 |
INTRODUCTION |
The Rho family of small GTPases, of which the best characterized
members include Rac, Rho, and Cdc42, are implicated in various cellular
processes including morphological reorganization and transcriptional
activation (1). Initial microinjection studies revealed that Rho drives
the assembly of filamentous actin into stress fibers, whereas Rac and
Cdc42 induce lamellipodia and filopodia, respectively (2-5).
Microinjected Cdc42G12V causes filopodia production and
neurite formation in neuroblastoma cells (6). In this context Cdc42
antagonizes the effects of RhoA, which needs to be down-regulated for
neurite formation (6). Recently Cdc42 has been found to associate with
the
-subunit of the coatomer complex; it is hypothesized that Cdc42
may compete with cargo receptors for binding to the coatomer protein
-COP subunit and facilitate the release of coatomer subunits
from transport vesicles (7).
Many targets for the Rho GTPases have now been identified (1). The
p21-activated serine/threonine kinases
(PAKs)1 have an N-terminal
high affinity Rac/Cdc42 binding domain that regulates the C-terminal
kinase domain (8). Besides PAK, other Cdc42-interacting proteins
include ACK (9), Wiskott-Aldrich syndrome protein (WASP) (10),
IQGAP (11) and mixed lineage kinase 3 (12). WASP and
IQGAPs regulate actin cytoskeletal reorganization (10, 11) and
WASP directs actin-based motility in cell extracts by stimulating actin
nucleation with the Arp2/3 complex (13).
ACK is the only tyrosine kinase known to interact with Cdc42 (9). ACK1
is a 1,036-residue protein with an N-terminal tyrosine kinase domain,
flanked by an SH3 and Cdc42 binding domain, and an extensive
proline-rich C-terminal region. ACK1 interacts specifically with Cdc42
and not Rac1 or Rho (9). The structure of Cdc42 complexed to the p21
binding domain of ACK indicates that hydrophobic residues in the
"effector loop" and also the C-terminal region of Cdc42 are
important in determining the specificity of effector binding (14). The
ACK-2 tyrosine kinase (a smaller spliced variant of ACK1) becomes
tyrosine phosphorylated upon stimulation by epidermal growth factor
(EGF) and bradykinin (15). Recently a Caenorhabditis elegans
homolog termed ARK-1, which also contains a CRIB domain, has
been reported to act as a negative regulator of EGF signaling downstream of let23 (16).
Here we report that a region of ACK1 specifically associates with the
N-terminal globular region of clathrin heavy chain in vitro.
This region has homology to sequences in
-arrestin, which provides a
link between clathrin and various serpentine receptors (17). Clathrin
is the major structural component of clathrin-coated vesicles,and
clathrin-mediated endocytosis plays a particularly important role in
receptor internalization and export of components out of the
trans-Golgi network. ACK1 is found here to co-localize with
a subset of brefeldin A-insensitive clathrin vesicles, and moderate
overexpression of GFP-ACK1 results in stimulation of transferrin
uptake, indicating that the kinase can participate in receptor-mediated endocytosis.
 |
MATERIALS AND METHODS |
Mammalian Cell Expression Vectors--
The pXJ-HA or pXJ-FLAG
and pXJ-GFP vectors containing the cytomegalovirus enhancer/promoter
and encoding the Kozak initiation and N-terminal HA or FLAG epitopes
were as described previously (9). GST-Nck and FLAG-Nck vectors
contained the full-length human cDNA. HA-ACK was constructed by
introducing a BamHI site adjacent to the initiation codon
and an HindIII site flanking the termination codon of ACK1
by polymerase chain reaction (using Vent polymerase, New England
Biolabs). The entire coding sequence was cloned into the pXJ-HA vector
via these unique sites. The kinase-inactive ACK1K158R
mutant was generated using the QuickChange protocol (Stratagene). ACK1
deletion (see Fig. 1A) and point mutants (see Fig.
4C) were constructed with BamHI- and
EcoRI-containing primers by polymerase chain reaction and
were cloned into pGEX vector for protein expression. The ACK1
C-terminal constructs were constructed by polymerase chain reaction
using BamHI and HindIII site linkers and were
cloned into pXJ-FLAG vectors for cell transfection. In Fig.
