From the Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135
Received for publication, October 9, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Membrane-associated guanylate kinase homologues
(MAGUKs) are generally found under the plasma membrane
of cell-cell contact sites and function as scaffolding proteins by
linking cytoskeletal and signaling molecules to transmembrane
receptors. The correct targeting of MAGUKs is essential for their
receptor-clustering function; however, the molecular mechanism of their
intracellular transport is unknown. Here, we show that the guanylate
kinase-like domain of human discs large protein binds directly within
the amino acids 607-831 of the stalk domain of GAKIN, a kinesin-like protein of broad distribution. The primary structure of the binding segment, termed MAGUK binding stalk domain, is conserved in
Drosophila kinesin-73 and some other motor and non-motor
proteins. This stalk segment is not found in GKAP, a synaptic protein
that interacts with the guanylate kinase-like domain, and unlike GKAP,
the binding of GAKIN is not regulated by the intramolecular
interactions within the discs large protein. The recombinant motor
domain of GAKIN is an active microtubule-stimulated ATPase with
kcat = 45 s Membrane-associated guanylate kinase homologues
(MAGUKs)1 are a family of
proteins composed of one or more PDZ domains, an SH3 domain, and a
guanylate kinase-like (GUK) domain (1). They are thought to play
scaffolding functions at specialized membrane sites, such as synaptic
membrane, tight junction, and adherens junction (2-4).
Drosophila Dlg is a MAGUK encoded by lethal (1) discs
large-1 tumor-suppressor gene (dlg), and mutations of
dlg cause neoplastic overgrowth of the imaginal discs (5).
The Dlg protein localizes to the septate junctions in epithelial cells where it regulates cell proliferation, apical-basal cell polarity, and
the organization of junctional structure (6-8). The human homologue of
Drosophila Dlg, termed hDlg, and its rat counterpart SAP97,
localize at the pre- and postsynaptic membrane sites as well as the
basolateral membrane of epithelial cells (9, 10) and are proposed to
perform scaffolding functions by linking cytoskeletal components to the
transmembrane proteins (11). In addition to the scaffolding function,
mounting evidence now indicates that hDlg regulates cell proliferation
and could be involved in tumorigenesis. For example, hDlg interacts
with viral oncoproteins such as high-risk human papillomavirus E6 and
human T-cell leukemia virus type 1 (HTLV-1) Tax (12-14). Similarly,
hDlg forms a complex with adenomatous polyposis coli (APC) tumor
suppressor gene product and negatively regulates cell cycle progression
(15, 16). The mechanism of how hDlg regulates cell proliferation is
still largely unknown.
Recently, we identified GAKIN (guanylate
kinase-associated kinesin), which is also
classified as human KIF13B (17), from Jurkat T lymphoma cells as a
binding partner for the GUK domain of hDlg (18). In T cells, hDlg
interacts with tyrosine kinase Lck and potassium channel Kv1.3 (19) and
translocates to the immune synapse-like structure upon cross-linking of
cell surface CD2 molecules (18). These observations suggest that hDlg
might play a role in the formation of physical contacts between T cells and antigen-presenting cells and regulate activation of T cells during
immune response. An intriguing possibility emerges suggesting the role
of GAKIN in the transport of hDlg to the immune synapse upon activation
of T cells. Since transcripts of GAKIN and hDlg are ubiquitously
expressed, it is conceivable that GAKIN-dependent trafficking is a widespread mechanism across species and tissues for
the transport of hDlg and other MAGUKs. Consistent with this paradigm
is the recent evidence indicating a role of soluble adaptor proteins in
the transport of cargo vesicles via kinesin-like motors. For example,
KIF17 motor interacts with mLin-10, which in turn mediates its
interaction with the cargo vesicles containing NMDA receptor subunits
(20). Similarly, KIF13A motor binds to a subunit of AP-1 complex
mediating its interaction with the vesicles containing mannose-6-phosphate receptor (21). Conventional kinesin, via its light
chain, binds to c-Jun N-terminal kinase-interacting proteins
that mediate interaction with specific cargo vesicles (22). Since hDlg
is a soluble scaffolding protein, it can in principle link cargo
vesicles to an intracellular motor, and therefore this property fits
well with the common paradigm of being a motor-cargo adaptor molecule.
