From the Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401
Received for publication, December 4, 2000, and in revised form, February 7, 2001
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ABSTRACT |
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The Ras-related GTP-binding protein Cdc42 has
been implicated in a diversity of biological functions including the
regulation of intracellular trafficking and endocytosis. While
screening for Cdc42 targets that influence these activities, we
identified the protein-tyrosine kinase ACK2 (for activated
Cdc42-associated kinase 2) as a new binding partner for
clathrin. ACK2 binds clathrin via a domain that is conserved
among a number of other clathrin-binding proteins including the
arrestins and AP-2. Overexpression of ACK2 in NIH3T3 cells results in
an inhibition of transferrin receptor endocytosis because of a
competition between ACK2 and AP-2 for clathrin. Activated Cdc42 weakens
the interaction between ACK2 and clathrin and thus reverses the
ACK2-mediated inhibition of endocytosis. Overexpression of ACK2
increases the amount of clathrin present in fractions enriched in
clathrin-coated vesicles. Taken together, our data suggest that
ACK2 may represent a novel clathrin-assembly protein and participate in
the regulation of receptor-mediated endocytosis.
The interactions of Cdc42 with its various effector proteins give
rise to actin cytoskeletal rearrangements that influence cell shape and
motility, as well as stimulate cell-cycle progression and under some
conditions, malignant transformation (1-3). Recently, it has become
apparent that Cdc42 also plays important roles in both early and late
stage exocytosis and in regulating endocytosis (4-7). The ACKs (ACK1
and ACK2)1 are
nonreceptor-tyrosine kinases that share some similarity with the focal
adhesion kinase (FAK) and protein-tyrosine kinase 2 (PYK2) and serve as
highly specific target/effectors for the Ras-related GTP-binding
protein Cdc42 (8, 9). They bind Cdc42 via a Cdc42/Rac interactive
binding (CRIB) domain that is also present in other Cdc42/Rac
effectors, including the PAKs (for p21-activated kinases), WASPs
(Wiscott-Aldrich syndrome proteins), and MLKs (mixed lineage kinases)
(10). ACKs have multiple known functional motifs such as SH3 and
proline-rich domains. Aside from Cdc42, there are no known binding
partners for ACKs. Although our previous studies indicate that ACK2 may
play a role in cell adhesion signaling (11), the exact function is
not known yet.
To better define the cellular role for ACK2, we have searched for novel
binding partners. In this study, we show that ACK2 directly interacts
with clathrin heavy chain via a conserved clathrin-binding motif shared
in all endocytic adaptor proteins and that Cdc42 can regulate the
ACK2-clathrin interaction. These findings now provide a potentially
interesting link between Cdc42 and one of its specific target/effectors
with clathrin-coated vesicle endocytosis.
Materials--
Anti-clathrin heavy chain antibody was purchased
from Transduction Laboratories. Anti-clathrin light chain antibody was
a generous gift from Dr. Tom Kirchhausen of Harvard Medical School. Anti-AP2 Cell Culture, Transfection, Immunoprecipitation, and
Immunoblotting--
NIH3T3 cells were cultured in DMEM plus 10% calf
serum. PC12 cells were cultured in DMEM plus 10% horse serum, 5%
fetal bovine serum, whereas C2C12, COS-7, and SK-N-DZ cells were
cultured in DMEM plus 10% fetal bovine serum. All cells were
maintained in 5% CO2 at 37 °C. The experimental
procedures for cDNA transfection, immunoprecipitation,
immunoblotting, and GST fusion protein precipitation were the same as
described previously (9). The immunofluorescent staining was performed
as described in Ref. 7.
Construction of the Plasmids Encoding ACK2 and Its
Mutants--
The construction of HA- or Myc-tagged ACK2 was described
previously (9). All of the ACK2 mutants were made by polymerase chain
reaction-directed mutagenesis, and the mutations were confirmed by DNA
sequencing. ACK2-2W2A, the SH3 domain mutant, was prepared by mutating
Trp-424 and Trp-425 to Ala-424 and Ala-425; ACK2-2H2A, the
Cdc42-binding defective mutant, was generated by mutating His-464 and
His-467 to Ala-464 and Ala-467; ACK2- Transferrin Receptor Endocytosis Assays--
The cells were
cultured to 80% confluency on coverslips in a 24-well plate. After the
medium was removed, the cells were incubated with warm (37 °C)
internalization medium (DMEM plus 1% bovine serum albumin) for 5-10
min. To initiate endocytosis, the cells were incubated with 200 µl of
transferrin conjugated with Cy3 in internalization medium (1:100-200)
at 37 °C for 15-30 min. Endocytosis was stopped by adding ice-cold
phosphate-buffered saline. After washing twice with ice-cold
phosphate-buffered saline, the cells were fixed with 3.7% formaldehyde
at 25 °C for 10 min and then washed with phosphate-buffered saline
(3×). The internalized Cy3-transferrin was monitored by fluorescence microscopy.
