GAKIN, a Novel Kinesin-like Protein Associates with theHuman Homologue of the Drosophila Discs Large TumorSuppressor in T Lymphocytes*

Received for publication, January 27, 2000, and in revised form, May 16, 2000Published, JBC Papers in Press, June 19, 2000, DOI 10.1074/jbc.M000715200

Toshihiko Hanada‡, Lunhui Lin‡, Elena V. Tibaldi§, Ellis L. Reinherz§, and Athar H. Chishti‡¶

From the ‡Section of Hematology-Oncology Research, Departments of Medicine, Anatomy, and Cellular Biology,St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135 and the §Laboratoryof Immunobiology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Reorganization of the cortical cytoskeleton is a hall-mark of T lymphocyte activation. Upon binding to anti-gen presenting cells, the T cells rapidly undergo cy-toskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, proteinsegregation, and cellular polarization. Previously, wereported cloning of the human lymphocyte homologueof the Drosophila Discs Large tumor suppressor protein(hDlg). Here we show that a novel protein termedGAKIN binds to the guanylate kinase-like domain ofhDlg. Affinity protein purification, peptide sequencing,and cloning of GAKIN cDNA from Jurkat J77 lympho-cytes identified GAKIN as a novel member of the kinesinsuperfamily of motor proteins. GAKIN mRNA is ubiqui-tously expressed, and the predicted amino acid se-quence shares significant sequence similarity with theDrosophila kinesin-73 motor protein. GAKIN sequencecontains a motor domain at the NH2 terminus, a centralstalk domain, and a putative microtubule-interactingsequence called the CAP-Gly domain at the COOH ter-minus. Among the MAGUK superfamily of proteins ex-amined, GAKIN binds to the guanylate kinase-like do-main of PSD-95 but not of p55. The hDlg and GAKIN arelocalized mainly in the cytoplasm of resting T lympho-cytes, however, upon CD2 receptor cross-linking thehDlg can translocate to the lymphocyte cap. We proposethat the GAKIN-hDlg interaction lays the foundation fora general paradigm of coupling MAGUKs to the micro-tubule-based cytoskeleton, and that this interactionmay be functionally important for the intracellular traf-ficking of MAGUKs and associated protein complexes invivo.

Dlg protein encoded by the Drosophila tumor suppressorgene lethal (1)discs large-1 (dlg) is located at the cytoplasmicsurface of septate junctions between epithelial cells. Loss offunction mutations of dlg result in the neoplastic overgrowthof imaginal discs and larval lethality (1). Dlg is also presentat the synaptic junctions of neurons and is required for the

development of synaptic structure at neuromuscular junc-tions (2). Dlg is a prototypical member of a growing family ofproteins termed membrane-associated guanylate kinase homo-logues (MAGUKs).1 MAGUKs are peripheral membrane pro-teins composed of either one or three PDZ domains, a singleSH3 domain, and a carboxyl-terminal domain that is homolo-gous to guanylate kinases (3, 4). Growing evidence indicatesthat MAGUKs function as scaffolding proteins necessary forthe assembly and organization of protein complexes at special-ized membrane sites (3–5). For example, the p55 MAGUK linksactin-spectrin-protein 4.1 complexes to the cytoplasmic face ofthe erythrocyte plasma membrane (6). The adaptor function ofp55 is mediated via the interaction of its single PDZ domainand HOOK domain to the cytoplasmic terminus of glycophorinC and the FERM domain of protein 4.1, respectively (7–9). Asimilar paradigm has been established for other MAGUKsalthough a detailed understanding of the scaffolding latticerequires isolation and identification of individual componentsof the MAGUK-associated protein complex in mammalian cells(8–11).

MAGUKs can be broadly classified into several subgroupsbased on their domain organization: Dlg-like, p55-like, Lin-2like, and ZO-1-like. Mammalian members of the Dlg subgroupinclude the PSD-95 (SAP90), Chapsyn-110 (PSD-93), NE-Dlg(SAP102), and hDlg (SAP97) (12–15). A hallmark of MAGUKsis the presence of a guanylate kinase-like domain of ;190residues (16). Both prokaryotic and eukaryotic guanylate ki-nases catalyze phosphorylation of GMP to GDP by transferringa phosphate from ATP (16). Initial recognition of the guanylatekinase-like (GUK) domain in MAGUKs raised an interestingpossibility that this domain may couple the guanine nucleotidemetabolism to the Ras signal transduction pathway (1). How-ever, subsequent studies revealed that the GUK domain ofhDlg is enzymatically inactive consistent with the three-aminoacid deletion found within its ATP binding A-motif (17, 18). Thecatalytically inactive GUK domain of SAP97, a rat homologueof hDlg, binds GMP/GDP in vitro but cannot rescue mutationsin the yeast guanylate kinase (18). These observations suggestthat the GUK domain of the Dlg subfamily of MAGUKs mayhave evolved to perform a specialized non-enzymatic functionin vivo.* This work was supported by National Institutes of Health Grants

CA66263 (to A. C.) and AI21226 (to E. R.). The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF279865.

¶ Established Investigator of the American Heart Association. Towhom correspondence should be addressed: St. Elizabeth’s MedicalCenter, CBR 404, 736 Cambridge St., Boston, MA 02135. Tel.: 617-789-3118; Fax: 617-789-3111; E-mail: achishti@semc.org or achishti@opal.tufts.edu.

1 The abbreviations used are: MAGUKs, membrane-associated gua-nylate kinase homologues; GUK domain, guanylate kinase-like domain;hDlg, human homologue of discs large protein; GAKIN, guanylate ki-nase associated kinesin; GST, glutathione S-transferase; PBS, phos-phate-buffered saline; aa, amino acid(s); PAGE, polyacrylamide gelelectrophoresis; EST, expressed sequence tag; PCR, polymerase chainreaction; kb, kilobase pair(s); RACE, rapid amplification of cDNA ends;mAb, monoclonal antibody.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 37, Issue of September 15, pp. 28774–28784, 2000© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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The first evidence showing GUK domains as specialized pro-tein recognition modules came from the observation that theGUK domains of PSD-95/SAP90 and hDlg/SAP97 associatewith a family of proteins termed SAPAPs/GKAP/DAP-1 (19–21). Transfection studies indicate that the GKAPs may beinvolved in the membrane targeting of MAGUKs in COS epi-thelial cells (20). Interestingly, the GKAP itself binds to theNH2-terminal guanylate kinase-like domain of a novel proteintermed S-SCAM (22) and both PSD-95 and S-SCAM in turnbind to yet another novel protein named MAGUIN (23). Be-cause the GKAPs, S-SCAM, and MAGUINs are expressed pre-dominantly in the brain, the proteins that bind to the GUKdomain of hDlg/SAP97 in non-neuronal cells remain to be iden-tified. It is noteworthy that the GUK domain of PSD-93/Chapsyn-110 binds to the microtubule-associated protein 1A,and the PSD-93/Chapsyn-110 localizes to the dendritic micro-tubules of cerebeller Purkinje neurons (24). Since the bindingaffinity of full-length PSD-93 to microtubule-associated protein1A is relatively weak, it was suggested that the accessibility ofthe GUK domain might be blocked by its intramolecular inter-actions within PSD-93 (24). Alternatively, the GUK domainmay also participate in intermolecular interactions amongMAGUKs as indicated by the interaction of SAP102/NE-dlgwith the GUK domain of PSD-95 (25). Recently, a novel proteinnamed BEGAIN (brain-enriched guanylate kinase domain-as-sociated protein) was identified that binds to the GUK domainof PSD-95/SAP90 (26). The physiological function of BEGAIN isnot yet known. Together, these studies suggest that the GUKdomains of MAGUKs function as specific protein-bindingmodules.