The guanylate kinase-like domains of MAGUKs exhibit little or no
guanylate kinase activity (23), and their principal function seems to
serve as a protein-protein interaction motif (18, 24). Besides GAKIN,
several proteins are reported to interact with the GUK domains of
MAGUKs. These GUK-binders include GKAP (GUK-associated protein) (24),
MAP1A (microtubule-associated protein 1A) (25), BEGAIN (brain-enriched
guanylate kinase-associated protein) (26), a Rap specific
GTPase-activating protein SPAR (27), and protein kinase A-anchoring
protein AKAP79 (28). At this stage, it is not clear how a single GUK
domain binds to so many seemingly non-related proteins and how these
interactions are regulated. Recent determination of the crystal
structures of PSD-95 revealed the existence of a novel mode of
intramolecular interactions between the SH3 and GUK domains, providing
a new perspective on the functional regulation of hDlg interactions
(29-32). Interestingly, the binding of GKAP to the GUK domain of SAP97
is regulated by a series of intramolecular interactions between the SH3
and GUK domains (33). The PDZ domains regulate MAP1A binding to the GUK
domain intramolecularly, although their mechanism of regulation seems
distinct from that of GKAP (25). In this manuscript, we provide
evidence for the existence of a novel protein-binding domain that links
GAKIN to the GUK domain of hDlg. The binding mode between GAKIN and
hDlg appears to be distinct from that of GKAP binding to hDlg/SAP97.
Our results also suggest that GAKIN mediates intracellular trafficking
of Dlg in epithelial cells.
Cell Culture--
Jurkat J77 cell line was maintained in RPMI
1640 (Sigma) medium supplemented with 10% fetal bovine serum
(HyClone), 2.0 mM glutamine (Sigma), and 1.0 mM
sodium pyruvate (Sigma). MDCK cells (a gift from Dr. Alan S. Fanning)
were maintained in Dulbecco's modified Eagle's medium
(Invitrogen) with 10% fetal calf serum.
Affinity Purification--
GST-GUK and control GST beads were
prepared by coupling the proteins to cyanogen bromide-activated
Sepharose 4B (Amersham Biosciences). Beads were incubated with Jurkat
cell lysate (5 × 108 cells) prepared in lysis buffer
(50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 2.0 mM phenylmethylsulfonyl fluoride,
1% Triton X-100, and 0.5% Nonidet P-40) for 18 h at 4 °C.
Beads were washed with lysis buffer and boiled in SDS-PAGE sample
buffer to elute bound proteins. Proteins were resolved by SDS-PAGE and visualized by silver staining.
In Vitro Binding Assay--
Segments of GAKIN were amplified by
PCR and cloned into the pET32a vector (Novagen). Radiolabeled proteins
were prepared using the STP3 in vitro translation system
(Novagen) in the presence of [35S]methionine. After the
completion of protein synthesis, lysate was diluted 10-fold in binding
buffer (phosphate-buffered saline with 1% Triton X-100) and incubated
with either GST or GST-GUK beads for 2 h. Beads were washed with
binding buffer, and protein was recovered and analyzed by SDS-PAGE and
fluorography. A direct binding assay was performed by incubation of
purified recombinant GAKIN-(487-989) with beads containing
either GST or GST-GUK in the binding buffer for 2 h. Beads were
recovered by centrifugation, washed with binding buffer, and analyzed
by SDS-PAGE and Coomassie staining.
Recombinant Protein Expression--
The motor domain of GAKIN,
corresponding to amino acids 1-368 (G368), was cloned in the pET32a
vector (Novagen) that contains a thioredoxin tag, an S tag, and a His
tag at the N terminus of the cloned fragment. Recombinant G368 was
expressed in Escherichia coli BL21-Gold (DE3) cells
(Stratagene) by induction with 0.05 mM
isopropyl-1-thio-
GAKIN-(487-989) was cloned into pRSETA (Invitrogen) that contains a
His tag at the N terminus. His-tagged GAKIN-(487-989) was expressed in
BL21-Gold (DE3) by induction with 0.1 mM
isopropyl-1-thio- Microtubule-stimulated ATPase Assay--
Tubulin was isolated
from cow brain following the method of Williams and Lee (34). This
method utilizes two cycles of polymerization and phosphocellulose
chromatography. Microtubules were obtained by polymerization of tubulin
with taxol (Calbiochem). Large protein aggregates were removed by
centrifugation, and microtubules were quickly frozen in liquid nitrogen
and stored at Transient Transfection Assay and Immunofluorescence
Analysis--
Constructs encoding full-length GAKIN and headless
GAKIN-(423-1826) were cloned into the pEGFP-C1 vector
(Clontech), which expresses the GFP at the N
terminus of the cloned fragment. MDCK cells were grown on glass
coverslips and transient transfections were performed using
LipofectAMINE 2000 (Invitrogen). Cells were incubated for 24 h
post-transfection, fixed with 4% paraformaldehyde, permeabilized in
0.1% Triton X-100, stained with an anti-hDlg monoclonal antibody
(2D11), and visualized using Texas Red-conjugated goat anti-mouse
secondary antibody (Molecular Probes). Images of GFP and Texas Red were
visualized by confocal microscopy.