Membrane Fractionation--
Membrane fractionations were
performed basically as adapted from Grimes et al. (13). The
cells were harvested by scratching with a cell spade in hypotonic cell
lysis buffer (40 mM Hepes, pH 7.4, 25 mM
To search for binding partners for ACK2, we prepared two GST
constructs that each contained one of the two proline-rich domains (PRDs) from ACK2 (amino acids 490-589 and 580-711, Fig.
1A); these were designated
GST·ACK2PRD1 and GST·ACK2PRD2, respectively. Following the
incubation of glutathione-agarose resins containing GST·ACK2PRD1 and
GST·ACK2PRD2 with lysates from the human neuroblastoma cell line
SK-N-DZ, an 180 kDa protein was co-precipitated with the GST·ACK2
constructs (Fig. 1B, lanes 1 and 2).
An apparently identical 180 kDa protein was precipitated following the
incubation of these GST·ACK2 fusion proteins with lysates from either
skeletal muscle C2C12 cells or from NIH3T3 cells transformed by
oncogenic Ras (data not shown). Microsequence analysis indicated that
the 180 kDa ACK2-binding protein was the clathrin heavy chain (Fig.
1C; Ref. 14). This was further confirmed by Western blot
analysis of the ACK2PRD1- and or ACK2PRD2-associated proteins with an
anti-clathrin heavy chain antibody (Transduction Laboratory) (Fig.
1B, lanes 5 and 6). Myc-tagged ACK2
was also co-immunoprecipitated with endogenous clathrin heavy chain
from COS-7 cells (Fig. 1D, lane 3).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
C subunit antibody was purchased from UBI. Cy3-conjugated transferrin was purchased from Molecular Probes. The GFP vector pGFPC1
was obtained from CLONTECH. The ACK2
tetracycline-inducible (Tet-off) expression cell line was established
as previously described (12).
N, the amino-terminal truncation mutant, was prepared by deleting the first 76 amino acid
residues; ACK2-
C, a carboxyl-terminal truncation mutant, was
prepared by deleting the last 241 amino acid residues.
-glycerophosphate, 1 mM sodium orthovanadate, 10 mM MgCl2, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin), and then homogenized manually with a 2-ml glass
homogenizer. The homogenate was centrifuged at 1,000 × g for 10 min to remove cell bodies, nuclei, and cytoskeletal
fractions. The supernatant was centrifuged at 8,000 × g for 10 min to pellet large vesicles. The clathrin-coated vesicles were pelleted from the 8,000 × g supernatant
by centrifugation at 300,000 × g for 30 min.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
ACK2 binds to clathrin heavy chain in
vitro and in vivo. A, the
ACK2 domains that were used for identifying ACK2-binding partners.
KD, kinase domain; SH3, Src homology 3 domain;
CRIB, Cdc42-binding domain; PRD, proline-rich
domain. B and C, ACK2 binds to clathrin heavy
chain in vitro. The GST·ACK2PRD1 and ACK2PRD2 fusion
proteins were incubated with SK-N-DZ cell lysates, and the interactive
proteins were precipitated, resolved by SDS-PAGE, transferred to
Immobilon membranes and visualized by Coomassie Blue staining
(left panel of B). A unique band at 180 kDa that
bound to both of the fusion proteins was subjected to microsequence
analysis. The sequences from two digested peptides are shown in
C. A BLAST search of the GenbankTM/EBI database
(14) indicated that the 180 kDa protein was the clathrin heavy chain.
