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INTRODUCTION |
Trafficking of membrane proteins within the secretory and
endocytic route of eukaryotic cells is characterized by the sorting of
the membrane proteins at sites where transport pathways diverge and the
packaging of the membrane proteins into vesicles occurs. The
adaptors AP1 and AP2 appear to play a key role in the sorting and
packaging of membrane proteins in clathrin-coated vesicles (CCVs).1 AP1-containing CCVs
are formed at the TGN and facilitate the transport of cargo from
the TGN to endosomes, whereas AP2-containing CCVs function in
receptor-mediated endocytosis at the plasma membrane. The adaptors are
heterotetrameric complexes composed of two 100-kDa subunits (designated
and
1 in AP1 and
and
2 in AP2; for review see Ref.
1). The
-,
1-, and
2-subunits have been implicated in clathrin
binding (2-4). Recognition of the membrane proteins is mediated by the
medium subunits µ1 and µ2 (5), but also the
-subunits have been
implicated in binding to membrane targets (6, 7). Tyrosine- and
leucine-based sorting motifs in cytoplasmic tails of membrane proteins
can serve as adaptor binding sites, and the tyrosine and leucine
residues have been shown to be critical for the sorting of the membrane
proteins, as well as for the adaptor binding (for review see Ref. 8). For noncanonical sequences mediating either sorting or adaptor binding,
this correlation, however, remains to be established. After formation
of the CCVs, the clathrin coat is removed by the uncoating ATPase
(hsc70) in a reaction depending on ATP hydrolysis (9-11). The adaptors
are removed by a yet uncharacterized reaction.
Thus adaptors associate with specific membranes such as the TGN or the
plasma membrane and dissociate from the vesicles budding from these
membranes. The mechanisms that control the association of adaptors with
and their dissociation from membranes are unknown. In principle three
different mechanisms can be envisaged. First, the adaptors may become
specifically modified before they bind to or dissociate from membranes.
Second the targets in membranes, to which adaptors bind, are
subject to a covalent modification prior to binding or to dissociation.
Third, a change of the internal milieu of CCVs,
e.g. by an ion pump, may translate into a
conformational change of the target e.g. by
altering its oligomeric state.
Several components of CCVs are subject to phosphorylation, including
clathrin, adaptors, and cytoplasmic tails of cargo proteins, which
serve as membrane targets (12-14). Furthermore several kinases are
associated with CCVs and purified adaptors (15-17). The
phosphorylation of membrane-associated and cytosolic AP2 has been shown
to be different, with cytosolic AP2 being the higher phosphorylated species (13). These observations point to a role of adaptor phosphorylation for regulating its binding to membranes. In the present
study we analyzed the effect of phosphorylation and dephosphorylation of purified adaptors on the binding to peptides representing adaptor binding sequences, and we also analyzed AP2 binding to membranes. We
observed that phosphorylation of AP2 enhances the association constant
for in vitro binding to various AP2 binding motifs and the
recruitment of AP2 to membranes, whereas dephosphorylation has the
opposite effect. The phosphorylation of the
-,
2-, and µ2-subunit is catalyzed by kinases copurifying with AP2. Furthermore, cytosolic AP2 is higher phosphorylated and has a higher binding affinity as compared with membrane-extracted AP2. Taken together these
data suggest that a cycle of phosphorylation and dephosphorylation of
AP2 is regulating the binding of AP2 to membranes.
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MATERIALS AND METHODS |
Cells and Antibodies--
NRK and MDBK cells were obtained from
ATCC (Manassas, VA) and cultured in Dulbecco's modified
Eagle's medium, 10% fetal calf serum. Antibodies specific for
subunits of AP2 were obtained from Sigma (anti-
and anti-
) and
from Transduction Laboratories (anti-
and anti-
). The antibodies
specific for µ2 and
2 were kindly provided by M. S. Robinson
(Cambridge, United Kingdom).
Preparation of AP1 and AP2--
Clathrin-coated vesicles were
prepared from porcine brain according to Keen et al. (18).
Coat proteins were extracted from CCVs with 0.5 M Tris-HCl,
pH 7.5, for 1 h under rotation. After centrifugation at
100,000 × g for 30 min, the material was applied to a
Superose-6 column (1.6 × 55 cm) connected to a fast protein liquid chromatography system (Amersham Pharmacia Biotech) at a flow rate of 0.3 ml/min. The column was equilibrated in 0.5 M Tris-HCl, pH 7.0, 0.2 mM dithiothreitol.