Direct Interaction of GAKIN with hDlg--
Previously we
identified GAKIN as a prominent phosphorylated substrate by an in
vitro kinase reaction of the protein complex that was isolated by
a pull-down assay with beads containing the GUK domain of hDlg (18). To
determine the molar stoichiometry of proteins pulled down by this
assay, we covalently conjugated the GST-GUK domain of hDlg to the
cyanogen bromide-activated Sepharose beads. This step was necessary to
eliminate the interference by GST-GUK fusion protein during SDS-PAGE
analysis. The GST-GUK-conjugated Sepharose beads were incubated with
detergent lysate of human T lymphocyte Jurkat cells, and bound proteins
were analyzed by SDS-PAGE. Silver staining of the pull-down complex
revealed a major protein of ~250 kDa, later identified as GAKIN, that
bound to the GUK domain of hDlg (Fig.
1A). Several minor bands
corresponding to the molecular mass of ~70-100 kDa (shown by
asterisk in Fig. 1A) were also present in the pull-down
complex recovered from Jurkat cell lysate. Since the molar amount of
these minor bands is relatively small as compared with GAKIN, it is
unlikely that these proteins function as adaptors to link GAKIN with
the GUK domain of hDlg. Since native GAKIN is intensely phosphorylated in the in vitro kinase reaction of the pull-down complex
(18), it is likely that one of these bands could represent a protein kinase that specifically phosphorylates GAKIN. In any event, the dominant stoichiometry of GAKIN in the pull-down complex suggested that
it might have a direct interaction with hDlg. To test this possibility,
we performed an in vitro binding experiment using purified
recombinant proteins. We have shown that a segment of GAKIN-(487-989)
expressed in reticulocyte lysate is sufficient to bind to the GUK
domain of hDlg (18). Recombinant GAKIN-(487-989) was expressed as a
His-tagged fusion protein in E. coli and purified to
homogeneity by nickel-agarose chromatography. Purified GAKIN-(487-989) was incubated with either GST or GST-GUK domain conjugated to glutathione-Sepharose beads. Bound proteins were recovered by centrifugation and analyzed by SDS-PAGE. Coomassie Blue staining revealed specific binding of GAKIN-(487-989) to GST-GUK beads but not
to the control GST beads (Fig. 1B). Western blotting
confirmed the identity of the bound protein as His-GAKIN-(487-989)
(Fig. 1C). These results demonstrate that GAKIN binds
directly to the GUK domain of hDlg.
A Novel Stalk Segment of GAKIN Binds to the GUK Domain of
hDlg--
To map the hDlg-binding site within GAKIN, a series of
truncated constructs of GAKIN were engineered and expressed in the rabbit reticulocyte transcription/translation system. The GUK-binding activity of these constructs was measured by the sedimentation assay as
described under "Materials and Methods." As shown in Fig.
2A, a specific region of GAKIN
corresponding to amino acids 607-831 emerged as the minimum binding
region required for binding to the GST-GUK domain of hDlg. We named
this 224-amino acid segment of the stalk domain of GAKIN as the MBS
(MAGUK binding stalk) domain. Sequence alignment analysis revealed that
the MBS domain is a fairly conserved module found in several proteins
(Fig. 2B). For example, the amino acid sequence of
Kinesin-73, a Drosophila orthologue of GAKIN, shows 60%
similarity with human GAKIN within the MBS domain (36). The MBS domain
is also found in the KIF13A (64% identity) and the KIF1 family of
kinesin-like motor proteins (37). A data base search identified two MBS
domains in tandem within the N-terminal half of RIM-BP1, a neuronal
protein that was isolated by a yeast two-hybrid screen using RIM1 as
bait (38). Invertebrate homologues such as Anopheles
gambiae ebiP9361 and Caenorhabditis
elegans UNC-104 and KLP-4 also contain a copy of the MBS domain.