Immunoblot analysis using the anti-clathrin heavy chain antibody
confirmed that the band bound to both of the ACK2 fusion proteins was
the clathrin heavy chain (right panel of B, lanes
5 and 6). D, ACK2 binds to the clathrin
heavy chain in vivo. Myc-tagged ACK2 (4 µg/60-mm dish) or
vector alone (4 µg/60-mm dish) was transfected into COS-7 cells and
immunoprecipitated using an anti-Myc antibody. The total cell lysate
proteins (lanes 1, 2) and the immunoprecipitated
proteins (lanes 3, 4) were immunoblotted with an
anti-clathrin heavy chain antibody (>120 kDa) and an anti-Myc antibody
(<120 kDa). Clathrin and Myc-ACK2 are indicated in the figure.
There are 10 amino acid residues that are shared by ACK2PRD1 and
ACK2PRD2 (Fig. 1A), and it therefore seemed likely that the clathrin-binding domain was contained within this overlapping region.
To confirm this, two additional GST·ACK2 fusion proteins were
prepared. The first, designated GST·ACK2PRD2-2, represented an
amino-terminal deleted version of ACK2PRD2 that lacked 8 of the
overlapping amino acids (i.e. residues that were also
present in ACK2PRD1), whereas the second, designated ACK2CBD,
essentially contained the region of overlap shared by ACK2PRD1 and
ACK2PRD2 but lacked either proline-rich domain (Fig.
2A). As shown in Fig. 2B, clathrin binds to ACK2CBD as well as to ACK2PRD2, but is
incapable of binding ACK2PRD2-2, thus indicating that the region shared between ACK2PRD1 and ACK2PRD2 does in fact contain the clathrin-binding site. Alignment of this region (amino acid residues 580-589 in ACK2)
with the clathrin-binding domains from endocytic adaptor proteins (15,
16) defines a conserved clathrin-binding motif, LIDF (Fig.
2C).
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Given that ACK2 is a specific target for Cdc42, an obvious question
concerned whether the interaction between ACK2 and clathrin is
influenced by activated Cdc42. To address this question, we transiently
expressed a Myc-tagged Cdc42-binding defective mutant, ACK2-2H2A (in
which two histidine residues from the CRIB motif of ACK2 were mutated
to alanine), in COS-7 cells. ACK2-2H2A is completely incapable of
binding activated Cdc42 (data not shown). However, the binding affinity
of this ACK2 mutant for clathrin was increased dramatically, as
indicated from co-immunoprecipitation experiments (Fig. 2D,
compare lanes 2 and 6, upper left
panel; the lower left panel compares the relative
levels of expression of the different Myc-tagged ACK2 constructs). In
addition, when we used GST·Cdc42Q61L, a GTPase-defective mutant, to
precipitate ACK2, clathrin was not complexed with ACK2 (Fig.
2D, upper right panel, compare lanes 2 and 3; NT designates an ACK2 construct that lacks an
amino-terminal positively charged cluster but still binds Cdc42; see
below). These data indicate that Cdc42 negatively regulates the
interaction of ACK2 with clathrin.
We examined whether the intramolecular interaction between the SH3 and proline-rich domains of ACK2 influenced its binding to clathrin. We mutated the two conserved tryptophan residues in the SH3 domain that are critical for binding proline-rich domains (Ref. 17; designated ACK2-2W2A) and tested the ability of this mutant to bind clathrin. As shown in Fig. 2D, the amount of clathrin that co-immunoprecipitated with Myc-tagged ACK2-2W2A was markedly decreased compared with the case for wild-type ACK2 (upper left panel, lanes 2 and 5), indicating that perturbations of the SH3 domain of ACK2 significantly reduced its binding affinity for clathrin. Apparently, the carboxyl-terminal proline-rich domain of ACK2, upon undergoing an intramolecular interaction with the SH3 domain (11), exposes a surface that constitutes the clathrin-binding site. When Cdc42 binds to the CRIB domain between the SH3 and proline-rich domains, this intramolecular interaction would then be reversed, such that the proper binding surface is no longer accessible to clathrin.
As expected, the removal of the carboxyl-terminal 241 residues
(ACK2-CT) that contains the clathrin-binding site prevents clathrin
from co-immunoprecipitating with Myc-tagged ACK2 (Fig. 2D,
upper left panel, lane 3). However, it is
interesting that the removal of the amino-terminal end of ACK2, which
contains a positively charged cluster of amino acids (yielding the
construct designated as ACK2-
NT) strongly increases the amount of
clathrin that co-precipitates with Myc-tagged ACK2 (Fig. 2D,
upper left panel, lane 4). This may reflect an
increased accessibility of the ACK2 mutant for clathrin, because we
have found that the amino-terminal truncation of ACK2 alters its
cellular location and increases the amount of the protein in the
soluble fraction (data not shown).