1.5-ml fractions were collected, and adaptor-containing fractions were
identified by SDS-PAGE. For the separation of AP1 from AP2, the
adaptor-containing fractions were pooled and applied to hydroxyapatite
chromatography according to Manfredi and Bazari (19). The purity of AP1
and AP2 was analyzed by SDS-PAGE and Western blotting using monoclonal antibodies to the
-subunit of AP2 or the
-subunit of AP1.
Peptide Synthesis--
Peptides were synthesized using amino
acids protected with Fmoc (N-(9-fluorenyl)methoxycarbonyl)
and activated with benzotriazol-1-yl-oxytripyrolidinophosphonium hexafluorophosphate and a 9050 peptide synthesizer (Millipore). After
cleavage from the resin and the protecting groups, peptides were
purified by reverse phase HPLC using Delta Pac C-18 column (Millipore)
and an elution from 0-50% acetonitrile in 0.1% trifluoroacetic, water for 50 min. Purity was confirmed by HPLC, UV spectrometry, and
mass spectrometry. Peptides corresponding to the tail sequences of the
following membrane proteins were used in this study: from lysosomal
acid phosphatase (RMQAQPPGYRHVADGQDHA), from the 67 residues comprising
MPR46 tail the residues 2-16 (RLVVGAKGMEQFPHL) and 49-67
(GDDQLGEESEERDDHLLPM), and from invariant chain
(MDDQRDLISNNEQLP-MLGRRPGAPESKCSR). The latter one was
produced as a glutathione S-transferase fusion protein and
was used as described (20).
Treatment of AP1 and AP2 with Alkaline Phosphatase--
AP1 and
AP2 were dialyzed into Buffer A (20 mM Hepes-NaOH, pH 7.0, 150 mM NaCl, 10 mM KCl, 2 mM
MgCl2, 0.2 mM dithiothreitol) and incubated
with various amounts of alkaline phosphatase (Roche Molecular
Biochemicals) for 30 min at 37 °C.
In VitroPhosphorylation of AP2 with
[
-32P]ATP--
AP2 was dialyzed against 50 mM
Hepes-KOH, pH 7.4, 5 mM MgCl2, 2 mM
MnCl2 and incubated with 1 µl of
[
-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech)
for 15 min at room temperature. The reaction was stopped by the
addition of sample buffer and boiling for 5 min at 95 °C. The probes
were analyzed by SDS-PAGE, autoradiography, and Western blotting with antibodies against the
-,
2- and µ2-subunits. For experiments including kinase inhibitors, a mixture of threonine and serine kinase
inhibitors was used (catalog number 539572; Calbiochem). For
phosphorylation with unlabeled ATP, AP2 was dialyzed into Buffer A and
incubated for 30 min at 37 °C in the presence of 2 mM
ATP. To test for phosphatases copurifying with AP2, the adaptor was
first incubated with [
-32P]ATP for 30 min at 37 °C,
was then treated with kinase inhibitors, and further incubated for up
to 2 h at 37 °C. Subsequently, the samples were resolved on
SDS-PAGE and analyzed after autoradiography. AP2 that was incubated in
parallel with unlabeled ATP was analyzed for tail binding by using the
biosensor. For phosphorylation with unlabeled ATP, AP2 was dialyzed
into Buffer A and incubated for 30 min at 37 °C in the presence of 2 mM ATP.
Preparation of NRK Membranes--
NRK cells were grown to
confluency on a 15-cm dish. The cells were washed two times with
phosphate-buffered saline, scraped in Buffer B (25 mM Hepes-KOH, pH 7.2, 125 mM KAc, 5 mM MgAc), and lysed by passing the suspension through a
22×g syringe 10 times. The cell lysate was centrifuged for 10 min at 1000 × g to remove intact cells and nuclei. The
supernatant was removed and centrifuged for 30 min at 100,000 × g to separate membranes (pellet) from the cytosol
(supernatant). The membrane pellet was incubated for 1 h with 0.5 M Tris-HCL, pH 7.5, to remove endogenous AP2. Subsequently
the membranes were recovered by centrifugation at 100,000 × g, solubilized in Buffer B, and stored in aliquots at
70 °C until use.