To define the physiological function of the GUK domain innon-neuronal cells, it is necessary to identify and characterizemolecules that specifically associate with the GUK domain ofMAGUKs. This information is likely to be critical for under-standing the role of MAGUKs in subcellular targeting, assem-bly of signaling complexes, and the regulation of proliferationpathways. In this paper, we demonstrate that a novel kinesin-like motor protein termed GAKIN (guanylate kinase associatedkinesin) binds to the GUK domain of hDlg in T lymphocytes.The direct association of hDlg with a ubiquitously expressedkinesin-like motor may reveal new aspects of hDlg function inneuronal as well as non-neuronal cells.

MATERIALS AND METHODS

T Cell Line and Monoclonal Antibody—Jurkat J77 cell line wasmaintained in RPMI 1640 (Life Technologies, Inc.) supplemented with10% fetal bovine serum (Sigma), 2.0 mM glutamine (Sigma), 50 units/mlpenicillin (Sigma), and 50 mg/ml streptomycin (Sigma). The monoclonalanti-hDlg antibody was produced using NH2-terminal domain of hDlgas an immunogen.2

Generation of GST Fusion Proteins—The following GST constructswere generated: hDlgDCT (aa 1–624), DNT (aa 201–926 containing theI2 insertion) (13), NT (aa 1–229), PDZ (aa 201–584), HOOK (aa 669–732), HOOK-GUK (aa 669–915), and GUK (aa 733–915) (Fig. 1). Thecorresponding cDNAs encoding these constructs were cloned intopGEX2T plasmid and expressed as GST fusion proteins in DH5a cells.The GUK domain of human p55 (aa 266–466) was cloned into pGEX2Tas described previously (8, 10). The GUK domain of human PSD-95 (aa574–767) was cloned into pGEX6P plasmid. Fusion proteins were af-finity-purified using glutathione-Sepharose beads as described previ-ously (8, 10).

GST Fusion Protein Pull-down and in Vitro Kinase Assays—J77 cellswere lysed in lysis buffer (1% Triton X-100, 0.5% Nonidet P-40, 50 mM

Tris-HCl, pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 2.0 mM phenylmethyl-sulfonyl fluoride, 10 mg/ml each of aprotinin, leupeptin, and pepstatin)and the cell lysate was precleared by centrifugation at 15,000 rpm for 30min. The GST fusion proteins encoding defined segments of hDlg wereimmobilized onto glutathione-Sepharose beads, and incubated with the

cell lysate for 4 h at 4 °C. Beads were centrifuged, washed six timeswith the lysis buffer, and two times with the kinase buffer (40 mM

HEPES, pH 7.4, 10 mM MgCl2, 3.0 mM MnCl2). The in vitro kinase assaywas performed at 25 °C by incubating the beads in 100 ml of kinasebuffer containing 10 mCi of [g-32P]ATP (Amersham Pharmacia Biotech)for 30 min. Radiolabeled proteins were resolved by SDS-PAGE andautoradiographed.

Purification of pp250—Cells were harvested from a 1-liter subcon-fluent culture of J77 cells and lysed in lysis buffer (see above). The celllysate was incubated with glutathione-Sepharose beads bearing theGST-GUK domain of hDlg for 4 h at 4 °C. Beads were recovered bycentrifugation, washed six times with the lysis buffer, and two timeswith the PBS. The proteins were resolved by SDS-PAGE. CoomassieBlue-stained bands corresponding to pp250 were cut out from eightlanes, washed three times with 50% acetonitrile/water mixture, andprocessed for peptide sequencing.

Tryptic Digestion, NanoLC Ion Trap Mass Spectrometry, and Se-quencing—Coomassie Blue-stained pp250 bands were subjected to in-gel reduction, carboxyamidomethylation, and tryptic digestion (Pro-mega). Multiple peptide sequences were determined in a single run byMicrocapillary Reverse-phase Chromatography directly coupled to aFinnigan LCQ quadrupole ion trap mass spectrometer. The ion trapwas programmed to acquire successive sets of three scan modes con-sisting of full scan MS over alternating ranges of 395–800 or 800–1300m/z, followed by two data-dependent scans on the most abundant ion inthose full scans. These data-dependent scans allowed the automaticacquisition of a high resolution (zoom) scan to determine charge stateand exact mass, and MS/MS spectra for peptide sequence information.MS/MS spectra were acquired with relative collision energy of 30% andan isolation width of 2.5 daltons. Interpretation of the resulting MS/MSspectra of the peptides was facilitated by programs developed in theHarvard Microchemistry Facility and by data base correction with thealgorithm Sequest (27, 28).

cDNA Cloning—Two peptide sequences (VTLQIPASSLDANR andVILNPVNTNLSK) were obtained by microsequencing of pp250 andwere found to match with two ends of one human EST clone (EST52797and EST52798). Based on the EST sequence, primers (Sense 59-TCAACGTTGCTTCTCTCTCCT and antisense 59-AGCCGAAGTGTTT-TGCTTATG) were designed to amplify the cDNA fragment from Hu-man Fetal Brain Marathon-Ready cDNA (CLONTECH Inc.). A singlePCR product of 1.8 kb was cloned using the TOPO-TA cloning kit(Invitrogen). Upstream (59) and downstream (39) RACE experimentswere performed using the same cDNA pool according to the manufac-turer’s instructions. The 59-RACE was performed using primers 59-GGGATTAATCCAGGTTGGTCAGCTGTG and AP1 for the first PCRand 59-CTGCAGGATATTCTCTCCAAGGCA and AP2 for the secondPCR. The 39-RACE was performed with primers 59-CGCTTAAGA-CAGTGGGCTGAGGAGAGAG and AP1 for the first PCR and 59-ACA-GATTCCAGCCTCCAGCCTGGATGCC and AP2 for the second PCR.The 39-RACE cDNA probe (1.6 kb) was used to screen a l ZAP II JurkatT cell cDNA library (Stratagene Inc.). Combined efforts of successiveRACE protocols and cDNA library screening identified overlappingpieces of the full-length cDNA sequence of pp250. The COOH-terminalportion of the coding sequence was found to be identical to theKIAA0639 (29). The overlapping segments of the full-length cDNA ofpp250 were amplified from the cDNA pool by PCR, cloned into a plas-mid vector, and sequenced. Several lines of evidence indicate that thereported cDNA encodes an authentic full-length pp250. First, the aminoacid sequence of more than 30 peptides derived from the tryptic digestof pp250 matched with the predicted primary structure of the pp250cDNA sequence. Second, the translation initiation codon of the pp250cDNA predicts a highly conserved NH2 terminus among the KIF1subfamily of kinesin superfamily of protein including the Drosophilakinesin-73. Third, the molecular weight of in vitro translated productsof defined segments of full-length cDNA predicts a protein of ;250 kDain agreement with the estimated mass of pp250 from gel electrophore-sis. Finally, the pp250 cDNA was amplified as three overlapping frag-ments by PCR using human fetal brain cDNA pool (CLONTECH Inc.),and these three cDNA segments predicted the amino acid sequence offull-length GAKIN as shown in Fig. 3.