4B human ACK1, rat arrestin-3, and human selenium-binding
protein (SBP) 56 sequences were cloned by annealing primers with a
BamHI site and EcoRI site overhang and cloned
into pGEX 2T. The sequences correspond to: ACK 19-mer (564), ACK
22-mer (561), SBP56 19-mer (437), SBP56 24-mer (432),
arrestin-3 19-mer (367), arrestin-3 26-mer (360).
Cell Culture--
COS-7, NIH 3T3, and A431 cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. For transfection studies cells were plated at low density
on glass chamber slides (Nunc) for 1-2 days. The cells were starved
for 1 h and then transfected by using LipofectAMINE (Life
Technologies, Inc.) using 0.5 µg of each plasmid and 3 µl of
LipofectAMINE/ml of medium. After an overnight period in 1% fetal
bovine serum cells were incubated with Texas Red-conjugated transferrin
(Molecular Probes) for 15 min at 37 °C, washed, and fixed for
indirect immunofluorescence.
Immunofluorescence and Imaging--
Cell staining was performed
as described previously (8). Primary antibodies were incubated in 0.5%
Triton X-100 for 2 h at 37 °C at the following dilutions:
anti-HA (Santa Cruz) or anti-FLAG monoclonal antibody (Sigma) was used
at 2.5 µg/ml and anti-clathrin (ICN) or anti-adaptin (Transduction
Laboratories) monoclonal antibody at a 1:20 dilution. The cells were
then washed twice with 0.1% Triton X-100 and incubated with secondary
antibody in 0.5% Triton X-100 for 1 h. Fluorescein
isothiocyanate-conjugated second antibodies at 1:100 dilutions and
rhodamine-conjugated second antibodies (Boehringer Manneheim) at 1:100
were used at room temperature. Photomicrographs were collected on an
Zeiss Axioplan microscope using a × 63 oil immersion objective.
Digitized data (8-bit) were subject to analysis performed using the
Macintosh version of the public domain NIH Image program (developed at
the U. S. National Institutes of Health and available on the Internet).
In Vitro Binding Assays--
GST fusion proteins were expressed
in Escherichia coli BL21 and purified as described (9). Rat
brain lysates were prepared in buffer A (40 mM HEPES pH
7.3, 0.1 M NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM sodium vanadate, 25 mM sodium fluoride, 5%
glycerol, 5 mM (dithiothrietol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin and leupeptin).
GST-ACK fusion proteins were loaded onto a glutathione-Sepharose column
(50 ml, final 2 mg/ml), and rat brain lysates (2 mg of protein in 0.5 ml) were cycled through these columns over 2 h at 4 °C. Columns
were washed extensively and bound proteins released by 10 mM glutathione. Crude coated vesicles were partially
purified from rat brain according to method by Kirchhausen and Harrison
(19).
Competition Assay--
In vitro translated clathrin
was prepared according to the manufacturer's protocol. Reticulolysates
containing in vitro translated [35S]Met
N-terminal clathrin was incubated with
-arrestin bound to
glutathione-Sepharose in the presence of either ACK peptides (19-mer),
pep-1 (normal), or pep-2 (reverse sequence). Bound proteins were eluted
with 10 mM glutathione.
Proteolytic Digestion--
2.5 ml of GST-ACK lysate was loaded
onto 100-µl glutathione-Sepharose 4B columns, washed with GST buffer,
and incubated with 0.25 ml of cytosol for 2 h. The column was
washed with GST buffer and then with 1 column volume of solution D (25 mM MES, pH 6.5, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM
CaCl2). 1 volume of subtilisin (2 µg/ml) in solution D
was added for 30 min at room temperature. 1 column volume of solution D
was defined as flow-through. Bound proteins were then eluted with
glutathione (referred to as bound fraction).
Western Analysis--
Proteins were transferred to
nitrocellulose filters, blocked in 5% skim milk for 1 h at room
temperature, and incubated with primary antibodies for 2 h at room
temperature. Mouse anti-clathrin (Transduction Laboratories) was used
at a 1:500 dilution in phosphate-buffered saline containing 1% milk.