Clathrin plays a pivotal role in receptor-mediated and synaptic vesicle
endocytosis as well as in trans-Golgi vesicle transport (18-24). To
examine the effects of ACK2 on clathrin-dependent receptor endocytosis, we used GFP·ACK2 fusion proteins to identify cells expressing ACK2, and Cy3-conjugated transferrin to monitor receptor endocytosis by the accompanying increases in Cy3 fluorescence. As shown
in Fig. 3A, the overexpression
of GFP alone did not affect the endocytosis of the transferrin receptor
(panels a and b; the arrows denote
cells expressing GFP). However, the overexpression of GFP·ACK2 in
NIH3T3 cells blocked receptor endocytosis (panels c and
d; the arrows point to cells expressing
GFP·ACK2). We obtained identical results in PC12 cells when
overexpressing ACK2 (data not shown). The inhibition of transferrin
receptor endocytosis was prevented when overexpressing a
carboxyl-terminal deletion mutant of ACK2 that lacked the
clathrin-binding site (panels e and f; the
arrows point to the ACK2-expressing cells), thus indicating that the ability of ACK2 to bind clathrin is necessary for the inhibition of endocytosis.
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The Cdc42-binding defective mutant, GFP·ACK2-2H2A, was also capable of mediating an inhibition of transferrin receptor endocytosis (Fig. 3A, panels g and h; the arrows point to cells expressing the GFP·ACK2 double mutant). This was consistent with our finding that the binding of Cdc42 is not necessary for the interaction of clathrin with ACK2. In fact, it would be expected that the expression of activated Cdc42, by interfering with the binding of ACK2 to clathrin, should reverse the ACK2-mediated inhibition of receptor endocytosis. This in fact turned out to be the case (Fig. 3B). When the GTPase-defective mutant, Cdc42Q61L, was co-expressed with GFP·ACK2, the endocytosis of transferrin receptors was essentially restored to that observed with control cells.
Adaptor protein-2 (AP-2) is a universal adaptor for plasma membrane
receptor endocytosis and is necessary for the cellular uptake of
transferrin receptors (25, 26). The subunit of AP-2 contains a
clathrin-binding site similar to that found on ACK2 (Fig.
2C). Thus, one plausible mechanism for the inhibition of
receptor endocytosis by ACK2 is that it competes with the AP-2
subunit for binding to clathrin. To examine this possibility, we
immunoprecipitated clathrin and AP-2 separately from the lysates of
cells transfected with an inducible (tetracycline-off) expression plasmid for ACK2 and then immunoblotted with either anti-AP-2 or
anti-clathrin antibody to analyze the association of AP-2 and clathrin
(Fig. 3C). Expression of Myc-tagged ACK2 was induced by
removing tetracycline from the culture medium (lane 1,
middle panel), whereas control (uninduced) cells did not
show a detectable expression of ACK2 (lane 2, middle
panel). Significantly less AP-2 was co-immunoprecipitated with
clathrin (using an antibody against the clathrin light chain) in cells
where the expression of ACK2 was induced, compared with cells showing
no detectable expression of Myc-tagged ACK2 (Fig. 3C,
compare lanes 3 and 4 in the lower
panel). Likewise, when using an anti-AP-2 antibody, there was a
significantly greater amount of clathrin co-immunoprecipitated with
AP-2 in cells that did not express Myc-tagged ACK2, compared with cells
that inducibly expressed ACK2 (compare lanes 5 and 6 in the upper panel). However, under the
conditions where the induction of ACK2 caused a marked reduction in the
amount of AP-2 associated with clathrin, a significant amount of ACK2
was co-immunoprecipitated with clathrin (Fig. 3C, compare
lanes 3 and 4 in the middle panel). These results demonstrate that the overexpression of ACK2 can effectively block the interaction of AP-2 with clathrin. This appears
to be translated into a change in the cellular localization of AP-2 and
its association with endocytotic vesicles. In control cells, the
staining of AP-2 shows the typical punctate appearance expected for
AP-2-containing vesicles (Fig. 3D, panel b; cell that lacks the arrow), whereas in cells overexpressing ACK2
(Fig. 3D, panel b, the arrow points to
the cell), there is a marked change in AP-2 staining such that there
appears to be less punctate vesicular structures and instead, AP-2
appears to be aggregated, particularly along the cell surface.