Recruitment of AP2 onto NRK Membranes--
AP2 was dialyzed in
Buffer B, pre-incubated with alkaline phosphatase or ATP, and incubated
with NRK membranes for 30 min on ice. The membranes were recovered by
centrifugation at 100,000 × g for 15 min, solubilized
in 2× sample buffer, resolved by SDS-PAGE, and analyzed by Western
blotting with an antibody against the
-subunit of AP2.
Surface Plasmon Resonance Interaction Analysis--
The
interaction between AP1, AP2, or MDBK cytosolic and membrane fractions
and the cytoplasmic tail peptides was analyzed in real time by surface
plasmon resonance using a BIAcore 2000 biosensor (BIAcore AB). The
peptides were coupled to a CM5 sensor chip via their primary amino
groups following the manufacturer's instructions (immobilized
ligands). Peptides with an isoelectric point below 3.5 could not be
immobilized to the CM5 sensor chip via amino coupling because of their
low pI. These peptides were immobilized to the sensor surface via the
thiol group of a cysteine residue that was synthesized at the amino
terminus (for details see Ref. 21). AP1 and AP2 (analytes) were
injected at a flow rate of 20 µl/min unless stated otherwise. The
adaptors were used at concentrations ranging from 50-350
nM to avoid mass transport effects that can occur if the
analyte concentration is low. For cytosolic and membrane-extracted
adaptors, concentrations were used ranging from 5-11 nM
because of the limited amount of adaptors. Association (1-2 min) was
followed by dissociation (2 min) during which Buffer A was perfused.
Subsequently bound APs were removed by a short pulse injection (15 s)
of 10 mM NaOH, 0.5% SDS.
Determination of Kinetic Rate Constants--
The rate constants
(ka for association and
kd for dissociation) of the interaction between
tail peptides and the adaptor complexes were calculated by using the
evaluation software of the BIAcore 2000. Association was determined
15-20 s after switching from buffer flow to adaptor solution to avoid
distortions because of injection and mixing. The dissociation rate
constants were determined 5-10 s after switching to buffer flow. The
association rate constant ka, the dissociation
rate constant kd, and the calculation of the
equilibrium rate constant, KD = kd/ka, were determined by assuming a first order kinetic A + B = AB. Further details are described elsewhere (22, 23).
Fractionation of MDBK Cells--
MDBK cells were harvested in
100 mM Mes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.2 mM dithiothreitol
and lysed by passing the suspension through a 22×g syringe 10 times.
The cell lysate was centrifuged at 1000 × g to remove
intact cells and nuclei. The supernatant was removed and centrifuged
for 30 min at 100,000 × g to separate membranes from
the cytosol. The pellet was extracted with 0.5 M Tris, pH
7.5, and centrifuged for 30 min at 100,000 × g.
Gel Filtration--
For gel filtration the cytosol and the
membrane extract (50-µl aliquots) were passed over a Superdex-200
column connected to a SMARTTM system (Amersham Pharmacia
Biotech), equilibrated, and eluted with Buffer A at a flow rate of 40 µl/min. Fractions of 50 µl were collected and analyzed by Western
blotting using an antibody against the
-subunit of AP2. The
AP2-positive fractions were combined and stored at
70 °C.
Isoelectric Focusing--
AP2 was dialyzed against Buffer A and
incubated with alkaline phosphatase for 30 min at 37 °C. AP2 was
precipitated with methanol/chloroform (24), subjected to
isoelectric focusing according to Braun et al. (25),
transferred onto nitrocellulose, and analyzed by Western blotting with
antibodies against the four subunits of AP2.
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RESULTS |
Dephosphorylation of AP2 Reduces in VitroBinding to the
Cytoplasmic Tail of MPR46--
AP1 and AP2 are phosphoproteins (13, 16,
17), and both kinases and phosphatases copurify with adaptor complexes
(15, 17, 18). To test whether dephosphorylation of adaptors affects their binding to the cytoplasmic tails of transmembrane cargo proteins
of CCVs, AP1 and AP2 purified from pig brain were treated with alkaline
phosphatase. Following this treatment, the binding of adaptors to a
peptide derived from the cytoplasmic tail of MPR46 was analyzed by
surface plasmon resonance. MPR46 is a cargo molecule of AP1 CCVs at the
TGN and of AP2 CCVs at the plasma membrane. The peptide representing
residues 49-67 of the cytoplasmic tail of MPR46 is known to bind AP1
and AP2 with high affinity (22). A corresponding peptide in which the
critical dileucine motif was mutated to a pair of alanines served as a
control for AP2 binding. Treatment with phosphatase had only a minor
effect on the affinity of AP1, whereas the affinity of AP2 was
decreased about 20-fold from a KD of 16 nM to a KD of 366 nM.