After washing, incubation with horseradish peroxidase-conjugated
secondary antibody (DAKO) was performed for 1 h. Signals were
visualized in the presence of luminol (Amersham Pharmacia Biotech).
 |
RESULTS |
A Clathrin Binding Region Is Present in ACK1--
The ACK1
cDNA was isolated from a human hippocampal expression library by
screening with [
32P]GTP·Cdc42 (9). A related human
kinase is TNK1 (20), which exhibits homology in the tyrosine kinase
domain, the flanking SH3 domain, and in a region at the extreme C
terminus of the two proteins (Fig.
1A). A bovine brain cDNA
encoding a related ACK isoform referred to as ACK2 which lacks
C-terminal regions has been isolated (15). Interestingly the "gene
33" protein binds Cdc42 through an ACK-related CRIB domain and
contains extensive homology to C-terminal regions also (21). To search
for interacting partners of ACK, a stable GST-ACK443-646
fusion protein (Fig. 1A, construct 1) was expressed and
used as an affinity matrix with rat brain lysate. An ~160-kDa protein that bound to GST-ACK443-646, but not GST, was determined
to be clathrin heavy chain by peptide microsequencing of the excised
band. Deletion analysis allowed us to map a minimal region required for
clathrin association (Fig. 1A). The p21 binding domain
(CRIB) was not required for clathrin association. A 32-residue
construct (ACK561-593) was sufficient for clathrin
binding, albeit with reduced efficiency.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 1.
Identification of a clathrin binding region
in mammalian ACK. Panel A, in
vitro binding assay to detect ACK interacting proteins. GST-ACK1
constructs as indicated were used as affinity matrices to bind proteins
from rat brain-soluble extract. After 2 h, columns were washed and
proteins eluted with glutathione. Proteins were separated on 9%
SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride
membranes. The 70-kDa protein that co-purifies with some GST proteins
is bacterial DnaK. The filters were Coomassie stained and probed with
anti-clathrin (lower frames). Corresponding bands were
excised from gels for microsequencing, which revealed the 160-kDa band
to be clathrin. Panel B, total brain lysate
(T) or that bound to ACK1443-646 (B)
were probed with anti-AP-180 or anti- -adaptin.
|
|
Clathrin-mediated endocytosis involves multiprotein components that
comprise the clathrin coat; accessory components include dynamin,
amphiphysin, adaptors, and AP-180. We did not detect other purified
proteins by Coomassie staining, nor were the coated vesicle proteins
AP-180 and
-adaptin enriched in the ACK-bound fraction (Fig.
1B).
ACK1 Co-localizes with Clathrin in Vivo--
COS-7 cells were
transiently transfected with FLAG-tagged ACK and stained for endogenous
clathrin. Clathrin typically shows punctate vesicular and perinuclear
staining in all cell types, reflecting its involvement in a variety of
membrane trafficking pathways (Fig.
2A, top left
frame). Transient (higher level) ACK1 expression caused
redistribution of clathrin that co-localized with ACK1 (Fig.
2A, top right panel). By contrast the
distribution of actin was unaffected by ACK1 expression (data not
shown). Various regions of ACK1 were then tested for their ability to
induce this alteration in clathrin distribution (as summarized in Fig.
2C). The N-terminal kinase/SH3 domains (1) did not
exhibit such an effect (not shown), whereas the C-terminal half of ACK1
(middle frames) induced clathrin redistribution to the same
extent as the full-length protein. A minimal clathrin binding domain
ACK1561-593 did not induce aggregation but did disrupt the
normal perinuclear distribution of clathrin (not shown). Thus, clathrin
redistribution (clustering) may require ACK1 C-terminal sequences to
form multivalent interactions with other proteins. This redistribution
of clathrin was reflected in the loss of receptor-mediated endocytotic
function as assayed by transferrin uptake in A431 cells (Fig.
2B). Similarly, ACK1514-1037 containing
sequences C-terminal to the p21 binding domain inhibited transferrin
uptake into transfected cells. However, smaller ACK constructs (compare
ACK514-927) apparently induced clathrin aggregation while
having little effect on transferrin uptake as summarized in Fig.