The presence of the MBS domain in a variety of proteins suggest that it
may represent a general GUK-binding motif, although we could not detect
an interaction between human KIF1A and the GUK domains of hDlg, human
PSD-95, and human p55 (data not shown). Clearly, further mapping of the
MBS domain as well as crystallographic determination of its
three-dimensional structure are required to determine the precise
boundaries of this newly identified protein-binding module for future
identification of novel binding partners.
Intramolecular Interactions Do Not Regulate GAKIN Binding to the
GUK Domain of hDlg--
The MBS domain of human GAKIN shows no
sequence similarity within the primary structure of human or rat GKAP,
another GUK-binding protein (24, 39, 40). This observation suggests
that the GKAP and GAKIN might bind to the GUK domain of hDlg by a
distinct mechanism. It is noteworthy here that the binding of rat brain GKAP to the GUK domain of SAP97, a rat homologue of hDlg, is regulated by intramolecular interactions of SAP97 (33). Binding of GKAP to the
GUK domain of SAP97 is inhibited by the presence of SH3 domain, and as
a consequence of this intramolecular regulation, GKAP does not bind to
a construct containing both SH3 and GUK domains (33). To test whether a
similar intramolecular mechanism modulates GAKIN binding to the GUK
domain of hDlg, we expressed full-length hDlg, an SH3-GUK construct,
and only the GUK domain of hDlg as GST fusion proteins in bacteria
(Fig. 3A). The radiolabeled GAKIN-(607-831) was expressed, and its binding was measured as described under "Materials and Methods." As shown in Fig.
3B, GAKIN binding to all three constructs of hDlg was
similar suggesting that, unlike GKAP, binding of GAKIN to the GUK
domain of hDlg is not regulated by the intramolecular interactions
between SH3 and GUK domains of hDlg.
GKAP Competes with GAKIN for Binding to the GUK Domain of
hDlg--
Since binding of GAKIN to the GUK domain of hDlg was not
regulated by intramolecular interactions between the SH3 and GUK domains, we speculated that the binding sites of GAKIN and GKAP might
be distinct within the GUK domain of hDlg. We examined this possibility
by testing whether GKAP could compete with GAKIN for binding to the GUK
domain of hDlg. Recombinant full-length GKAP of rat origin was
expressed in Sf9 cells and purified (see "Materials and
Methods"). Similarly, the recombinant GAKIN-(487-989) was expressed
in bacteria and purified (see "Materials and Methods"). Unlabeled
GAKIN-(487-989), designated as cold GAKIN, was added to the reaction
mixture to displace/inhibit binding of 35S-labeled
GAKIN-(607-831) to the GUK domain of hDlg. Binding of 35S-labeled GAKIN-(607-831) to the GUK domain of hDlg was
inhibited by recombinant cold GAKIN-(487-989) indicating successful
displacement of the binding reaction under these conditions (Fig.
4A). Contrary to our
expectations, recombinant GKAP quantitatively competed with
35S-labeled GAKIN-(607-831) for binding to the GUK domain
of hDlg (Fig. 4B). This result suggests that both GKAP and
GAKIN presumably bind to the same region on the GUK domain of hDlg.
Alternatively, GKAP and GAKIN could bind to distinct sites within the
GUK domain of hDlg, but their physical proximity is such that the
binding of one occludes the binding of other under the present
experimental conditions. It is noteworthy here that we could not
express 35S-labeled full-length GKAP or its shorter
N-terminal half containing the putative GUK domain binding site using
the reticulocyte lysate in vitro expression system and
therefore could not perform the reverse competition experiment. Future
structural studies of the binary complex of each with the GUK domain
may provide further insights into their binding mode in solution.
Microtubule-stimulated ATPase Activity of GAKIN Motor
Domain--
Our next objective was to examine whether GAKIN is a
functional microtubule-dependent motor. To accomplish this
goal, we expressed the putative motor domain of GAKIN spanning amino
acids 1-368, termed G368. Recombinant G368 domain was expressed in
E. coli and purified to homogeneity as described under
"Materials and Methods" (Fig.