The decrease in affinity was because of a
lower association rate constant (see Fig.
1 and Table
I). The effect of alkaline
phosphatase treatment was dependent on the concentration of
alkaline phosphatase (Fig. 2) and
sensitive to the inhibition by 1 mM pyrophosphate or 1 mM vanadate (data not shown).

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Fig. 1.
Effect of phosphatase treatment on binding of
AP1 and AP2 to MPR46 peptide 49-67. Binding of AP1 and AP2
purified from pig brain to the MPR46 tail peptide 49-67 was recorded
in real time using a biosensor as described under "Materials and
Methods." A mutant tail peptide in which the critical dileucine motif
was substituted for a pair of alanines served as a control for binding
specificity. The adaptors were used at a concentration of 350 nM and injected for 2 min at a flow rate of 20 µl/min.
Prior to binding to the tail peptide, the adaptors were incubated in
the presence or absence of 2 units of alkaline phosphatase (30 min at 37 °C). It is notable that the incubation with phosphatase
did only significantly decrease the affinity of AP2 for the MPR46 tail
peptide. For calculation of the binding constants see Table I.
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Table I
Rate constants for the binding of AP1 and AP2 to the MPR46 tail peptide
49-67
Purified AP1 and AP2 were incubated for 30 min at 37 °C in the
absence or presence of 2 units of alkaline phosphatase. Subsequently,
binding of AP1 and AP2 to the MPR46 tail peptide 49-67 was recorded
with a biosensor as described under Fig. 1. The rate constants for
association (ka), dissociation
(kd), and the equilibrium rate constant
(KD = kd/ka)
were determined from the sensorgrams shown in Fig. 1.
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Fig. 2.
Dose-dependent effect of alkaline
phosphatase on AP2 binding. AP2 was incubated with various amounts
of alkaline phosphatase prior to recording AP2 binding to the MPR46
tail peptide 49-67 as described in the legend to Fig. 1. The
equilibrium rate constants for the AP2 binding to the tail peptide was
determined (KD = kd/ka), and the values
are plotted against the amount of phosphatase.
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In bovine kidney cells all subunits of AP2 except for the
2-subunit
are known to exist as phosphoproteins (13). Isoelectric focusing of AP2
prior to and after phosphatase treatment confirmed these data for AP2
subunits from pig brain. The
-,
2-, and µ2-subunit exist as
multiple forms, whereas the
2-subunit exists as a single form.
Phosphatase treatment shifted the pattern of the
-,
2-, and
µ2-subunits toward more basic forms, whereas it did not affect the
isoelectric point of
2 (shown for
,
2, and
in Fig.
3). We conclude from these data that
three of the four subunits of AP2 are phosphorylated and that
dephosphorylation of AP2 reduces its high affinity in vitro
binding to a peptide derived from a transmembrane cargo protein of
CCVs.

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Fig. 3.
Effect of alkaline phosphatase on AP2
subunits. 20 µg of purified pig brain AP2 were incubated in the
presence (+) or absence (-) of 2 units of alkaline phosphatase for 30 min at 37 °C. Subsequently the samples were precipitated and
subjected to isoelectric focusing, followed by transfer onto
nitrocellulose. The different AP2 subunits were analyzed by Western
blotting using subunit-specific antibodies. The solid
triangles point to the subunit isoforms that decrease or increase
in concentration because of shifting from a more acidic pH value to a
more basic pH value upon incubation with phosphatase. The open
triangle indicates the position of the 2-subunit, which is the
only AP2 subunit resistant to alkaline phosphatase. The asterisk (*)
indicates a non-specific signal present in the 2 blot.