2C.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
ACK can cause reorganization of clathrin
in vivo. Panel A, fluorescence
micrograph of COS-7 cells showing the distribution of ACK1 and of
clathrin in untreated cells. COS-7 cells were transfected with
full-length ACK1 and immunostained with affinity-purified anti-ACK1
(right frames) and anti-clathrin antibody (left
frames). Panel B, fluorescence micrograph of A431 cells
showing that overexpression of ACK1 blocks transferrin uptake. A431
cells were transfected with FLAG-tagged ACK1 and incubated with Texas
Red-conjugated transferrin for 15 min. Localization of the FLAG epitope
(ACK1, right frame) and labeled transferrin (left
frame) is shown. Panel C, summary of the effects of
various ACK constructs on clathrin redistribution in COS-7 cells or
transferrin uptake in A431 cells. The data represent observations made
on at least 30 cells in two separate sets of transfection
experiments.
|
|
ACK Provides a Link between Clathrins and Signaling Adaptor
Proteins--
The tyrosine phosphorylation of ACK2 in response to
integrin (15) and of ACK1 in response to melanoma chondroitin sulfate proteogylcan has been noted (22), where the kinase is linked to these
pathways via p130Cas (22). This important adaptor is known to provide
docking sites for other key SH2-containing partners including Nck and
Src (23). A striking feature of ACK is the presence of multiple
proline-rich motifs in the C-terminal half with substantial similarity
to the gene 33 protein, being potential binding sites for SH3 or
WW domain-containing proteins. The adaptor Nck (containing three SH3
domains and one SH2 domain) was tested for an association with ACK1.
This interaction has recently been documented between
Drosophila DACK and Dock, the fly Nck homolog (24). The
striking segregation of ACK1 and clathrin in cultured cells (Fig. 2)
allows in vivo association to be tested by co-localization. As shown in Fig. 3A, the
distribution of Nck was shifted dramatically by co-expression of ACK1,
indicating an association of the proteins through SH3 rather than SH2
domains. This was confirmed by blot overlay (18) using the Nck SH3
domains as a probe (data not shown). Consistent with this,
-PAKK298A (the catalytic inactive form that binds most
tightly to the second SH3 domain of Nck) did not segregate with ACK1
complexes present in the cell (Fig. 3A). Most likely a
trimeric ACK·Nck·PAK complex is not seen because the second SH3
domain of Nck is involved in binding both partners.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 3.
ACK1 can associates with Nck and Src.
Panel A, wild type or kinase-inactive ACK1K158R
was co-transfected with an expression vector encoding GST-Nck into
COS-7 cells. Both ACK constructs caused Nck redistribution from
peripheral and diffuse intracellular locations (left frames)
into punctate vesicles (right frames) that co-localized with
ACK (not shown). In cells expressing ACK1, Nck, and kinase-inactive
PAKK298A, PAK did not relocalize to the
ACK/Nck-containing vesicles, as would be expected if the three formed a
complex; rather the PAK remained in elongated structures corresponding
to focal complexes. Panel B, various GST-ACK proteins
(numbered 1-5) were separated on 9% SDS-polyacrylamide
gels and transferred onto polyvinylidene difluoride membranes. The
filters were denatured and renatured (9) and incubated with
125I-labeled GST/Src-SH3 protein (10 µg/ml) for 2 h,
washed, and exposed to film overnight. Stars show positions
of the nonbinding proteins. The right side shows
schematically the ACK1 constructs used to test Src SH3 binding. The
filled boxes (gray) represent positive
association, clathrin binding domain, CBD.
|
|
An interaction of Src SH3 with arrestin is implicated in the coupling
of signals between G protein-coupled receptors and downstream pathways
(25), indicating that Src kinases may play a general role in
down-regulation of receptors. We also detected a co-localization of
ACK1 and Src (data not shown) as for Nck. From analysis of various ACK
constructs, at least two proline-rich Src-SH3 interacting regions
(490-646 and 705-787) were identified by overlay (Fig. 3B). Because a significant fraction of cellular Src in
fibroblasts is associated with endosomes and can enhance EGF receptor
internalization (26), a cooperative role of Src with ACK1 is clearly a
possible mechanism of action.