5A). Purified G368 protein
showed microtubule-stimulated ATPase activity with
kcat = 45 s GAKIN Transports Dlg to the Tip of Epithelial Projections--
To
investigate the functional significance of the direct interaction
between GAKIN and mammalian Dlg, we transiently expressed full-length
GAKIN as a GFP fusion protein in MDCK epithelial cells. The expression
of heterologous GFP-GAKIN induced long process structures in many
transfected cells, and interestingly, the GFP-GAKIN was concentrated
heavily at the tip of these projections (Fig. 6A). Similar accumulation of
motor proteins has been observed in overexpressed KIF13A and
conventional kinesin (21, 42), both of which are microtubule plus
end-directed kinesin-like motor proteins. Since the topology of
microtubules dictates that the tip of the cellular projection contains
the plus end of microtubules, the localization of GAKIN at the tip of
these projections suggests that GAKIN is also a microtubule plus
end-directed motor protein. In the non-transfected MDCK cells,
endogenous Dlg is localized mainly at the plasma membrane where cells
form contacts (Fig. 6B), as reported previously (9, 43).
However, endogenous Dlg accumulated at the tip of cellular projections
in the GAKIN transfected MDCK cells where it showed strong
co-localization with GFP-GAKIN (Fig. 6, B and C,
arrows). This result indicates that the localization of
endogenous Dlg was altered by the overexpression of GAKIN, possibly due
to the dysregulation of Dlg transport by the overexpressed motor. To
further confirm that the accumulation of Dlg at the tip of long
projections depends on intact functional GAKIN, we expressed a mutant
form of GAKIN that lacks its motor domain (headless GAKIN). The MDCK
cells expressing headless GAKIN also generated extended projections,
but there was no significant accumulation of headless GAKIN at the tip
of these projections (Fig. 6D, arrows). This
result suggests that the accumulation of GAKIN at the tip of these
projections requires the motor activity of the intact protein.
Consistent with this observation, the accumulation of endogenous Dlg
was not observed at the tip of these projections (Fig. 6, E
and F, arrows). It is noteworthy here that a
GFP-motor domain construct (1-557) of GAKIN failed to induce long
projections in the transfected MDCK cells suggesting the importance of
the C terminus of GAKIN in the induction of these processes in
vivo (data not shown). Together, our data indicate that Dlg is
transported to the tip of epithelial projections by GAKIN in a
motor-dependent manner and suggest that Dlg is a cargo
molecule of GAKIN in vivo.
Human Dlg protein and its rat orthologue SAP97 are localize at
specialized membrane regions where cells form contacts such as the
synaptic membrane in neuronal cells (10), adherens junctions of
epithelial cells (9, 43), and contact sites between T lymphocyte and
antigen-presenting cells (18). In contrast, hDlg/SAP97 is present
predominantly in the cytoplasm and appears to attach to intracellular
membranes in cells that do not display cell-cell contacts (43, 44). In
addition to the PDZ domains that link hDlg to the cytoplasmic domains
of transmembrane receptors (4), the primary structure of hDlg contains
multiple protein-protein interaction domains that interact with
cytoskeletal components and signaling molecules (9, 19). These multiple
protein interactions presumably permit hDlg to function as a
scaffolding protein by forming large protein complexes at the interface
of the membrane-cytoskeleton (11). A major remaining issue of
fundamental importance pertains to the mechanism of hDlg trafficking
and its delivery to specialized sites. Our original identification of
GAKIN was made by virtue of its association with the GUK domain of hDlg
in the context of whole cell lysate (18), therefore a possibility
remained that the GAKIN-hDlg interaction might not be direct. In this
manuscript, we demonstrate that GAKIN interacts directly with the GUK
domain of hDlg (Fig. 1). A unique feature of this interaction is the unusual location of the hDlg-binding region within the stalk domain of
GAKIN. The traditional view of kinesin motors implies that their
globular C-terminal tails usually serve as the cargo binding modules
(45). However, in the case of GAKIN, the MBS domain that binds to hDlg
is located within the N-terminal half of GAKIN downstream of its
N-terminal motor domain (Fig. 2). Thus, the C-terminal half of GAKIN
with a single copy of CAP-Gly domain either serves a regulatory region
for cargo binding and/or binds to distinct cargo molecules. It is
noteworthy here other intracellular motors, such as the Rab6-binding
kinesin Rab6-KIFL, have been speculated to harbor cargo-binding domains
in their coiled-coil regions (46). In any case, the presence of the MBS
domain in a variety of motor and non-motor proteins suggests that this
region might represent a novel cargo-binding motif with implications in
linking soluble adaptors to motor proteins within the scaffolding complex. For example, the KIF13A motor transports cargo vesicles via
its C-terminal tail that interacts with The MBS domain of GAKIN does not share any sequence similarity with
proteins that bind to the GUK domains of MAGUKs. Indeed, our results
indicate that binding of GAKIN to the GUK domain of hDlg is not
regulated by intramolecular interactions of the SH3 and GUK domains
(Fig. 3). Based on our observation that GKAP competes with the MBS
domain of GAKIN for binding to the GUK domain of hDlg (Fig. 4), we
speculate on a model that offers an explanation for at least one
function of the intramolecular interactions of MAGUKs. According to
this model, the MBS domain of GAKIN interacts with a "folded" state
of hDlg in the cytoplasm and transports it to specialized membrane
sites. This folded and thus closed state of hDlg does not permit
binding with other GUK domain binders such as GKAP. Once the hDlg cargo
reaches the target membrane sites, other membrane and protein
interactions "unfold" the closed SH3-GUK conformation thus
permitting the transfer of hDlg to another GUK domain binder such as
GKAP. The final assembly of the mature scaffolding complex occurs at
this site by recruitment of additional binding partners to the
"open" conformation of hDlg. Further experimental verification of
this model would require identification of other cargo molecules of the
GAKIN-hDlg complex and further investigation as to whether novel
segments of GAKIN regulate protein-protein interactions of the
scaffolding complex at or during the assembly process.