Northern Analysis—Human Multiple Tissue Northern blot I (CLON-TECH Inc.) was used for Northern blot analysis according to the man-ufacturer’s protocol. A cDNA fragment of 628 base pairs (nucleotides3813–4441) of pp250 (GAKIN) cDNA sequence was radiolabeled usingDECAprime II kit (Ambion), and used as a probe. Jurkat cell mRNAwas isolated using FastTrack 2.0 Kit (Invitrogen Inc.) and a total of 5.0mg of mRNA was analyzed per lane. For Human Multiple Tissue North-ern blot II (CLONTECH Inc.) and Jurkat mRNA, a different cDNA2 A. H. Chishti, unpublished data.

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probe (nucleotides 1674–3920 of GAKIN cDNA) was used. The probewas also radiolabeled using DECAprime II kit (Ambion) and purifiedusing Sephacryl S-200HR column (Amersham Pharmacia Biotech). Hy-bridization reaction was performed at 65 °C using ExpressHyb Hybrid-ization Solution (CLONTECH Inc.).

Antibody Production—Two polyclonal antibodies were raised againstthe NH2 and COOH termini of GAKIN. For the NH2-terminal antibody,a GST fusion protein encoding 53–487 amino acids of GAKIN wasexpressed in bacteria. Inclusion bodies containing GST fusion proteinwere recovered from bacterial lysate and analyzed by SDS-PAGE. Thecorresponding protein band was visualized by staining with 4.0 M so-dium acetate and excised from the gel, and used to immunize rabbits.For affinity purification of antibodies, the GST fusion protein wassolubilized in 6.0 M urea and renatured by dialyzing in decreasingconcentrations of urea. The renatured fusion protein was coupled toCNBr-activated Sepharose 4B beads (Amersham Pharmacia Biotech).To generate polyclonal antibodies against the COOH terminus ofGAKIN, the GST fusion protein encoding 1414–1826 amino acids ofGAKIN was expressed in bacteria and affinity purified using glutathi-one-Sepharose beads. The GST portion of the fusion protein was cleavedby digestion with thrombin. Purified protein without GST was used toimmunize rabbits. The GST fusion protein was coupled to CNBr-acti-vated beads, and used to affinity purify antibodies from whole serum.

Immunoprecipitation, GST Pull-down, and Immunoblot Assay—J77cells were lysed in lysis buffer and the cell lysate was precleared asdescribed before. Anti-GAKIN (COOH terminus) serum (10 ml) or anequivalent amount of preimmune serum was incubated with the J77lysate for 4 h at 4 °C. Immune complexes were recovered by centrifu-gation after incubation with Protein A-Sepharose CL-4B (AmershamPharmacia Biotech) beads for 1.0 h. Beads were washed extensivelywith lysis buffer and solubilized by boiling in SDS buffer. The GSTfusion protein pull-down assay was performed by incubating the celllysate with 10 ml of glutathione-Sepharose beads that were saturatedwith GST fusion proteins for 4 h at 4 °C. Beads were recovered bycentrifugation, washed extensively, and analyzed by SDS-PAGE. West-ern blotting was carried out using an affinity purified polyclonal anti-body against the NH2 terminus of GAKIN and a monoclonal antibodyagainst hDlg. Blots were developed using LumiGLO kit (Kirkegaardand Perry Laboratories, Inc.).

In Vitro Binding Assay—Defined segments of GAKIN cDNA werecloned into pCITE-2a vector and in vitro transcription and translationreactions were performed using Single Tube Protein System 3 (Nova-gen). This system utilizes rabbit reticulocyte lysate for in vitro proteinsynthesis. Protein products were radiolabeled by the addition of[35S]methionine (NEN Life Science Products Inc.) in the translationreaction. After the completion of protein synthesis, 1.0 mg of RNase Awas added to each sample and incubated for 5 min at room temperature.The mixture containing translated polypeptides was diluted 10-foldwith PBS, 1.0% Triton X-100, divided into two tubes, and incubatedwith either GST or GST-GUK fusion protein bound to glutathione-Sepharose beads for 2.0 h at 4 °C. Beads were recovered by centrifuga-tion and analyzed by SDS-PAGE. The gel was stained with CoomassieBlue, incubated in Amplify solution (Amersham Pharmacia Biotech) for15 min, and radiolabeld proteins were detected by fluorography.

Immunofluorescence Analysis—To examine the distribution patternof CD2 molecules, 4 3 106 J77 cells were incubated with PE-conjugatedanti-T11-1 mAb (5–10 ml of T11-RD1, Coulter Immunology) in FACSbuffer (PBS 1 2% FCS) at 4 °C for 30 min followed by two washes in thesame buffer. For CD2 cross-linking, 4 3 106 J77 cells were preincubatedwith a pair of anti-CD2 mAbs (10 mg/ml anti-T11-2 and 10 mg/mlanti-T11-3) for 30 min at 37 °C, washed two times with FACS buffer,and then stained for CD2 as described above. The cells were then fixedwith 3.7% paraformaldehyde in PBS at 4 °C for 1.0 h and permeabilizedwith permeabilization buffer (0.1% saponin (Sigma) in FACS buffer) at4 °C for 30 min. Subsequently, the cells were stained with fluoresceinisothiocyanate-labeled anti-hDlg mAb (0.6 mg/ml) in permeabilizationbuffer at 4 °C for 30 min. After three washes in permeabilization bufferand one wash in PBS, the cells were mounted on microscope slides andanalyzed under 3 40 magnification on a Nikon Diaphot 300 fluoromi-croscope equipped with a photometric PXL cooled CCD camera linked toan Oncor imaging analysis system. To analyze the distribution ofGAKIN, 4 3 106 J77 cells were fixed with paraformaldehyde in PBS for1.0 h at 4 °C, washed two times with PBS, and permeabilized in 0.1%saponin in PBS 1 1% bovine serum albumin (Sigma) for 1.0 h. The cellswere subsequently incubated overnight at 4 °C with affinity purifiedanti-COOH terminus GAKIN polyclonal antibody diluted in permeabi-lization buffer. The cells were then washed twice in permeabilizationbuffer and incubated with fluorescein isothiocyanate-conjugated goat

anti-rabbit mAb (Pierce, 1:700 dilution in permeabilization buffer) for30 min at 4 °C. After three washes with the permeabilization buffer andone wash in PBS, the cells were analyzed by microscopy as describedabove. A control for the specificity of the antibody binding was per-formed using anti-COOH terminus GAKIN antibody previously ab-sorbed with GST fusion protein encoding 1414–1826 amino acids ofGAKIN.