Identifying Key Residues in ACK1 Required for Clathrin
Binding--
-Arrestin and arrestin-3 have been found to contain a
common region that acts as a clathrin adaptor and which is not present in the visual arrestins (17). This region of
-arrestin has sequence
similarity to part of ACK561-593 and the "clathrin
box" of other proteins (Fig.
4A), including the
1,
2,
and
3 adaptins, epsin, AP-180, and amphiphysins (27, 28). Although
only leucine is completely conserved among these binders, other
candidate clathrin binders including SBP56 (29), SH3 and
ankyrin repeat containing SHANK isoforms (30) were
identified. Interestingly, SBP56 has recently been purified as a factor
that can stimulate intra-Golgi transport (31).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4.
Residues required for efficient clathrin
binding. Panel A, sequence comparison of the core
clathrin binding sequences derived from ACK, rat arrestin-3, human
selenium-binding protein (SBP56), amphiphysin-2, the AP-3 subunit
3A, yeast Ent1p, and rat epsin 1. The two putative clathrin binding
sequences in Drosophila ACK and C. elegans ARK-1
are shown below. Panel B, various GST-ACK, GST-SBP56, or
GST-arrestin-3-derived peptides were expressed, immobilized on a
glutathione-Sepharose 4B column, and tested for clathrin binding as for
Fig. 1. Proteins were separated on 9% SDS-polyacrylamide gels,
transferred to polyvinylidene difluoride membranes, Coomassie stained
(upper frame), and probed with anti-clathrin monoclonal
antibody (lower frame). Exposures shown were for 1 min. The
constructs correspond to residues of human sequences (in parentheses):
ACK1 19-mer (564), ACK1 22-mer (561), SBP56 19-mer
(437), SBP56 24-mer (432), arrestin-3 19-mer (367), and
arrestin-3 26-mer (360). Panel C,
GST-ACK564-582 containing single point substitutions as
indicated above each lane were analyzed for clathrin binding
as for Fig. 1 using anti-clathrin antibodies.
|
|
GST fusion proteins displaying peptides corresponding to sequences from
ACK1,
-arrestin, and SBP56 were generated and assessed for their
clathrin binding potential (Fig. 4B). An ACK 19-mer bound
efficiently to clathrin, but the
-arrestin 19-mer or an SBP56 19-mer
interacted only weakly. Although a larger
-arrestin 26-mer bound
clathrin, the SBP56 24-mer contained core sequences essentially
identical to those of ACK1, it interacted only weakly under these
conditions. The clathrin binding of SBP56 may require flanking
sequences to adopt a higher affinity binding state. The suggestion that
SBP56 stimulates intra-Golgi transport (31) via a clathrin-binding
protein is intriguing.
Site-directed mutagenesis was used to introduce amino acid
substitutions within GST-ACK1561-593 in the core clathrin
box (Fig. 4A). Substitutions of four of the core residues
prevented clathrin binding (Fig. 4C), whereas the fifth
position was unaffected by the glycine
alanine change. The
hydrophobic residues within the core motif (i.e. ACK1
Leu-570, Ile-571, and Phe-573) play similarly important roles in other clathrin binders (32). We conclude that a central motif (LIDF) corresponding to residues 575-580 is indeed essential for clathrin binding, as has been found with other adaptors (28). Further, we were
able to identify similar motifs (Fig. 4A) in
Drosophila ACK and the recently described C. elegans homolog ARK-1 (16), although the C-terminal halves of
these proteins are otherwise dissimilar.
ACK and Arrestin Compete for a Common Binding Site on the
N-terminal Head Region of Clathrin--
Clathrin forms a trimeric
complex of three heavy and light chains in solution. These assemble
into cage-like structures under appropriate condition with the globular
head domain facing inward (19). To confirm that ACK interacted with
this head domain, clathrin immobilized on GST-ACK443-646
was subjected to proteolytic digestion with subtilisin in
situ, then flow-through and bound fractions were analyzed (Fig.
