Membrane-associated Guanylate Kinase Homologs Research Articles

MPP1 Determines the Mobility of Flotillins and Controls the Confinement of Raft-Associated Molecules.

MPP1 (membrane palmitoylated protein 1) belongs to the MAGUK (membrane-associated guanylate kinase homologs) scaffolding protein family. These proteins organize molecules into complexes, thereby maintaining the structural heterogeneity of the plasma membrane (PM). Our previous results indicated that direct, high-affinity interactions between MPP1 and flotillins (raft marker proteins) display dominant PM-modulating capacity in erythroid cells. In this study, with high-resolution structured illuminated imaging, we investigated how these complexes are organized within erythroid cells on the nanometer scale. Furthermore, using other spectroscopic techniques, namely fluorescence recovery after photobleaching (FRAP) and spot-variation fluorescence correlation spectroscopy (svFCS), we revealed that MPP1 acts as a key raft-capturing molecule, regulating temporal immobilization of flotillin-based nanoclusters, and controls local concentration and confinement of sphingomyelin and Thy-1 in raft nanodomains. Our data enabled us to uncover molecular principles governing the key involvement of MPP1-flotillin complexes in the dynamic nanoscale organization of PM of erythroid cells.

MPP1-based mechanism of resting state raft organization in the plasma membrane. Is it a general or specialized mechanism in erythroid cells?

Biological membranes are organized in various microdomains, one of the best known being called membrane rafts. The major function of these is thought to organize signaling partners into functional complexes. An important protein found in membrane raft microdomains of erythroid and other blood cells is MPP1 (membrane palmitoylated protein 1)/p55. MPP1 (p55) belongs to the MAGUK (membrane-associated guanylate kinase homolog) family and it is a major target of palmitoylation in the red blood cells (RBCs) membrane. The well-known function of this protein is to participate in formation of the junctional complex of the erythrocyte mem-brane skeleton. However, its function as a "raft organizer" is not well understood. In this review we focus on recent reports concerning MPP1 participation in membrane rafts organization in erythroid cells, including its role in signal transduction. Currently it is not known whether MPP1 could have a similar role in cell types other than erythroid lineage. We present also preliminary data regarding the expression level of MPP1 gene in several non-erythroid cell lines.

Trans-homophilic interaction of CADM1 activates PI3K by forming a complex with MAGuK-family proteins MPP3 and Dlg.

CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently downregulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the trans-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that trans-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.

Trans-homophilic interaction of CADM1 activates PI3K by forming a complex with MAGuK-family proteins MPP3 and Dlg.

CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently down-regulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the trans-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that trans-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.

ZO‐2, a tight junction scaffold protein involved in the regulation of cell proliferation and apoptosis

ZO-2 is a membrane-associated guanylate kinase homologue (MAGUK) tight protein associated with the cytoplasmic surface of tight junctions. Here, we describe how ZO-2 is a multidomain molecule that binds to a variety of cell signaling proteins, to the actin cytoskeleton, and to gap, tight, and adherens junction proteins. In sparse cultures, ZO-2 is present at the nucleus and associates with molecules active in gene transcription and pre-mRNA processing. ZO-2 inhibits the Wnt signaling pathway, reduces cell proliferation, and promotes apoptosis; its absence, mutation, or overexpression is present in various human diseases, including deafness and cancer.

Expression pattern of membrane-associated guanylate kinases in interneurons of the visual cortex

GABAergic interneurons are key elements regulating the activity of local circuits, and abnormal inhibitory circuits are implicated in certain psychiatric and neurodevelopmental diseases. The glutamatergic input that interneurons receive is a key determinant of their activity, yet its molecular structure and development, which are often distinct from those of glutamatergic input to pyramidal cells, are poorly defined. The membrane-associated guanylate kinase (MAGUK) homologs PSD-95/SAP90, PSD-93/chapsyn110, SAP97, and SAP102 are central organizers of the postsynaptic density at excitatory synapses on pyramidal neurons. We therefore studied the cell-type-specific and developmental expression of MAGUKs in the nonoverlapping parvalbumin (PV)- and somatostatin (SOM)-positive interneurons in the visual cortex. These interneuron subtypes account for the vast majority of interneurons in the cortex and have different functional properties and postsynaptic structures, being either axodendritic (PV(+)) or axospinous (SOM(+)). To study cell-type-specific MAGUK expression, we used DIG-labeled riboprobes against each MAGUK along with antibodies against either PV or SOM and examined tissue from juvenile (P15) and adult mice. Both PV(+) and SOM(+) interneurons express mRNA for PSD-95, PSD-93, and SAP102 in P15 and adult tissue. In contrast, these interneuron subtypes express SAP97 at P15, but for adult visual cortex we found that most PV(+) and SOM(+) interneurons show low or no expression of SAP97. Given the importance of SAP97 in regulating AMPA receptor GluA1 subunit and NMDA receptor subunits at glutamatergic synapses, these results suggest a developmental shift in glutamate receptor subunit composition and regulation of glutamatergic synapses on PV(+) and SOM(+) interneurons.

CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

Insights into Regulated Ligand Binding Sites from the Structure of ZO-1 Src Homology 3-Guanylate Kinase Module

Tight junctions are dynamic components of epithelial and endothelial cells that regulate the paracellular transport of ions, solutes, and immune cells. The assembly and permeability of these junctions is dependent on the zonula occludens (ZO) proteins, members of the membrane-associated guanylate kinase homolog (MAGUK) protein family, which are characterized by a core Src homology 3 (SH3)-GUK module that coordinates multiple protein-protein interactions. The structure of the ZO-1 SH3-GUK domain confirms that the interdependent folding of the SH3 and GUK domains is a conserved feature of MAGUKs, but differences in the orientation of the GUK domains in three different MAGUKs reveal interdomain flexibility of the core unit. Using pull-down assays, we show that an effector loop, the U6 region in ZO-1, forms a novel intramolecular interaction with the core module. This interaction is divalent cation-dependent and overlaps with the binding site for the regulatory molecule calmodulin on the GUK domain. These findings provide insight into the previously observed ability of the U6 region to regulate TJ assembly in vivo and the structural basis for the complex protein interactions of the MAGUK family.

Erythrocyte Scaffolding Protein p55 Functions as An Essential Regulator of Neutrophil Polarity

Erythrocyte p55 is a prototypical member of a family of scaffolding proteins known as Membrane Associated Guanylate Kinase Homologues (MAGUKs). MAGUKs are multi-domain proteins that couple signals from specialized sites at the plasma membrane to intracellular signal transduction pathways and the cytoskeleton. P55 was originally identified in the erythrocytes as part of a ternary complex with protein 4.1R and glycophorin C, providing a critical linkage between the actin cytoskeleton and the plasma membrane. Although p55 is expressed in a variety of tissues, especially hematopoietic cells, its biological function is unclear. Here, using a p55 knockout mouse model, we show that p55 plays a prominent role in the regulation of neutrophil polarization. Neutrophils are the first respondents during infection and injury, adopting a highly polarized morphology when stimulated with chemotactic factors. G proteincoupled surface receptors recognize the external chemotactic gradient and translate it into an internal gradient of signaling molecules. At the front of the cell, accumulation of the lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP3), activation of the small GTPase Rac, and polymerization of F-actin stimulates a positive feedback loop promoting pseudopod formation. Here, we show that neutrophils lacking p55 form multiple transient pseudopods at the sides and back of the cell upon stimulation. P55 is required for limiting the pseudopod in the direction of chemoattractant. As a result, these neutrophils do not migrate efficiently up a chemotactic gradient in vitro. Biochemical analysis indicates that total F-actin polymerization and total Rac activation is similar between wild type and p55 knockout neutrophils. However, we found that phosphorylation of AKT, the major kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-PIP3 pathway, is almost completely blocked in p55 knockout neutrophils. This finding suggests that p55 exerts its functional effect by regulating PIP3 accumulation or its localization at the membrane, which is responsible for amplification of the frontness signal and stability of the leading edge pseudopod. Consistent with this finding, the p55 null mice are significantly more susceptible to spontaneous and induced infections. Taken together, we have identified p55 as a novel mediator of the frontness signal in neutrophils that promotes polarization and efficient chemotaxis.

Structural requirements for calmodulin binding to membrane‐associated guanylate kinase homologs