The data presented in this manuscript suggest that human GAKIN is a
biologically active kinesin motor, providing a molecular basis for the
intracellular trafficking of hDlg in mammalian cells. Our results also
suggest that direct binding of GAKIN to the GUK domain of hDlg could
permit transport of a soluble multiprotein complex to specialized
cell-cell contact sites. Alternatively, the GAKIN-hDlg interaction may
also allow trafficking of the scaffolding complex attached to the
intracellular vesicles. The hDlg-bearing vesicles are then delivered to
the plus end of microtubules by GAKIN. The proposed model of
GAKIN-dependent transport of intracellular vesicles is also
consistent with the observed punctate and vesicular distribution of
hDlg/SAP97 in neuronal, epithelial, and lymphoid cells (18, 44, 47). In
addition to hDlg, the GAKIN-dependent trafficking could
also play a pivotal role for the transport of PSD-95, based on the fact
that GAKIN binds to the GUK domain of PSD-95 and is expressed
abundantly in neuronal cells (18).
The proposed model adds a new example to the emerging general paradigm
that scaffolding adaptor proteins act as molecular links between
specific motor proteins and cargo vesicles. Recent examples supporting
this paradigm include the mLin-10 adaptor that links KIF17 motor to
vesicles via mLin-2, mLin-7, and
N-methyl-D-aspartic acid receptor subunit
complex (20), the role of JIPs in connecting the conventional kinesin
to cargo vesicles via the Reelin receptor (22), and also the GRIP1
mediated linkage of kinesin heavy chains to vesicles via an AMPA
receptor subunit (48). A more recent demonstration of SAP97 binding to
the minus end-directed actin motor myosin VI also suggests a mechanism
for the reversible translocation of MAGUKs in vivo (49). In
conclusion, the identity of various components of the hDlg and/or
PSD-95 cargo complex transported by GAKIN could open an area of
considerable interest for the assembly, transport, and regulation of
this complex and more importantly could provide insights into the
mechanism of dynamic regulation of the cell-cell contact structure in
normal and disease states.
1,
K0.5 (MT) = 0.1 µM.
Overexpression of green fluorescent protein-fused GAKIN in Madin-Darby
canine kidney epithelial cells induced long projections with both GAKIN
and endogenous discs large accumulating at the tip of these
projections. Importantly, the accumulation of endogenous discs large
was eliminated when a mutant GAKIN lacking its motor domain was
overexpressed under similar conditions. Taken together, our results
indicate that discs large is a cargo molecule of GAKIN and suggest a
mechanism for intracellular trafficking of MAGUK-laden vesicles to
specialized membrane sites in mammalian cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside at 25 °C for
14 h. Cells were recovered by centrifugation and resuspended in
lysis buffer (20 mM Tris-HCl, pH 8.0, 4 mM
MgCl2, 150 mM NaCl, 0.1 mM ATP, 0.01%
-mercaptoethanol) containing 0.5 mM
phenylmethylsulfonyl fluoride and 0.25 mg/ml lysozyme. After incubation
for 10 min at 4 °C, cells were lysed by sonication and lysate was
cleared by centrifugation. The supernatant was applied to a column of ProBond Nickel-Agarose (Invitrogen) and washed with lysis buffer containing 500 mM NaCl. Bound protein was eluted with lysis
buffer supplemented with 500 mM Imidazole. Eluted fractions
were diluted with ten volumes of buffer A (4 mM
MgCl2, 0.1 mM ATP, 0.01%
-mercaptoethanol) and applied to a column of P11 Phosphocellulose (Whatman). Following washing of the column with A25 buffer (25 mM ACES/KOH, pH
6.9, 2.0 mM magnesium acetate, 2.0 mM EGTA, 0.1 mM EDTA, 0.01%
-mercaptoethanol), protein was eluted
with A25 buffer supplemented with 800 mM NaCl. Peak
fractions were pooled and dialyzed against A25 buffer containing 50 mM KCl and 50% glycerol and stored at
80 °C.