5A). After the ~160-kDa
clathrin heavy chain (Fig. 5A, second
lane) was fragmented by subtilisin, and the ~50-kDa
N-terminal globular "head" domain (19) remaining in the bound
fraction (fourth lane) was detected by an anti-clathrin (N
terminal) antibody.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
ACK and arrestin compete for a common binding
site in the N-terminal head region of clathrin. Panel
A, proteolytic digest of clathrin bound to GST-ACK. Clathrin bound
to GST-ACK561-593 was untreated (left side) or
subjected to proteolysis by subtilisin in situ, then washed
with buffer alone (FT) or containing 10 mM
glutathione (B). Anti-clathrin (N-terminal domain) was
detected by Western analysis. Panel B, competition assay of
ACK and arrestin for clathrin. In vitro translated
clathrin1-579 was incubated with
-arrestin360-385 bound to glutathione-Sepharose in the
presence of either ACK564-582 pep-1 (SGAEVTLIDFGEEPVVPAL)
or using the reverse sequence pep-2. Eluted proteins were analyzed by
Western blotting with anti-clathrin antibody.
|
|
Residues within the globular clathrin N-terminal domain are
involved in associating with
-arrestin (17). Because ACK and
-arrestin probably bind to the same surface we next tested whether ACK and
-arrestin compete for clathrin binding. Synthetic peptides (19-mers) corresponding to the ACK clathrin binding sequence pep-1 and
a control reverse peptide (pep-2) were synthesized. In vitro translated clathrin1-579 was passed through columns
containing immobilized GST/
-arrestin360-385, in the
presence of these peptides (Fig. 5B). ACK pep-1 but not pep-2 (reverse sequence) could compete with
-arrestin for binding to
both clathrin1-579 (head) and full-length clathrin (from
the reticulolysate). The latter binds more efficiently as a
consequence of its multivalent character. The high peptide
concentration required for competition probably reflects the dimeric
nature of the immobilized GST-arrestin360-385 and the
smaller length of competitor (i.e. GST-26-mer
versus 19-mer competitor). Thus both ACK and
-arrestin
bind to the same site on the clathrin head domain.
Localization of GFP-ACK1 in NIH 3T3 Cells--
Because transient
overexpression of ACK leads to deleterious effects on the general
distribution and function of clathrin, we sought to localize directly a
GFP-tagged version of ACK1 protein expressed at low level in cultured
cells. Antibodies raised against the conserved N-terminal region of
ACK1 recognized an endogenous ACK species in COS-7 and NIH 3T3 cells
that co-migrated with transfected HA-ACK1 (Fig.
6A), suggesting that this is
the endogenous species not the smaller ACK2 protein. However, our
antibody did not detect ACK by immunofluorescence efficiently.
Therefore, a pool of NIH 3T3 cells stably expressing GFP-ACK was
selected and passaged six times (~12 divisions) to confirm viability;
among this population ~20% of the cells stained positive for
anti-GFP, although the GFP fluorescence itself was barely detectable.
The distribution of GFP-ACK1 in these cells (using anti-GFP antibodies)
was punctate with no overall disruption to the perinuclear and
vesicular clathrin localization, compared with the nonexpressing cells
(Fig. 6B). At high magnification ACK1 indeed co-localized to
a subpopulation of clathrin vesicles (Fig. 6C), although we
noted that most of the vesicles showed only weak clathrin staining
(marked by arrows). Thus in the merged image
(right) it appears that many (green) ACK vesicles
lack clathrin.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 6.
GFP-ACK1-expressing NIH 3T3 cells exhibit
normal clathrin distribution. Panel A, NIH 3T3
(lane 1) and COS-7 cells (lanes 2 and
4) express an endogenous ACK protein with mobility of ~150
kDa which matches that of transient HA-ACK1 in COS-7 cells (lane
3, marked by an asterisk). Lane 5 shows that
GFP-ACK1 migrates with the expected size of ~175 kDa. Panel
B, stable GFP-ACK1-expressing NIH 3T3 cells exhibit punctate
staining for GFP-ACK1, whereas the perinuclear clathrin distribution is
normal compared with nonexpressing neighbors. Bar = 10 µM. Panel C, higher magnification showing
co-localization of GFP-ACK1 vesicles with clathrin. Many vesicles
exhibit only weak clathrin staining, as indicated by the
arrowheads. In the merged image these appear
green rather than yellow.
|
|
Two major populations of clathrin-containing vesicles termed AP-1 and
and AP-2 complexes are well characterized. AP-1-containing vesicles,
which are marked by
-adaptin, are thought to be primarily trans-Golgi originating vesicles that are sensitive to
brefeldin A. As seen in Fig.