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Dephosphorylation of AP2 Reduces Binding to Tyrosine- and
Leucine-based and Noncanonical Sorting Motifs--
AP2 recognizes in
cytoplasmic tails of transmembrane proteins tyrosine- and leucine
based- and noncanonical sorting motifs (8, 26). The tail peptide 49-67
of MPR46 contains a leucine-based and a noncanonical AP2 binding motif
that can substitute each other (22). To determine whether
dephosphorylation of AP2 reduces binding to either of these motifs,
binding to the lysosomal acid phosphatase (LAP) tail peptide, the
invariant chain tail peptide, and the MPR46 tail peptide 2-16 was
determined. These peptides are known to bind AP2 via a tyrosine-based
signal (LAP, 27), a leucine-based signal (invariant chain, 20),
or a noncanonical sorting motif MPR46, 22). Alkaline phosphatase
treatment reduced the affinity to any of the three classes of
AP2 binding motifs by 4- to 15-fold (Table
II). As for the MPR46 peptide 49-67 this decrease in affinity resulted almost exclusively from lower association rate constants (not shown).
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Table II
AP2 binding to tail peptides containing different sorting signals
Peptides corresponding to the cytoplasmic tails of LAP, invariant
chain, and MPR46 were immobilized on a sensor surface and probed for
AP2 binding as described above. Prior to tail peptide binding, AP2 was
kept untreated or incubated with ATP (2 mM) or alkaline
phosphatase (2 units) for 30 min at 37 °C. The equilibrium rate
constants (KD) for the AP2 interaction with the
various tail peptides were calculated from the BIAcore sensorgrams. The
decrease or increase of the KD caused by
preincubation with ATP or alkaline phosphatase resulted from changes of
the association rate constants (not shown).
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Phosphorylation by AP2-associated Kinases Increases in Vitro
Binding of AP2 to Sorting Motifs--
Endogenous kinases associated
with purified AP2 are known to phosphorylate AP2 subunits (17,
19). When AP2 purified from pig brain was incubated in the
presence of [
-32P]ATP, incorporation of
32P into the
-,
2-, and µ2-subunits was observed
(Fig. 4).

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Fig. 4.
Phosphorylation of AP2 by associated
kinases. 0.5 µg of AP2 was incubated with 1 µl of
[ -32P]ATP (specific activity, 3000 Ci/mmol) for 15 min
at 20 °C. Subsequently, the samples were resolved on urea containing
7.5% SDS-PAGE, followed by detection of the incorporated radioactivity
via autoradiography (A) or by transferring the proteins onto
nitrocellulose and Western blot analysis of the different AP2 subunits
(B).
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To test the influence of AP2 phosphorylation and dephosphorylation more
precisely, we purified AP2 and devided the preparation into three
aliquots. One aliquot was treated with alkaline phosphatase, one was
incubated in the presence of ATP to allow phosphorylation by copurified
kinases, and one aliquot served as a nontreated control. One-third of
each sample was subsequently analyzed for binding to the MPR46 tail
peptide 49-67 (Fig. 5B),
whereas the remaining sample was subjected to isoelectric focusing,
followed by Western blotting and detection of the AP2
-subunit (Fig.
5A). As shown in Fig. 5A, the purified AP2
-subunit (mock) exists as two major forms with pIs of 6.5 and 7.2 (Fig. 5A, lane 1). Incubation with
alkaline phosphatase resulted in a shift of almost all
2 to the
basic form (lane 2). When incubated in the presence of ATP,
2 was converted to more acidic forms (lane 3). A similar result was obtained for the
-subunit (data not shown). This
indicates that the bulk of isolated AP2 represents a partially
phosphorylated form (dash), which is converted by
dephosphorylation or phosphorylation into more basic (open
triangle) or acidic (filled triangle) forms, respectively. In conclusion, dephosphorylation of AP2 with alkaline phosphatase or phosphorylation by copurifying kinases changes the
phosphorylation state of the entire AP2 pool and not just of a fraction
of it.

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Fig. 5.
Effect of AP2
phosphorylation/dephosphorylation on binding to MPR46 peptide
49-67. AP2 was incubated with 2 mM ATP for 30 min at
37 °C with 2 units of alkaline phosphatase or kept untreated
(mock). Subsequently, two-thirds of the sample was subjected
to isoelectric focusing followed by Western blotting and detection of
the AP2 -subunit (A). The remaining sample was
analysed for binding to the MPR46 tail peptide 45-67 as described
above (B). Note that tail binding of AP2 was of higher
affinity upon preincubation with ATP, whereas dephosphorylation of AP2
reduced its binding affinity by more than 20-fold (see Table II for the
numbers).