RESULTS

Identification of a Novel hDlg-binding Protein—While inves-tigating the phosphorylation status of hDlg in T lymphocytesby GST pull-down and in vitro kinase assays, we detected aprominent phosphorylated protein of 250 kDa associated withthe hDlg protein complex. GST fusion proteins of hDlg lackingeither its COOH terminus (GST-DCT) or the NH2 terminus(GST-DNT) were immobilized on glutathione-Sepharose beadsand incubated with J77 cell lysate (Fig. 1). Protein complexesbound to the beads were collected, washed extensively, andincubated with [g-32P]ATP to examine for the presence of pro-tein kinase activity associated with the protein complex. Pre-viously we have shown that the NH2 terminus domain of hDlgbinds to p56lck tyrosine kinase in T cells (30). Consistent withthis observation, the GST-DCT fusion protein of hDlg wasphosphorylated by the associated protein kinase (Fig. 2A). Incontrast, the GST-DNT fusion protein that has similar mobilityon SDS gels to that of GST-DCT protein but lacks the NH2-terminal p56lck-binding domain was not phosphorylated underthese conditions (Fig. 2A). Instead, a prominent phosphoryl-ated band of 250 kDa (pp250) was detected in the lane contain-ing the GST-DNT protein complex (Fig. 2A). Based on theseresults, we concluded that a phosphorylated protein of ;250kDa specifically associated with the COOH terminus of hDlg inT lymphocytes. To determine the pp250-binding domain withinhDlg, a series of fusion protein constructs of hDlg were made(Fig. 1). Only those fusion proteins that included the GUKdomain of hDlg bound to pp250 as tested by the GST pull-downand in vitro kinase assays (Fig. 2B). These results indicate thatthe pp250 interacts with the GUK domain of hDlg and may bea substrate of the protein kinase activity associated with theprotein complex. Since the mobility of pp250 was similar tospectrin, filamin, CBP, P300, mTOR, and ankyrin, proteinsthat are likely to be involved in hDlg cytoskeletal and signaltransduction pathways, we attempted to establish the identityof pp250 by Western blotting. Specific antibodies against theproteins mentioned above failed to identify pp250 suggestingthat the pp250 might be a novel protein (data not shown).

Isolation of pp250—To purify sufficient quantity of pp250 forbiochemical analysis, we used an affinity column of GST-GUKfusion protein of hDlg to isolate pp250 from J77 cells. In vitroprotein kinase assays confirmed the presence of pp250 in theGST-GUK protein complex (data not shown). The Coomassie

FIG. 1. Schematic representation of GST fusion proteins ofhDlg. DCT (aa 1–624) and DNT (aa 201–926), the DNT constructcontains the I2 insertion sequence instead of the I3 insertion. NT (aa1–229), PDZ (aa 201–584), and HOOK (aa 669–732), the HOOK con-struct contains the I3 insertion sequence. HOOK-GUK is aa 669–915.The HOOK-GUK construct contains the I3 insertion sequence, GUK (aa739–915).

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Blue-stained band of pp250 was excised and processed forpeptide sequences as described under “Materials and Meth-ods.” Two peptide sequences that were derived from pp250matched with a cDNA clone in the human EST data base. Acombination of polymerase chain reaction, conventional cDNAlibrary screening, and 59-, 39-RACE protocols identified an openreading frame of a novel protein of 1826 amino acids (Fig. 3B)(see “Materials and Methods” for details). To confirm the iden-tity of the cDNA clone, additional peptide sequences were ob-tained from the tryptic digest of pp250 (Table I). More than 30peptide sequences matched completely with the predictedamino acid sequence of pp250 cDNA (Table I). Sequence com-parison analysis of the pp250 cDNA revealed that it encodes anovel member of the kinesin superfamily of motor proteins. We

therefore renamed pp250 as GAKIN (guanylate kinase associ-ated kinesin) to highlight its unique association with the gua-nylate kinase-like domain of hDlg and PSD-95.

Properties of GAKIN—GAKIN encodes a protein of 1826amino acids with a calculated molecular mass of 202,650 Da

FIG. 2. Identification of pp250. A, GST pull-down and in vitrokinase assays revealed that a specific phosphorylated band of 250 kDawas associated with the GST-DNT pull-down complex. GST-DCT andGST-DNT fusion proteins were immobilized on glutathione-Sepharosebeads and used in pull-down experiments to identify interacting pro-teins from the J77 cell lysate. Beads were washed with lysis buffer, andthe pull-down complexes were incubated with [g-32P]ATP to detectprotein kinase activity associated with the complex. PhosphorylatedGST-DCT protein is indicated by an arrowhead. Note that the phospho-rylated 250-kDa band (asterisk) was observed only in lanes containingthe GST-DNT pull-down complex. B, the presence of GUK domain isnecessary to pull-down pp250 from J77 cell lysate. GST fusion proteinsencoding defined segments of hDlg were used for pull-down and in vitrokinase assays. Fusion proteins that contained the GUK domain of hDlg(DNT and HOOK-GUK) precipitated phosphorylated pp250.

FIG. 3. Primary structure and sequence analysis of pp250(GAKIN). A, schematic diagram of the domain organization of GAKIN.B, deduced amino acid sequence of GAKIN. The motor domain is aconserved motif of the kinesin superfamily (boxed and shaded). TheCAP-Gly domain located at the COOH-terminal end is underlined. C,protein matrix alignment analysis of human GAKIN with Drosophilakinesin-73 protein (MacVector 6.5). Note that the sequence identityextends essentially throughout the length of two proteins except nearthe end of the COOH terminus but retains the conserved CAP-Glydomain. The nucleotide sequence of GAKIN has been deposited with theEBI/GenBank™ data libraries with the accession number AF279865.

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and an isolectric point of 5.44. A consensus ATP/GTP bindingmotif (GQTGSGKS) is located within the NH2-terminal motordomain (Fig. 3). There is no detectable repeat structure in theprimary sequence of GAKIN. Many serine/threonine phospho-rylation sites are found throughout the primary structure ofGAKIN and the MacVector computer program predicts onerather weak tyrosine phosphorylation site (residue 1004: EN-GEY). The NH2-terminal motor domain of GAKIN has thehighest sequence homology (98% identity) to mouse KIF13B(31). The amino acid sequence of only the motor domain ofKIF13B has been published to date (31). GAKIN also containsa recognizable motif called the CAP-Gly domain at its COOHterminus (Fig. 3). The CAP-Gly domain is found in proteinssuch as CLIP170/restin, dynactin, and kinesin-73, componentsof the microtubule-based cytoskeleton (32–34). Drosophila ki-nesin-73 shares significant sequence homology with GAKINsequence, thus making it the most likely Drosophila homologueof GAKIN (Fig. 3C). Although the physiological function ofmouse KIF13B and Drosophila kinesin-73 is unknown, bothhave been classified as homologues of the KIF1A/Unc104 sub-family of kinesin-like motor proteins that function as antero-grade vesicle transporters (35). A stalk domain composed ofseveral short a-helical segments is located between the motordomain and the CAP-Gly domain of GAKIN (Fig. 3). The stalkdomain is known to mediate formation of coiled-coil dimer inmany kinesin-like proteins (36). A comparison of the aminoacid sequence of GAKIN with the KIF1A/Unc104 motor pro-teins indicates that the sequence homology extends from theNH2 terminus motor domain to a segment of the stalk domain(Fig. 3). Because the KIF1A and KIF1B kinesin motors exist asmonomers in solution (37, 38), it is possible that the GAKIN

may function as a monomeric motor in vivo.Northern blot analysis indicated ubiquitous expression of

GAKIN mRNA with relative abundance in kidney, pancreas,brain, and testis (Fig. 4). A single mRNA band of ;8.5 kb wasdetected in all tissues. Consistent with the abundant expres-sion of GAKIN in T cells, the Jurkat cells also showed a singleband of ;8.5 kb by Northern blot analysis. The ubiquitousexpression of GAKIN is consistent with the ubiquitous expres-sion profile of hDlg (13) thus implying a functionally conservedinteraction between hDlg-GAKIN in these tissues.