7A, brefeldin A causes loss of
punctate
-adaptin distribution but had no effect on ACK. At higher
magnification it was clear that the predominantly perinuclear
-adaptin never co-localized with GFP-ACK. By contrast there was
significant co-localization with the
-adaptin in the perinuclear
region, although it was apparent that many of the peripheral located
AP-2 vesicles did not contain ACK.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7.
ACK1 is present in a brefeldin-A
(BFA)-insensitive pool of vesicles and co-localizes
with -adaptin. Panel A, the
punctate perinuclear distribution of -adaptin-containing AP-1
vesicles was disrupted by a 30-min brefeldin A treatment, but GFP-ACK1
distribution was unaffected. Bar = 10 µM.
Panel B, magnified view of GFP-ACK1 vesicles
co-stained with -adaptin (arrows), which marks AP-2
complexes.
|
|
Because AP-2 is involved in receptor-mediated endocytosis and the
related
-arrestin acts as a clathrin adaptor (17) it seems likely
that ACK1 affects clathrin-mediated endocytosis. In the stable GFP-ACK
cell lines we noted enhanced transferrin-receptor endocytosis relative
to control cells (data not shown). To confirm this effect we analyzed
cells expressing moderate levels of transiently expressed ACK1 or the
kinase-inactive ACK1-K168R. Texas Red uptake over a 15-min labeling
period was monitored in transiently transfected COS-7 cells and
quantified from the fixed cell images (using NIH Image software). This
revealed that both ACK1 constructs could stimulate transferrin uptake
as illustrated in Fig. 8, A
and B. Thus the stimulatory effect was not dependent on
kinase activity.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of GFP-ACK1 on transferrin
uptake. Panel A, uptake of Texas Red-labeled
transferrin (Tr-transferrin) into COS-7 cells can be
increased by expression of ACK1. Transiently transfected cells were
stained for anti-ACK1; the nuclear staining is background. The
photomicrograph illustrates inhibition of Texas Red-labeled transferrin
uptake at higher levels of GFP-ACK1 expression (1) and
enhanced uptake at lower levels (2) compared with control
cells (stars). Panel B, distribution of
transferrin uptake in cells expressing low levels of GFP-ACK or
kinase-inactive (KD) ACK(K158R) is similar. Amounts of Texas
Red-labeled transferrin in individual COS-7 cells transiently
expressing low levels of kinase (obtained in three separate
experiments) are expressed as total pixel density/cell. The profile
indicates that both active and inactive kinase can stimulate uptake,
although inhibition is observed in a portion of cells.
n = total number of cell counted.
|
|
 |
DISCUSSION |
Several Cdc42 targets have been identified, and possible roles of
these effectors have been reported (12). The Cdc42 binding region
(present also in Drosophila and C. elegans
ACK-related kinases) is thought to differ somewhat from those of WASP
and PAK where residues C-terminal to the GTPase binding domain regulate intramolecular interactions (33). Thus it is possible that Cdc42·GTP targets ACK but plays no role in modulating kinase function.
Taking our data with the recent report that an ACK1 homolog ARK-1
inhibits EGF receptor signaling in C. elegans (16), it is
tempting to speculate that this occurs by a stimulation of clathrin-mediated receptor endocytosis. This idea is strengthened by
the observation that ARK-1 kinase activity is dispensable for this
function which instead resides in the C-terminal half of ARK-1 (16).
Similarly expressing ACK1 or a kinase-inactive mutant alters
endocytosis similarly. We have suggested a region of ARK-1 to be
involved with binding clathrin (Fig. 4A), but this remains to be validated. ARK-1 is also implicated in an interaction with the
Grb2 homolog SEM-5 thereby linking to the let-23 EGF-like receptor. Multiple proline-rich domains in the C-terminal half of ACK1
appears to provide SH3 docking sites for other adaptors, including Nck
and Src as we show here (Fig. 3) potentially linking to tyrosine kinase
receptors via SH2 interactions. Another negative regulator of EGF
receptor signaling is the unc-101 gene encoding a homolog of
the clathrin-associated AP-47, a medium sized component of the AP-1
complex (34). One can envisage that either a defective trans-Golgi transport system (in unc-101 mutants)
or endocytic system (in ark-1 mutants) might lead to the
potentiation of EGF signaling because of a sustained presence of the
active receptor on the cell membrane.