Based on the observation that overexpression of ACK2 inhibited
transferrin receptor endocytosis, and competed with AP-2 for binding
clathrin, we expected that the immunostaining of clathrin-coated vesicles would be significantly decreased in ACK2-expressing cells. However, as shown in Figs.
4A-C, this is not the case.
First, it appears that the overall staining of clathrin-associated
structures is actually increased upon overexpression of ACK2 (Fig.
4A, panels b and d, and g and
h). Note that it is possible to still detect overlap between
ACK2 and clathrin staining (Fig. 4B), as well as a high
degree of overlap between clathrin and phosphotyrosine staining in
cells expressing ACK2 (Fig. 4A, panels a and
d, and c and f); the latter is likely
because of autophosphorylated ACK2 associated with clathrin based on
anti-phosphotyrosine Western blot analysis (data not shown). Second,
whereas roughly equivalent amounts of clathrin were found in the
1,000 × g and 8,000 × g supernatants
and pellets, both in the absence and presence of ACK2 (Fig.
4C, lanes 1-6, upper panel), the
amount of clathrin present in the 300,000 × g pellet,
which is enriched in clathrin-coated vesicles, was consistently
increased (by at least 2-fold) in cells overexpressing ACK2 relative to
control cells (compare lanes 7 and 9). Identical
results were obtained when PC12 cells were used (data not shown). Fig.
4C also shows that ACK2 was predominantly present in the
300,000 × g pellet. Taken together, the
immunofluorescence data and the results obtained from cell
fractionation experiments lead us to suspect that ACK2 may promote,
rather than inhibit, clathrin assembly, as has been suggested for the
adaptor proteins AP-2 and AP-3 (27).
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Previous studies have shown that Rac and Rho inhibited
receptor-mediated endocytosis (28); however, our findings provide, to
our knowledge, the first demonstration that a Cdc42 target/effector (ACK2) is a clathrin-binding protein and raise interesting
possibilities for Cdc42 in the regulation of clathrin assembly and/or
clathrin-coated vesicle endocytosis. Although ACK2 is a
nonreceptor-tyrosine kinase, we have not yet found any requirement for
its tyrosine kinase activity in the regulation of receptor endocytosis.
This either means that the tyrosine kinase activity of ACK2 serves
another function and/or has only modulatory effects on an endocytic
process. Our finding that ACK2 can negatively regulate transferrin
receptor endocytosis is likely a direct outcome of its ability to bind clathrin, competitively versus AP-2 in cells, rather than a
reflection of its true physiological function. It is especially
attractive to consider a role for ACK2 as an adaptor of clathrin-coated
vesicles in brain or neuronal cells, as it is highly expressed in these cell types (9). Whereas early work implicated Cdc42 in actin cytoskeletal rearrangements and cell shape changes, more recent studies
have provided strong indications for the involvement of Cdc42 in the
stimulatory regulation of membrane trafficking, exocytosis, and
endocytosis (4-7). The endocytosis of neurotrophic factors has been
suggested to be necessary for neurite extension and signaling that
leads to differentiation (29, 30), and Cdc42 has been implicated to
play positive roles in neurite extension (31, 32). ACK2 may serve to
link Cdc42 effects on neuronal differentiation with the endocytosis of
neurotrophic factors and/or to the assembly of neurotransmitter
vesicles, which is an essential function of differentiated neuronal cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tom Kirchhausen of Harvard Medical School for sending us anti-clathrin light chain antibody (CON.1). We thank Dr. Jun-Lin Guan for reading this manuscript and providing helpful comments. We also thank Cindy Westmiller for her excellent secretarial help.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM47458 and GM40654.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: Veterinary Medical
Center C3-155, Ithaca, NY 14853. Tel.: 607-253-3888; Fax: 607-253-3659; E-mail: rac1@cornell.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M010893200
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ABBREVIATIONS |
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The abbreviations used are: ACK2, activated Cdc42-associated kinase 2; CRIB, Cdc42 interactive binding domain; DMEM, Dulbecco's modified Eagle's medium; PRD, proline-rich domain; GFP, green fluorescent protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
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