Specificity of Interaction between GAKIN and hDlg—A poly-clonal antibody was raised against the NH2-terminal domain ofGAKIN and affinity purified against the immobilized antigen(see “Materials and Methods”). The affinity purified antibodydetected a single ;250-kDa band in the lysate of Jurkat cells(Fig. 5A). This affinity purified antibody was used to detectGAKIN in protein complexes pulled down from J77 lysate byglutathione-Sepharose beads with immobilized GST fusion pro-teins (Fig. 5B). GAKIN specifically associated with beads con-taining either the DNT fusion protein or the GUK domain ofhDlg (Fig. 5B, lanes 2 and 5). In contrast, the GAKIN failed toassociate with beads containing either the DCT fusion proteinor PDZ domains of hDlg (Fig. 5, lanes 3 and 4). It is noteworthyhere that the binding of GAKIN to the GUK domain is rela-tively more efficient than the DNT fusion protein of hDlg pre-sumably because of the better folded conformation of the GUKdomain, alternatively, the steric hindrance via intradomaininteractions within hDlg could account for the reduced bindingunder these conditions. The specificity of binding betweenGAKIN and the GUK domain of hDlg was further demon-strated by the observation that the GUK domain of p55MAGUK failed to associate with GAKIN under identical bind-ing conditions (Fig. 5B, lane 1). Unlike hDlg, the GUK domainof p55 is prototypical of the GUK domains of MAGUKs thatretain residues necessary for ATP binding (16). To furtherestablish an association between hDlg and GAKIN in vivo,GAKIN was immunoprecipitated from J77 cell lysate using apolyclonal antibody raised against the COOH-terminal domainof GAKIN. The presence of hDlg in GAKIN immunoprecipi-tates was established by Western blotting using a hDlg-specificmonoclonal antibody (Fig. 5C). Successful immunoprecipita-tion of GAKIN was confirmed by probing the same blot usingan NH2 terminus antibody against GAKIN as described earlier(Fig. 5C). These results demonstrate the existence of hDlg-GAKIN complex in human T lymphocytes and suggest that theGUK domain of hDlg mediates this interaction.

Mapping of the Binding Domains within GAKIN and hDlg—Binding between hDlg and GAKIN was investigated by ex-pressing defined segments of GAKIN in vitro using the rabbitreticulocyte lysate transcription/translation system (Fig. 6).The longest GAKIN construct (Gakin 1–1335) containing theentire motor domain and most of the stalk domain bound to theGST-GUK protein of hDlg but not to control GST (Fig. 6C). Thisobservation suggested that the interaction between hDlg andGAKIN might be direct. However, we cannot exclude the pos-sibility that an undefined protein present in the reticulocytelysate may facilitate this interaction. The hDlg-binding sitewas further mapped using smaller segments of GAKIN, and asegment of the stalk domain in close proximity to the motordomain emerged as the most likely binding site within GAKIN(Fig. 6C). This segment of the stalk domain of GAKIN sharessequence similarity with the KIF1A/Unc104 subfamily of kine-sin-like motor proteins. However, it remains to be ascertainedwhether other MAGUKs bind to this family of motor proteins.Sequence comparison analysis indicated that the hDlg-bindingregion of GAKIN is not present in other GUK domain-binding

TABLE ITryptic peptide fragment sequences of pp250 (GAKIN) identified by

ion trap mass spectrometryThe details of peptide isolation and sequencing are described under

“Materials and Methods.”

Residue number Peptide sequence

35–46 VILNPVNTNLSK73–81 YAGQDIVFK

126–133 LCSGLFER146–157 VEVSYMEIYNEK249–259 LSLVDLAGSER279–297 SLTTLGLVISALADQSAGK307–315 DSVLTWLLK428–442 QLESLGISLQSSGIK448–468 CFLVNLNADPALNELLVYYLK513–531 TFVNGSSVSSPIQLHHGDR573–593 VDGDSSSEVSSEVNFNYEYAQ600–623 ALGSNDPMQSILNSLEQQHEEEKR676–685 EATLNNSLMR715–728 VTLQIPASSLDANR731–742 GSLLSEPAIQVR765–781 DLYQEWKECEEDNPVIR772–781 ECEEDNPVIR833–839 LHVEVMR874–891 ILQATGLPQHLSHFVFCK961–974 KNPALWDLGIIQAK988–1013 KLEFWVQILEQNENGEYCPVEVISAK

1095–1101 WLNALTK1102–1112 RQEYLDQQLQK1135–1141 LTLTEER1223–1239 AEASWDSAVHGCPQLSR1309–1331 PEDAQGVEEREALARMAANVENP1346–1358 SVLAVENLLTLDR1359–1366 LRQEVAVK1406–1421 GRWESQQDVSQTTVSR1422–1455 GIAPAPALSVSPQNNHSPDPGLSNLAASYLNPVK1479–1490 RPSPLAHQPVPR1491–1501 IMVQSASPDIR1703–1713 EGEFVTVGAHK1719–1741 YVGPADFQEGTWVGVELDLPSGK1791–1810 SATLSGSATNLASLTAALAK

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proteins such as GKAP/SAPAP/DAP1 and BEGAIN. These ob-servations suggest that a novel protein domain may mediateinteraction of GAKIN with hDlg and other members of theMAGUK superfamily.

We have also examined whether the GAKIN can interactwith the GUK domains of other MAGUKs. We used the GUKdomain of human PSD-95, which shares significant sequencehomology with the GUK domain of hDlg (78% identity), and arelatively distant GUK domain of p55 (27% identity). As shownin Fig. 7, the GUK domain of human PSD-95 binds to theGAKIN segment expressed in vitro using the rabbit reticulo-cyte transcription/translation system. Consistent with the re-sults of the GST pull-down assay (Fig. 5), the GUK domain ofp55 failed to associate with GAKIN (Fig. 7). Recently, a func-tional role has been assigned to the COOH-terminal GUKdomain of PSD-95. This domain has been implicated in thesubcellular targeting of PSD-95 to synaptic sites in neuronalcells (39). It was suggested that the GUK domain of PSD-95harbor a protein-protein interaction site that may be requiredfor the correct targeting of PSD-95 in vivo (39). Because theGUK domains of PSD-95 and hDlg share significant sequenceidentity, a possibility exists that the GAKIN may bind to thesame site within hDlg and PSD-95. Our initial mapping resultsshow that the last 11 amino acids of hDlg do not participate inthe binding of GAKIN since the GST-GUK domain construct ofhDlg lacking the last 11 amino acids is able to bind GAKIN(Fig. 6B). We attempted to introduce sequential deletions fromthe COOH terminus of hDlg but the corresponding fusion pro-teins were completely insoluble as GST fusion proteins in bac-teria (data not shown). We also constructed a GST fusion pro-tein encoding the last 25 amino acids of hDlg. This fusionprotein did not bind to GAKIN in vitro (data not shown). Thisresult suggests that the folding of the binding site either mayrequire an additional portion of the GUK domain or the bindingsite lies elsewhere within the GUK domain of hDlg. Precise invitro mutagenesis, peptide scanning, and crystallographic ap-proaches will be needed to identify critical residues for theGAKIN-binding site within the GUK domain of hDlg.

