.
Both Eps15 and epsin are also shown here to be regulated
phosphoproteins in nerve terminals. They join the list of several other
proteins implicated in the clathrin-dependent endocytosis of synaptic vesicles that undergo stimulation-dependent
dephosphorylation (22, 23, 25, 30, 31). Based on the recent study of
Slepnev et al. (25), it would appear that triggered
dephosphorylation, which favors the assembled state of cytosolic
endocytic proteins, represents a general and characteristic feature of
"endocytic" proteins of nerve terminals. A reduced state of
phosphorylation of endocytic proteins may increase the efficiency of
the synaptic vesicle membrane internalization reaction. This effect may
account for the enhanced rate of endocytosis observed in cultured
neurons after incubation with the protein kinase inhibitor
staurosporine (32) and is consistent with the inhibition of synaptic
vesicle endocytosis produced by inhibitors of the
Ca2+-dependent phosphatase calcineurin (32).
Finally, dephosphorylation by a Ca2+-dependent
phosphatase may play a role in the block of synaptic vesicle
endocytosis produced by depletion of internal Ca2+
(34). Eps15 is a substrate for EGF receptor kinase (8). We have
found that epsin as well undergoes phosphorylation in response to EGF
stimulation of fibroblastic cells, although this phosphorylation does
not occur on tyrosine residues, indicating an indirect effect of EGF
receptor kinase.2 Based on
our preliminary experiments, however, the EGF-dependent phosphorylation of Eps15 and epsin does not appear to decrease their
interaction with AP-2, suggesting that these phosphorylation reactions
play a role in cell physiology different from those described in this study.
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FOOTNOTES |
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* This study was supported in part by Grants CA46128 and NS36251 from the National Institutes of Health, by the Human Frontier Science Program and the U. S. Army Medical Research and Development Command (to P. D. C.), and by grants from the Associazione Italiana per la Ricerca sul Cancro, European Community (Biomed-2), the Armenise-Horrad Foundation, and the Ferrero Foundation (to P. P. D. F.).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: Dept. of Cell
Biology, Howard Hughes Medical Inst., 295 Congress Ave., New Haven, CT
06510. Tel.: 203-737-4465; Fax: 203-737-1762; E-mail: pietro.decamilli{at}yale.edu.
The abbreviations used are: EGF, epidermal growth factor; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.
2 S. Fré, M. R. Capua, H. Chen, P. Di Fiore, and P. De Camilli, unpublished observations.
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REFERENCES |
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