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When the same aliquots were analyzed for binding to the MPR46 tail
peptide 49-67 (see Fig. 5B and Table II), the
phosphorylation of AP2 by endogenous kinases led to a 2.5-fold enhanced
association rate to the peptide 49-67 of MPR46 (Fig. 5B,
+ATP) as compared with untreated AP2 (mock). On
the other hand, the phosphatase-treated AP2 sample exhibited a 20-fold
decreased affinity for the tail peptide, clearly demonstrating that the
phosphorylation status of AP2 is a critical determinant for the binding
to sorting signals.
Phosphorylation also enhanced the affinity of AP2 to peptides
representing tyrosine- and leucine-based and noncanonical AP2 binding
motifs 2- to 4-fold (Table II). The presence of 150 µg/ml polylysine
during the phosphorylation reaction affected neither the incorporation
of 32P into nor the affinity of AP2 (not shown). We
conclude from these data that in vitro phosphorylation of
AP2 by associated kinases enhances the affinity of AP2 to the different
classes of AP2 binding motifs.
When phosphorylated AP2 was incubated in the presence of general
protein kinase inhibitors for 30 min at 37 °C, no dephosphorylation was detectable (not shown). This suggests that in contrast to kinases,
endogenous phosphatases are not associated with purified AP2.
Phosphorylation and Dephosphorylation of AP2 Modulates the
Recruitment of AP2 to Membranes--
The experiments described above
had demonstrated the dependence of in vitro binding of AP2
to immobilized peptides by the phosphorylation status of AP2. Next we
assayed whether phosphorylation or dephosphorylation of AP2 affects its
binding to membranes. A membrane fraction was prepared from NRK cells
and stripped from endogenous adaptors by treatment with 0.5 M Tris, pH 7.5. The purified AP2 was incubated for 30 min
at 37 °C in the presence of ATP, and subsequently the AP2 was placed
on ice and incubated for 30 min in the presence of membranes.
Membrane-associated AP2 was then pelleted and quantified by Western
blotting (Fig. 6). Incubation of AP2 in
the presence of ATP increases the amount of membrane-bound AP2 as
compared with a control in which AP2 was incubated in the absence of
ATP by 4.1 ± 1.0-fold (n = 5). Under the
experimental conditions used, the ATP that was used to stimulate the
phosphorylation of AP2 may also affect the phosphorylation status of
some membrane components even at 4 °C. To rule out this possibility,
AP2 was cleared from ATP by centrifugation through a high molecular
weight cut-off filter and then incubated with the membranes. The
stimulation of AP2 binding was similar as before (data not shown),
indicating that it is the phosphorylation of AP2 that increases the AP2
association with membranes. The amount of membrane binding of AP2 was
lowered by 2.0 ± 1.1-fold (n = 4) when the
complex was treated with alkaline phosphatase for 30 min at 37 °C
and then incubated on ice 30 min with membranes in the presence of 1 mM pyrophosphate/1 mM vanadate as
compared with control AP2. This clearly indicates that recruitment of
AP2 to membranes is controlled by the phosphorylation of AP2.

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Fig. 6.
Recruitment of AP2 to membranes. AP2 (3 µg) was incubated with ATP (2 mM; upper panel)
or alkaline phosphatase (2 units; lower panel) for 30 min at
37 °C. As a control, AP2 was kept at 37 °C without any additives.
Subsequently, AP2 was incubated with crude membranes (10 µg) prepared
from NRK cells for 30 min on ice. The membranes had been treated with
0.5 M Tris to remove endogenous AP2 prior to the incubation
with bovine AP2. Following the incubation, the membranes were pelleted,
subjected to SDS-PAGE, and analyzed for the amount of bound AP2 by
Western blotting using an -adaptin-specific antibody. Whereas
preincubation of AP2 with ATP stimulated the binding to membranes,
preincubation with phosphatase decreased the amount of AP2 bound to
membranes. Quantification is given in "Results."