Translocation of hDlg to Lymphocyte Cap upon CD2 Cross-linking—Our earlier results indicate that hDlg is localized atthe cell-cell contact sites in confluent epithelial cells (13). How-ever, its subcellular localization in non-adherent cells like lym-phocytes has yet to be studied. In resting T lymphoma cells(Jurkat J77), the hDlg localization showed a largely diffusedpattern throughout the cytoplasm (Fig. 8B). T lymphocytesform cell-cell contacts with their cognate interactor cellswhether they be target cells, in the case of cytotoxic T lympho-cytes, or antigen presenting cells in the case of helper T lym-phocytes. Upon activation through the T cell receptor, addi-

tional membrane and intracellular protein alterations occur,resulting in the formation of what some refer to as an “immu-nological synapse” (40). To examine the possibility that lym-phocyte hDlg translocates to a cell-cell contact site upon T cellactivation, we cross-linked the cell surface CD2 molecules tomimic the contact made between antigen-presenting cells withT lymphocytes (41). The CD2 molecules distributed uniformlyon the plasma membrane in non-cross-linked T cells (Fig. 8C)but formed a distinct patch-like structure after cross-linkingwith a mitogenic pair of anti-CD2 monoclonal antibodies (Fig.8G, note that CD2 molecule is clustered in 3 out of 4 Jurkat J77cells). Similar clustering is observed during T cell conjugateformation with CD58 expressing antigen presenting cells (41)(42). Interestingly, immunolocalization studies indicated a sig-nificant translocation of hDlg molecules to sites where CD2molecules clustered into patches (Fig. 8F). Although hDlg par-tially co-localized with CD2 (Fig. 8H), we failed to coprecipitatethe CD2-hDlg complex from J77 cells after cross-linking of theCD2 receptor (data not shown). These results suggest that thetranslocation of hDlg to immune synapse is not likely to bemediated by the direct interaction between hDlg and CD2 cellsurface receptor. Since the T cell surface cross-linking via CD2adhesion receptor causes polarization and reorganization ofmicrotubule structures (43), we speculate that the transloca-tion of hDlg to immune synapse is mediated by the microtu-bule-based transport mechanism which requires participationof GAKIN.

Localization of GAKIN in T Lymphocytes—We used an affin-ity purified polyclonal antibody against the COOH terminusportion of GAKIN to localize GAKIN in resting T lymphocytes.This antibody detected a single GAKIN band of ;250 kDa inthe Jurkat cell lysate (Fig. 5A). The GAKIN was localized in thecytoplasm of resting J77 cells (Fig. 9D) consistent with thelocalization pattern of hDlg in resting T cells (Fig. 8B). A finermicroscopic examination of GAKIN distribution in resting Tcells indicated that it is localized as distinct punctate foci orvesicles of unknown identity (Fig. 9F). This vesicular pattern ofGAKIN disappeared upon preabsorption of the affinity purifiedantibody with an immobilized GST fusion protein encoding theCOOH terminus segment of GAKIN, confirming the specificityof the signal for GAKIN. We then attempted to visualize theGAKIN redistribution in J77 cells after cross-linking with anti-CD2 monoclonal antibodies as shown for hDlg. These attemptsproved unsuccessful because of nonspecific cross-reactivity be-tween the secondary goat anti-rabbit IgG used to detect GAKINand the anti-CD2 monoclonal antibodies used to induce recep-tor cross-linking (data not shown). Currently we are developinga GAKIN-specific monoclonal antibody that will be directlyconjugated to an appropriate fluorescent probe. The develop-

FIG. 4. Northern blot analysis of GAKIN. A, Human Multiple Tissue Northern blot (CLONTECH Inc.) was hybridized with a GAKIN cDNAfragment of 628 base pairs (nucleotides 3814–4441). B, Human Multiple Tissue Northern blot II (CLONTECH Inc.) was hybridized with theGAKIN cDNA fragment encoded by nucleotides 1674–3920. C, the Jurkat mRNA was isolated using FastTrack 2.0 kit (Invitrogen Inc.) and 5 mgof purified mRNA was hybridized with the GAKIN cDNA probe described in panel B. A single band of GAKIN mRNA (;8.5 kb) was detected inall tissues.

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ment of fluorescent antibody against GAKIN will allow us tovisualize GAKIN distribution upon T cell activation in thecontext of hDlg targeting to the immune synapse as well aswith other known markers of T cells.

DISCUSSION

Binding studies have been successful in predicting a scaffold-ing function of hDlg/SAP97, however, its precise physiologicalfunction in vivo remains unknown. Yeast two-hybrid experi-

ments revealed that hDlg/SAP97 binds to COOH termini ofShaker-type K1 channels (44). This interaction conforms to thegeneral binding paradigm of PDZ domains to the COOH ter-mini of transmembrane channels and receptors (45–47). Sub-sequent transfection studies have indicated that the co-expres-sion of SAP97 (rat homologue of hDlg) and Kv1.4 potassiumchannels in COS-7 epithelial cells results in the formation oflarge intracellular aggregates (14). Interestingly, these aggre-gates were morphologically distinct from the PSD-95 inducedclusters of potassium channels formed at the cell surface (14,48). More recent evidence indicates that the hDlg/SAP97 pro-tein also clusters NR2 subunits of N-methyl-D-asparatate anda-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-typeglutamate receptors at synaptic sites (49). The differential clus-tering activity of hDlg/SAP97 and PSD-95 MAGUKs may re-flect their specialized roles in transfected epithelial cells, and isconsistent with their distinct expression pattern in vivo. Forexample, the PSD-95 expression is restricted to the neuronalcells and the protein is localized to the postsynaptic density of

FIG. 5. Specific association of GAKIN with hDlg. A, character-ization of anti-GAKIN antibodies. Rabbit polyclonal antisera againstthe NH2 and COOH termini of GAKIN (see “Materials and Methods”)were affinity purified using immobilized recombinant proteins, respec-tively. Western blot of Jurkat cell lysate was probed with an anti-COOHterminus antibody. Similar results were obtained with an anti-NH2terminus antibody (not shown). Note that a single band of GAKIN wasdetected in the lysate of Jurkat J77 cells. B, immobilized GST fusionproteins were used to pull-down GAKIN from J77 cell lysate. GSTfusion proteins containing defined segments of hDlg and p55 wereincubated with J77 lysate, and the presence of GAKIN in the pull-downcomplexes was examined by immunoblotting using an affinity purifiedantibody against the NH2-terminal domain of GAKIN. Note that theGST-GUK domain fusion protein of hDlg was significantly more effi-cient in pulling down GAKIN. C, co-immunoprecipitation of hDlg withGAKIN. GAKIN was immunoprecipitated from J77 cells using a poly-clonal antibody raised against the COOH-terminal domain of GAKIN orwith the preimmune serum (PI). Co-precipitation of hDlg was detectedby immunoblotting using a monoclonal antibody against hDlg. Success-ful immunoprecipitation of GAKIN was confirmed by immunoblottingof the same membrane with an affinity purified antibody raised againstthe NH2-terminal domain of GAKIN.

FIG. 6. Mapping of the hDlg-binding site within GAKIN. De-fined segments of GAKIN were expressed and 35S-radiolabeled using invitro rabbit reticulocyte lysate system (“Materials and Methods”). Ly-sate samples expressing segments of GAKIN were diluted in PBS (1%Triton X-100) and incubated with either GST or GST-GUK domainfusion proteins immobilized on glutathione-Sepharose beads. Beadswere extensively washed and bound proteins were analyzed by SDS-PAGE and fluorography. A, schematic representation of GAKIN con-structs. The numerals indicate the number of amino acids in GAKIN. B,fluorogram of total cell lysate of various GAKIN constructs. C, fluoro-gram of the binding assay. Note that the GAKIN segment encoded bythe GAKIN 487–989 construct specifically associated with the GST-GUK domain of hDlg.