The ability of overexpressed ACK to induce clathrin aggregation relates
to activities associated with its C-terminal region (i.e.
kinase-independent). As a consequence transient overexpressed ACK
blocks transferrin uptake (Fig. 2). Such general disruption to
clathrin-mediated endocytic events as has also been seen with epsin
over expression (28). Gene 33 also known as receptor-associated late
transducer (RALT), which is homologous to ACK1, has recently been found to bind to ErbB2 receptor complexes (21), consistent with it
providing a negative feedback loop via receptor down-regulation.
The clathrin binding region in ACK1 is almost identical to a sequence
in SBP56 (Fig. 4A), and yet it binds poorly to clathrin, perhaps because of the presence of lysine C-terminal to this box (acidic residues are common here). Porat et al. (31)
conclude from their studies that SBP56 is required at a late stage of
intra-Golgi transport (i.e. docking or fusion), although the
protein is primarily cytosolic (31). Binding of ACK (or SBP56) to
clathrin is perhaps controversial because the recent structure of the
clathrin head with peptides derived from
-arrestin-2 or the
-subunit of AP-3 reveals significant interactions of their fifth
acidic residue in the clathrin box with basic residues in clathrin
(35). This position in ACK contains a glycine, however, but one should
note that an equivalent residue in epsin is alanine (Fig. 4A
and Ref. 28). Further, our mutagenesis data confirm that this glycine can be replaced by alanine with no effect on binding, whereas substitution of the other four residues essentially leads to complete loss of interaction. This suggests that binding of the ACK peptide to
the major groove in the clathrin head occurs through a distinct set of
interactions, perhaps involving a different peptide conformation. Certainly the ACK 24-mer binds to clathrin as efficiently as the arrestin 26-mer peptide (Fig. 4B).
Clathrin adaptors are not necessarily concentrated in clathrin-coated
vesicles, for example neither amphiphysin nor epsin is enriched in
purified clathrin-coated vesicles (36). Although the specific role of
ACK in clathrin-coated vesicle-mediated endocytosis remains to be
established, the observation that ACK is associated with vesicles
relatively depleted in clathrin (Fig. 6) suggests an association with
partially uncoated vesicles, an event occurring prior to docking or
fusion with endosomes. ACK-2 is responsive to signaling by receptor
tyrosine kinases or heterotrimeric G protein-coupled receptors (15).
The nonvisual arrestins,
-arrestin and arrestin-3, promote G
protein-coupled receptor internalization by binding to clathrin and
promoting assembly of endocytic vesicles (17). We show here that
elevated ACK1 can both stimulate and inhibit the rate of transferrin
uptake (depending on the level of the protein). Such stimulated
transferrin uptake might result from a general stimulation of clathrin
assembly on the plasma membrane, similar to overexpression of
transferrin receptor itself (36) or HIV Nef protein (37). Although
tyrosine phosphorylation of integral membrane proteins can mediate
endocytosis (38) we do not observe any requirement for ACK1 catalytic activity.
Although only Rho and Rac were previously implicated in
receptor-mediated endocytosis (39), the involvement of Cdc42 is not
unexpected. It is already known that disruption of the Golgi apparatus
by brefeldin A blocked cell polarization and directed cell migration
(40), processes under the control of Cdc42. This GTPase is
predominantly localized to the Golgi (41) and associated in a
GTP-dependent manner with the coatomer protein
-COP (7). The serine/threonine kinase PAK4 preferentially
interacts with Cdc42·GTP and is recruited to the Golgi apparatus (42)
where it might also contribute to coatomer function. The recent
observation that DACK is implicated upstream of Drosophila
SH3PX1 (24), a mammalian protein related to the sorting nexins, again
implicates ACK in membrane trafficking. The possibility that mammalian
SH3PX1 forms a complex with ACK1 is under investigation.