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Cytosolic and Membrane-associated AP2 Differ in Phosphorylation and
Affinity to Cytoplasmic Tail Peptides--
In MDBK cells the
2-subunit of cytosolic AP2 is higher phosphorylated than the
2-subunit of membrane-associated AP2. In addition the incorporation
of 32P into the
- and
2-subunits is higher for
cytosolic than for membrane-associated AP2 (13). Isoelectric focusing
of cytosolic and membrane-extracted AP2, followed by Western blot
analysis for the
-subunit, showed that cytosolic AP2 is enriched in
acidic
-subunit forms. This indicates that the steady state
phosphorylation of the
-subunit is higher for cytosolic AP2 than for
membrane-extracted AP2 (Fig. 7).

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Fig. 7.
Isoelectric focusing pattern of the
-subunit of cytosolic and membrane-extracted
AP2. Cytosol and crude membranes were prepared from MDBK cells in
the presence of phosphatase inhibitors. The membranes were then
incubated with Tris to extract AP2. This membrane-derived extract and
the cytosol was passed over a Superdex-200 gel-filtration column
connected to a SMARTTM system. AP2-enriched fractions were adjusted to
equal concentrations of AP2, subjected to isoelectric focusing, and
analyzed by Western blotting for the AP2 -subunit. Note that the
isoelectric focusing pattern of the -subunit from the bovine kidney
cell line differs from that of the -subunit from pig brain (see Fig.
3). The triangles indicate the two major -subunits
isoforms, whose relative abundance is different in cytosolic and
membrane-extracted AP2.
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The cytosolic and membrane-derived extracts contain both AP1 and AP2.
To distinguish AP2 binding from that of AP1, we used the MPR46 tail
peptide 2-16, which binds AP2 but not AP1 as an affinity matrix (22).
Binding was determined after adjusting the extracts to 10 nM AP2 (Fig. 8). The affinity
of the cytosolic AP2 (KD = 11 nM)
was about 5-fold higher than that of the membrane-derived AP2
(KD = 61 nM). As observed for the
in vitro binding of phosphorylated and dephosphorylated AP2,
the difference in AP2 affinity resulted mainly from different
association rate constants (Ka = 2.8 × 105 × M
1 × s
1 for cytosolic
AP2 versus a Ka = 0, 23 × 105 × M
1 × s
1 for
membrane-extracted AP2).

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Fig. 8.
Binding of cytosolic and membrane-extracted
AP2 to the MPR46 peptide 2-16. AP2 was prepared from the cytosol
and from membranes of MDBK cells as described in the legend to Fig. 7.
Subsequently, equal amounts of the cytosolic and the membrane-extracted
AP2 were passed over a sensor surface to which the AP2 binding peptide
2-16 from MPR46 was immobilized. For clarity, AP2 binding to the
mutated tail peptide 2-16, which was not detectable, is not shown.
Note that binding of AP2 prepared from cytosol was of higher affinity
as compared with membrane-extracted AP2. Binding constants are
given in "Results."
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DISCUSSION |
The results reported in this study support a cycle of
phosphorylation and dephosphorylation of AP2 as a mechanism to regulate the cycle of association and dissociation of AP2 with membranes during
receptor-mediated endocytosis. Phosphorylation increases the affinity
of AP2 for sorting signals in cargo proteins segregating into CCVs. The
affinity of phosphorylated and dephosphorylated AP2 for different
sorting signals differed by a factor of 15 to 33. The recruitment of
phosphorylated and dephosphorylated AP2 to membranes differed by a
factor of 8. Thus, the affinity to sorting signals does not exactly
parallel that to membranes. Although phosphorylation increased the
affinity of AP2 for sorting signals (2- to 4-fold) and to membranes
(4-fold) to a similar extent, dephosphorylation decreased the binding
to membranes only by 2-fold, whereas the affinity to sorting signals
decreased 3- to 15-fold. This suggests that lowering the affinity of
AP2 for sorting signals below a certain threshold does not further
decrease AP2 recruitment to membranes. The residual recruitment of
dephosphorylated AP2 to membranes (see Fig. 6) is therefore likely to
be because of the interaction of AP2 with other membrane components
such as membrane-associated proteins or lipids.
AP2 binds at the cytoplasmic face of the plasma membrane to membrane
proteins that contain the appropriate signals for sorting into CCVs. In
addition, AP2 interacts with a number of proteins involved in the coat
assembly, the vesicle fission, and coat disassembly, including clathrin
(2, 3), amphiphysin (28), synaptotagmin (29), Eps15 (30, 31), Shc (32),
and epsin (33). Furthermore, AP2 interacts with polyphosphoinositides,
which supports the recruitment of AP2 to sites of CCV formation (34).