FIG. 7. Binding of GAKIN to other MAGUKs. The 35S-labeledGAKIN construct (GAKIN 487–989) was expressed in vitro and itsbinding to fusion proteins was examined. Lane 1, GST; Lane 2, GUKdomain of hDlg (hDlg); Lane 3, GUK domain of human PSD-95 (PSD-95); Lane 4, GUK domain of human p55 (p55). Immobilized GST fusionproteins were incubated with the lysate containing 35S-labeled GAKIN487–989 protein, and bound GAKIN 487–989 was detected byfluorography.

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FIG. 8. Translocation of hDlg upon CD2 cross-linking of Jurkat T lymphocytes. Panels A-D represent J77 cells that were not cross-linkedwith anti-CD2 monoclonal antibodies, and panels E-H represent J77 cells that were cross-linked via CD2 as described under “Materials andMethods.” Panels D and H represent merged images of photographs shown in panels B/C and F/G, respectively. The hDlg protein was visualizedusing a monoclonal antibody specific for the NH2-terminal domain of hDlg (see “Materials and Methods”). Note that the clustering of hDlg occurredat sites of CD2 cross-linking (panels F/H).

FIG. 9. Immunofluorescent localization of GAKIN in the cytoplasm of Jurkat T lymphocytes. GAKIN was visualized in J77 cells usingan anti-GAKIN polyclonal antibody (COOH terminus). Panel A, phase-contrast picture of J77 cells (360 magnification). Panel B, immunofluo-rescence image of the same field stained with the goat anti-rabbit fluorescein isothiocyanate antibody alone. Similar results were obtained withan anti-GAKIN antibody that was pre-absorbed with the COOH terminus fusion protein of GAKIN. Panels C and D are phase and immunoflu-orescence images of J77 cells which were incubated with an affinity purified polyclonal antibody against the COOH terminus of GAKIN. PanelsE and F correspond to a magnified view of panels C and D, respectively. Note that the GAKIN is localized throughout the cytoplasm of J77 cellsin a punctate/vesicular pattern.

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forebrain synapses (12, 50, 51). In contrast, hDlg/SAP97 isdistributed widely including cells of neuronal, epithelial, andhematopoietic origin (13, 52). In neuronal cells, SAP97 is local-ized predominantly at the axonal and presynaptic regions(53). In confluent epithelial cells, hDlg/SAP97 localizes at thebasolateral membrane, however, it is found in the cytoplasmof nonconfluent epithelial cells (13, 52, 54). Together, theseresults suggest that the subcellular targeting of hDlg/SAP97in neuronal as well as non-neuronal cells may be modulatedby interactions mediated by specific motifs present in thehDlg/SAP97.

The PDZ domains of MAGUKs have been extensively char-acterized (45, 46, 55), however, the biochemical basis of GUKdomains remains unclear. The first evidence for the physiolog-ical function of the GUK domain came from genetic studies ofDlg tumor suppressor protein in Drosophila. Woods and Bryant(1, 56) identified several mutant alleles affecting the GUKdomain of Dlg tumor suppressor: dlgsw, dlg1P20, dlgv59, anddlgXI-2 alleles result in the progressive deletion of the GUK ofDlg causing tissue overgrowth and suggesting a role in tumorsuppression and signal transduction in Drosophila (1, 56). Al-ternatively, the proliferation defect observed in the GUK do-main mutants of Dlg tumor suppressor could be a secondaryconsequence of altered subcellular targeting of Dlg and abreakdown of septate junction structure (56). Recessive allelesthat completely truncate the GUK domain of Dlg produce a fulltumorous phenotype in imaginal discs, suggesting a require-ment of the GUK domain in cell proliferation events in thistissue. Hough et al. (57) demonstrated that a Dlg derivativewithout the GUK domain is localized normally in a dlg1 back-ground, and can rescue epithelial structure and growth controlphenotypes in a dlg-null background. The most likely explana-tion for this observation is that in a dlg1 background theD-GUK derivative is able to localize normally through multim-erization of PDZ domains with the endogenous full-length Dlg.In the dlg-null background, the D-GUK derivative is able torescue the mutant phenotype because it is expressed at veryhigh levels, which can provide normal genetic function evenwithout normal localization and transport requirements. Morerecent evidence indicates that the GUK domain of DrosophilaDlg plays an essential role in the development of boutonpostsynaptic morphology (58). Mammalian SAP97/hDlg cansuppress the tumorigenic growth in dlg-1 flies and substitutesfor Dlg at the neuromuscular junctions thus implying that themammalian SAP97/hDlg may perform a similar function invivo (59).

Further evidence for the protein binding function of the GUKdomain of MAGUKs comes from studies of the LIN-2 protein inCaenorhabditis elegans. A minigene of lin-2 was engineeredthat lacked residues essential for the enzymatic function of theGUK domain of LIN-2 (60). This mutant lin-2 allele can rescuethe lin-2 Vul phenotype in C. elegans thus indicating that theclassical enzyme function of the GUK domain of LIN-2 is notrequired for vulval induction (60). Together, these results sup-port the hypothesis that the protein binding properties of theGUK domain of LIN-2 may be sufficient to restore its signalingfunction in vulval induction.

The central objective of the present investigation was toelucidate protein-binding interactions of the GUK domain ofhDlg in human T lymphocytes. Here we report identification,cloning, and partial characterization of a novel kinesin-likemotor protein (GAKIN) that interacts with the GUK domain ofhDlg. To our knowledge, this is the first report of a biochemicalassociation between a kinesin-like motor protein and the GUKdomain of MAGUKs. Kinesin-like motor proteins play criticalroles in the anterograde transport (movement toward the plus

end of microtubules) of synaptic vesicle precursors, mitochon-dria transport, spindle function, and chromosome segregation(61–64). The membrane and secreted proteins are sorted intoseveral types of vesicles, which then are translocated alongaxonal microtubules to their target sites (37). Sequence com-parison analysis of GAKIN with the kinesin superfamily sug-gests that it may play a role in the anterograde transport ofsynaptic vesicle precursors in vivo. The closest homologue ofGAKIN in Drosophila is kinesin-73 (34). Although mutantanalysis of kinesin-73 has not been conducted yet, the geneticsof a similar kinesin-like gene (unc104) has been investigated inC. elegans. The unc104 mutants show fewer synaptic vesiclesand make few small synapses (65). Interestingly, a large num-ber of vesicles are found tethered together in the cytoplasm ofcell bodies indicating that Unc104 protein is an anterogrademotor for translocation of synaptic vesicles along axonal micro-tubules. It is relevant to mention here that the unc104 gene ofC. elegans is not the closest homologue of Drosophila kinesin-73. In fact, another gene termed CeF58E3.3 has been recentlyidentified during the C. elegans genome-sequencing projectwhich appears to be the true homologue of human GAKIN andDrosophila kinesin-73. Therefore, the functional consequencesof genetic inactivation of the closest homologue of GAKIN in C.elegans and Drosophila remain to be elucidated.