-D-galactopyranoside at 37 °C for
6 h. Recombinant protein was recovered from inclusion bodies by
solubilizing the bacterial pellet in lysis buffer B (20 mM
Tris-HCl, pH 7.9, 500 mM NaCl, 5.0 mM
imidazole, 6.0 M urea) and applied to nickel-agarose
column. Bound protein was eluted using elution buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 500 mM imidazole, 6.0 M urea) and renatured by a
series of dialysis steps against phosphate-buffered saline containing
decreasing concentrations of urea. Full-length GST-hDlg (amino acids
1-989), GST-SH3-GUK (amino acids 566-989), and GST-GUK (amino acids
733-989) of hDlg were cloned into pGEX2T (Amersham Biosciences), and
GST fusion proteins were expressed in E. coli and purified
using glutathione-Sepharose beads. Rat brain GKAP cDNA (kindly
provided by Dr. Craig C. Garner) was cloned into pFASTBACHTb for
BAC-TO-BAC baculovirus expression system (Invitrogen). Full-length
His-tagged GKAP was expressed in Sf9 cells according to the
manufacturer's instructions and purified using ProBond
nickel-agarose chromatography (Invitrogen).
80 °C. Microtubule-stimulated ATPase activity was
measured as described previously (35). Motor protein (20 nM) was mixed with microtubules in A25 buffer containing 8.0 µM taxol and 1.0 mM ATP followed by
incubation at 25 °C. Aliquots of 20 µl were taken out at defined
time intervals, and the amount of Pi was determined using
the malachite-green method. The ATPase rate was calculated from the
linear portion of the slope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Direct interaction of GAKIN with hDlg.
A, beads containing GST (left panel) or GST-GUK
(right panel) were incubated with Jurkat cell lysate. After
extensive washing, beads were recovered by centrifugation, and bound
protein was analyzed by SDS-PAGE and silver staining. The
asterisk, *, represents unidentified bands recovered by the
GST-GUK beads. Note that a small amount of GST-GUK protein was eluted
from the affinity beads during the boiling process. B,
His-tagged GAKIN-(487-989) was expressed in E. coli BL21
(DE3), purified by nickel-agarose chromatography, and incubated with
either GST or GST-GUK immobilized on glutathione-Sepharose beads. Beads
were washed, and bound protein was resolved by SDS-PAGE. Coomassie
Brilliant Blue staining of the gel. C, the same panel was
analyzed by Western blot analysis to detect His-tagged GAKIN-(487-989)
using nickel horseradish peroxidase ExpressDetector (KPL).
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Fig. 2.
Mapping of the Dlg-binding site within
GAKIN. A, defined segments of 35S-labeled
GAKIN were expressed in vitro and tested for binding to
GST-GUK by pull-down assay. Upper panel indicates the
boundaries of constructs, left panel indicates 10% input of
expressed proteins used in the binding assay, and right
panel shows the binding of radiolabeled GAKIN to GST-GUK by
fluorography. Note that the similar size of the GST-GUK protein
somewhat interferes with the migration of the radiolabeled protein
produced by the T2 construct. B, schematic representation of
proteins containing the MBS domain.
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Fig. 3.
Intramolecular interaction of hDlg does not
regulate GAKIN binding. A, schematic representation of
GST-hDlg fusion proteins used in the binding experiment. B,
in vitro binding of 35S-labeled GAKIN MBS domain
(607-831) to GST-hDlg fusion proteins. Fluorography shows the
35S-labeled GAKIN-(607-831) recovered by GST-hDlg
fusion proteins immobilized on glutathione-Sepharose beads. GST
alone does not bind to GAKIN-(607-831), whereas full-length hDlg,
SH3-GUK, and GUK domains bound GAKIN-(607-831) with comparable
efficiency (upper panel). Coomassie Blue staining shows the
GST fusion proteins used for the binding experiment (lower
panel).
