Direct Interaction with a Kinesin-related Motor Mediates Transport of Mammalian Discs Large Tumor Suppressor Homologue in Epithelial Cells*

Noriyuki AsabaDagger, Toshihiko HanadaDagger, Atsuko Takeuchi, and Athar H. Chishti§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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.

    MATERIALS AND METHODS
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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-beta -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% beta -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% beta -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% beta -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.

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-beta -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).

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 -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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.


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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).

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.


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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.

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.


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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).

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.


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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.

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-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.

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.


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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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 beta 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.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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