Phosphorylation of AP2 has been shown to inhibit its binding to
clathrin (13). Most of the proteins involved in clathrin-mediated
endocytosis are known to exist as phosphoproteins, and their assembly
and disassembly has been shown to be controlled by phosphorylation.
Phosphorylation of amphiphysin inhibits its binding to AP2 and to
clathrin, whereas the assembly of amphiphysin, dynamin1, and
synaptojanin into complexes that include clathrin and AP2 from brain
extracts is promoted by dephosphorylation (35). This has led to the
concept that dephosphorylation regulates the assembly of cytosolic
proteins to endocytic coat complexes (35).
The data of this study suggest a modification of the concept that
dephosphorylation promotes coat assembly in general. Phosphorylation of
AP2 was found to promote its binding to the sorting signals present in
the cytoplasmic domains of membrane proteins to be integrated in
endocytic CCVs and its recruitment to membranes. As AP2 binding to the
cargo proteins of CCVs is considered to represent one of the initial
steps in formation of endocytic CCVs (36), the following sequence of
phosphorylation and dephosphorylation of AP2 is proposed.
Phosphorylated AP2 is recruited to the cytoplasmic face of the plasma
membrane. After binding to the membrane, it is dephosphorylated. This
allows recruitment of clathrin and of other components assisting coat
assembly and CCV fission, such as amphiphysin, synaptojanin, and
dynamin1. Their assembly is supported by dephosphorylation. The
uncoating of endocytic CCVs is initiated by the removal of the clathrin
lattice, which is catalyzed by the hsc70 uncoating ATPase and auxilin
(37, 38). The dissociation of AP2 and presumably also of the other
accessory proteins assisting coat assembly and vesicle fission is
promoted by their phosphorylation.
This model resembles that which has been proposed for the initial steps
of clathrin-mediated endocytosis of ligand-activated G protein-coupled
receptors, where the nonvisual arrestins replace AP2 and link the
receptors to the clathrin coat (39-41). The cytoplasmic nonvisual
-arrestin1 is constitutively phosphorylated. Its binding to
agonist-occupied receptors is promoted by the phosphorylation of the
receptors by receptor kinases, and the subsequent binding of clathrin
requires the dephosphorylation of
-arrestin1 (42).
It remains to be determined whether phosphorylation of cytoplasmic AP2
is constitutive as that of cytoplasmic
-arrestin1. Kinases that
convert AP2 into its high affinity form for membrane binding were found
to be closely associated with AP2. Although known for many years
(15-17, 43), these kinases remain to be identified. Although
adaptor-phosphorylating kinases that are influenced by polylysine have
been reported for than 10 years (16, 44), the kinases associated with
AP2 purified from pig brain CCVs were found to be
polylysine-insensitive. If the kinases are constitutively active in the
cytoplasm, AP2 recruitment to the membrane would probably need to be
controlled by a mechanism that modifies its membrane targets as has
been established for the binding of
-arrestin1 to phosphorylated G
protein-coupled receptors.
With respect to AP2-specific phosphatases, it has recently been shown
that dephosphorylation of the AP2
-subunit is mediated by a PP2A
phosphatase (43). Endocytosis was blocked if the phosphatase was
inhibited. Interestingly, the authors observed that in cells where PP2A
was blocked, still a large fraction of AP2 was membrane-associated. This is in line with our results, showing that the initial membrane association is phosphorylation-dependent. In this context
one can speculate whether the AP2-specific phosphatase is activated by
the membrane binding of AP2, by a conformational change of AP2, or by
recruiting AP2 to the site of the phosphatase. Moreover we still do not
know whether a single phosphatase is able to dephosphorylate the
-,
-, and µ-subunits of AP2 simultaneously or whether the dephosphorylation is subunit-specific. Additionally, the
phosphorylation, as well as the dephosphorylation of the individual
subunits, is likely to be dependent on the functional status of AP2 and
can be influenced by other known or unknown AP2 binding factors,
offering the possibility of a tight regulation of the adaptor function.
The identification and characterization of the kinases and the
phosphatases controlling the phosphorylation of AP2 and other proteins
involved in formation of endocytic CCVs will deepen our understanding
of how the assembly and disassembly of the endocytic machinery is regulated.