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Fig. 4.
GKAP competes with GAKIN for binding to the
GUK domain of hDlg. A, cold recombinant GAKIN competes
35S-labeled GAKIN MBS domain for GUK domain binding.
Increasing amounts of recombinant GAKIN-(487-989) were added to the
1.0-ml binding reaction containing 35S-labeled
GAKIN-(607-831) and GST-GUK immobilized on glutathione-Sepharose
beads. Beads were recovered by centrifugation, and bound
35S-labeled GAKIN-(607-831) was detected by fluorography.
This control experiment established that recombinant GAKIN effectively
competes with 35S-labeled GAKIN-(607-831) under these
experimental conditions. B, recombinant GKAP expressed in
Sf9 cells also competed with 35S-labeled
GAKIN-(607-831) for binding to the GUK domain of hDlg.
1,
K0.5 (MT) = 0.1 µM (Fig.
5B) values that are similar to the reported values for the
bacterially expressed motor domain of Drosophila kinesin
(kcat = 80 s
1,
K0.5 (MT) = 0.16 µM) (41). This
result suggests that GAKIN is a functional
microtubule-dependent motor protein.
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Fig. 5.
Characterization of microtubule-stimulated
ATPase activity of GAKIN. A, Coomassie Blue staining of
purified motor domain, G368, of GAKIN expressed in E. coli.
B, microtubule-stimulated ATP hydrolysis of G368 in the
presence of 1.0 mM MgATP.
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Fig. 6.
GAKIN transports Dlg in
vivo. MDCK cells were transiently transfected with
full-length GFP-GAKIN (A-C). Arrows indicate the
tip of extended projections where GAKIN and Dlg accumulate.
D-F show MDCK cells expressing a mutant
headless GFP-GAKIN construct. Arrows show the tip of
extended projections where neither mutant GAKIN nor Dlg accumulate.
A and D, GFP-GAKIN; B and
E, endogenous Dlg stained with an anti-hDlg monoclonal
antibody; and C and F, overlay images. It is
noteworthy here that endogenous Dlg is predominantly cytoplasmic in
non-confluent cells and is localized to the plasma membrane at the
regions of cell-cell contact in confluent cells. The induction of long
projections was abolished when cells were treated with nocodazole (0.05 µg/ml) for 2 h (data not shown) suggesting the dependence of
GAKIN and Dlg transport on the presence of intact microtubule
network.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-adaptin and
mannose-6-phosphate receptor (21). Our identification of the MBS domain
in KIF13A raises the possibility that these motors could potentially
bind additional cargoes within their long stalk domains. Similarly, the
presence of two MBS domains in RIM-BP1 also implicates recruitment of
additional proteins in the assembly of RIM and Rab3-based trafficking machinery in the brain (38). In summary, our mapping data on the direct
interaction between hDlg and GAKIN reveals a novel protein-binding
domain that could mediate similar interactions with a large number of
GUK domain proteins.
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ACKNOWLEDGEMENTS |
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We thank Drs. Craig Garner and Alan Fanning for sharing their GKAP construct and MDCK cells, respectively. We are grateful to Donna-Marie Mironchuk for assistance with the artwork, Caroline Walsh for excellent editorial assistance, and Dr. Steven Oh for valuable suggestions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA 94414, HL60755, and Tufts University Earl P. Charlton Award (to T. H.).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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: St. Elizabeth's Medical Center, CBR404, 736 Cambridge St., Boston, MA 02135. Tel.: 617-789-3118; Fax: 617-789-3111; E-mail: Athar.Chishti@Tufts.edu.
Published, JBC Papers in Press, December 21, 2002, DOI 10.1074/jbc.M210362200
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ABBREVIATIONS |
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The abbreviations used are: MAGUKs, membrane-associated guanylate kinase homologues; SH, Src homology; GUK, guanylate kinase-like; Dlg, discs large protein; GAKIN, guanylate kinase-associated kinesin; GKAP, GUK-associated protein; MAP, microtubule-associated protein; MDCK, Madin-Darby canine kidney; GST, glutathione S-transferase; ACES, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid; GFP, green fluorescent protein; MBS, MAGUK binding stalk; SAP97, synapse-associated protein of 97 kDa.
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