A conserved feature of the kinesin-like proteins is the pres-ence of a motor domain that confers microtubule binding andATP hydrolysis (36). The GAKIN sequence contains an NH2-terminal motor domain and a single copy of the CAP-Gly do-main at the COOH terminus. The CAP-Gly domain has beenpreviously identified in Drosophila kinesin-73 (34), CLIP170/Restin (33, 66), Drosophila Glued (67), rat dynactin (32), hu-man tubulin folding cofactors B and E (68, 69), and Alp11(fission yeast homologue of cofactor B) (70). Functionally, theCAP-Gly domain mediates direct binding of CLIP170 to micro-tubules and presumably serves as a linking module to endo-cytic vesicles (33, 66). The identification of CAP-Gly domains inproteins important for microtubule-dependent processes sug-gest that its presence at the COOH termini of GAKIN andDrosophila kinesin-73 may signify a similar role in microtu-bule-based functions in vivo. The predicted motor domains ofGAKIN and kinesin-73 are homologous to the motor domain ofKIF1A/Unc104 subfamily of kinesin-like motor proteins (37,65). The KIF1A/Unc104 subfamily of kinesin-like motor pro-teins mediates specific motile events and functions as organelleand vesicle transporters (65). GAKIN also shares sequencesimilarity with the KIF1A/Unc104 near a segment of the stalkdomain that is proximal to the motor domain. Interestingly, thehDlg-binding site in GAKIN was localized within this region(Fig. 6) indicating that the interaction between MAGUKs andKIF1A/Unc104 subfamily of motor proteins may be conservedin vivo. Our results also demonstrate that the GUK domains ofhDlg and PSD-95 bind to GAKIN whereas the GUK domain ofp55 does not (Figs. 5 and 7). Based on these observations, wespeculate that the KIF1A/Unc104 subfamily of motor proteinsmay bind to the p55 subfamily of MAGUKs. Similarly, theGUK-binding domain of GAKIN shares sequence identity withthe corresponding domain in kinesin-73 suggesting that themammalian GAKIN-hDlg interaction may be evolutionary con-served in Drosophila.

In addition to their function in intracellular transport, somekinesin-like motor proteins are also involved in mitotic spindleformation, chromosome segregation, and control of cell division(62, 71). For example, Drosophila KLP38B which is homolo-gous to KIF1A/Unc104 is known to play a role in mitosis (72,73). Therefore, a functional role of the GAKIN-hDlg complex incell division cannot be excluded at this stage. In fact, several

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hDlg-interacting proteins are known to interact with compo-nents of the cell cycle. For example, the HTLV Tax oncoproteinthat binds to the PDZ domain of hDlg (74) interacts with thehuman mitotic checkpoint protein MAD1 and abrogates themitotic checkpoint (75). Adenomatous polyposis coli (APC) co-lon tumor suppressor protein binds to the PDZ domains of hDlg(76) and blocks progression from G0-G1 to S phase of the cellcycle (77, 78). Similarly, protein 4.1 that binds to the HOOKdomain of hDlg (10, 13) has been localized in the centrosome,nucleus, and mitotic spindles (79, 80). Protein 4.1 also associ-ates with the nuclear mitotic apparatus (NuMA) protein (81)and is believed to play a role in cell division. Whether thehDlg-GAKIN complex interacts with these proteins in vivo andplays a role in the regulation of cell division is an issue ofconsiderable significance for future studies.

The differential expression pattern of hDlg/SAP97 in conflu-ent and non-confluent epithelial cells indicates that an activetargeting mechanism must exist for the correct localization ofhDlg/SAP97 in vivo. Recent studies on synaptic targeting ofPSD-95 revealed a 12-amino acid sequence located within theGUK domain, which plays a critical role in the subcellulartargeting of PSD-95 in vivo (39). In contrast, the deletion of theGUK domain of SAP97 only slightly reduced its targeting effi-ciency to the lateral plasma membrane in CACO-2 colon carci-noma epithelial cells (52). A limitation of these studies is thatthe CACO-2 cells contain a significant amount of endogenoushDlg thus complicating the interpretation due to potential oli-gomerization between transfected and endogenous molecules.Indeed, we have recently shown that a unique amino-terminaldomain mediates multimerization of hDlg into dimeric andtetrameric species in solution (82). Another important issue isthe characterization of the protein kinase activity associatedwith the GAKIN-hDlg complex. Initially, we identified GAKINas a prominent phosphorylated protein by in vitro kinase as-says (Fig. 2). The GAKIN sequence has no putative proteinkinase domain, hence it is likely that a distinct protein kinaseis associated with the GAKIN-hDlg complex. GAKIN is theonly protein in the complex that is phosphorylated, therefore,the associated protein kinase has strict substrate specificity forGAKIN. Several kinesin-like proteins are subject to regulationby phosphorylation. For example, phosphorylation of kinesinby cAMP-dependent protein kinase reduces its binding to syn-aptic vesicles (83), and phosphorylation by cdc2 kinase regu-lates spindle association of mitotic kinesins, HsEg5 andCENP-E (84, 85). Another mitotic kinesin, CHO1/MKLP-1, in-teracts with a protein kinase Plk and is phosphorylated (86).These observations suggest that the protein kinase associatedwith the GAKIN-hDlg complex may be a specific regulator ofGAKIN function and microtubule-based cellular dynamics invivo. Identification of the protein kinase activity and othercomponents of the GAKIN-hDlg complex will be essential fordefining the physiological basis of MAGUKs in cellular trans-port and motile functions.

The rapid translocation of hDlg from T cell cytoplasm to thesite of CD2 receptor cross-linking is consistent with the role ofMAGUKs as modulators of signaling pathways that exist be-tween membrane receptors and cytoskeleton. CD2 is a trans-membrane receptor expressed in T cells, and binds to the CD58ligand expressed on antigen presenting cells (87). Upon bindingto CD58, the CD2 receptor facilitates cell-cell contact betweenT lymphocytes (both helper and cytotoxic) and CD58-bearingantigen presenting cell. This in turn optimizes the ability of Tcell receptor to recognize the specific antigen bound to majorhistocompatibility complex molecules (88, 89). The T lympho-cyte-antigen presenting cell contact site, a specialized junctionoften called an “immunological synapse,” contains T cell recep-

tors, adhesion molecules, cytoskeletal proteins, and intracellu-lar signaling molecules (40, 90). Following CD2 cross-linking, alarge patch of clustered CD2 molecules accumulates on the Tcell surface, and the microtubule organizing center (MTOC)moves to the site of CD2 cross-linking, resulting in cell polar-ization (41, 43). We have previously shown that hDlg interactswith p56lck tyrosine kinase and potassium channel Kv1.3 in Tlymphocytes (30). Whether the translocation of any of thesemolecules requires the presence of hDlg and GAKIN remains tobe investigated. Alternatively, the translocation of hDlg to theimmune synapse may be a consequence of T cell polarizationand cytoskeletal reorganization at the contact site. To resolvethis issue, it would be essential to use selective inhibitionstrategies to decipher the role of a novel kinesin-like motorprotein and MAGUKs in cellular transport (including cytokinesecretion, granular cytotoxin release etc.), receptor clustering,and the formation of immune synapse in the broader context ofT cell signaling and activation. This may also give us someclues about the function of MAGUKs in receptor clustering andsignaling pathways in other cell types.

Acknowledgments—We thank William S. Lane of the Harvard Mi-crochemistry Facility for the peptide sequence analysis and Dona-MarieMironchuk for graphic technical assistance. We are grateful to Dr.Mohini Lutchman, Brenda Riley, and Dr. Gautam Sondarva for sug-gestions and editorial assistance.

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Toshihiko Hanada, Lunhui Lin, Elena V. Tibaldi, Ellis L. Reinherz and Athar H. Chishti Discs Large Tumor Suppressor in T Lymphocytes Drosophila

GAKIN, a Novel Kinesin-like Protein Associates with the Human Homologue of the

doi: 10.1074/jbc.M000715200 originally published online June 19, 20002000, 275:28774-28784.J. Biol. Chem. 

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