Effector molecules such as calmodulin modulate the interactions of membrane-associated guanylate kinase homologs (MAGUKs) and other scaffolding proteins of the membrane cytoskeleton by binding to the Src homology 3 (SH3) domain, the guanylate kinase (GK) domain, or the connecting HOOK region of MAGUKs. Using surface plasmon resonance, we studied the interaction of members of all four MAGUK subfamilies--synapse-associated protein 97 (SAP97), calcium/calmodulin-dependent serine protein kinase (CASK), membrane palmitoylated protein 2 (MPP2), and zona occludens (ZO) 1--and calmodulin to determine interaction affinities and localize the binding site. The SH3-GK domains of the proteins and derivatives thereof were expressed in E. coli and purified. In all four proteins, high-affinity calmodulin binding was identified. CASK was shown to contain a Ca2+-dependent calmodulin binding site within the HOOK region, overlapping with a protein 4.1 binding site. In ZO1, a Ca2+-dependent calmodulin binding site was detected within the GK domain. The equilibrium dissociation constants for MAGUK-calmodulin interaction were found to range from 50 nM to 180 nM. Sequence analyses suggest that binding sites for calmodulin have evolved independently in at least three subfamilies. For ZO1, pulldown of GST-calmodulin was shown to occur in a calcium-dependent manner; moreover, molecular modeling and sequence analyses predict conserved basic residues to be exposed on one side of a helix. Thus, calmodulin binding appears to be a common feature of MAGUKs, and Ca2+-activated calmodulin may serve as a general regulator to affect the interactions of MAGUKs and various components of the cytoskeleton.

Somatostatin receptor subtype 1 is a PDZ ligand for synapse-associated protein 97 and a potential regulator of growth cone dynamics

We report that somatostatin receptor subtype 1 (sst1) associates in vivo and in vitro with synapse-associated protein SAP) 97, a membrane-associated guanylate kinase homolog implicated as a scaffolding protein in the structural organization of specialized membrane complexes in various tissues, including the CNS. SAP97 and sst1 were coimmuno-precipitated from rodent brain and from transfected human embryonic kidney (HEK) 293 cells, and pull-down experiments demonstrated that the interaction is dependent on the class I PDZ binding motif in sst1 carboxyterminus. Calorimetric titration indicated that the postsynaptic density-95/discs large/zona occludens-1 (PDZ) 2 domain of SAP97 provides the main contribution to the interaction. We noticed substantial sst1 immunoreactivity in differentiating cortical neurons in culture which declined as the cultures matured. The sst1 immunoreactivity extended, together with SAP97 to neuronal growth cones. Somatostatin (1 μM) triggered retraction of the filopodia and lamellipodia in the growth cones. This growth cone collapse was enhanced by overexpression of green fluorescent protein–tagged sst1, whereas sst1 mutant lacking the PDZ binding motif had no effect. These findings suggest a role for somatostatin signaling in the regulation of growth cone stability, which may involve PDZ domain proteins interacting with sst1 and/or other somatostatin receptors. Consistent with a developmental role, sst1 immunoreactivity was present transiently in the developing mouse cortex, peaking at postnatal day 5 and declining thereafter to low levels in the adult cortex.

ZO-1 and ZO-2 Independently Determine Where Claudins Are Polymerized in Tight-Junction Strand Formation

A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

Interaction between SAP97 and PSD-95, Two Maguk Proteins Involved in Synaptic Trafficking of AMPA Receptors

Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.

Histochemical and immunohistochemical investigation of guanase and nedasin in human tissues

Guanase is known as an enzyme released from the liver. Recently, cloning and sequencing of the guanase gene were reported. In addition, almost simultaneously, it was reported that an unknown protein that binds to neuronal and endocrine lethal(1)-discs large (NE-dlg), one of the membrane-associated guanylate kinase homologues (MAGUK) family proteins involved in synaptic connection between neurons, was cloned and named nedasin (NE-dlg associated protein), whose sequence was almost identical to that of guanase. We immunostained fresh frozen sections of surgically removed human liver, kidney, and small intestine with anti-nedasin antibody, and simultaneously performed histochemical staining for guanase for comparison. Histochemically, guanase activity was observed in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the mucosal epithelium on the small intestine, and in the proximal tubule on the kidney. Immunohistochemically, a brown discoloration due to DAB oxidation was seen in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the proximal tubule but in the distal tubule a little on the kidney, in the mucosal epithelium on the small intestine. The stained region of the liver and the small intestine were different from that of the kidney. The different staining properties dependent on the organs were considered to be due to different isozymes. The physiological significance of guanase may vary with the isozymes, further studies are considered necessary.

Tumor Suppressor in Lung Cancer (TSLC)1 Suppresses Epithelial Cell Scattering and Tubulogenesis

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.

Involvement of a cell adhesion molecule, TSLC1/IGSF4, in human oncogenesis

The occurrence of aberrations in cell adhesion is a critical phase in the invasion and metastasis of human cancer. A tumor suppressor gene, TSLC1/IGSF4, from chromosomal region 11q23 was identified in non-small cell lung cancer (NSCLC) by its tumor suppressor activity in nude mice. TSLC1/IGSF4 is expressed in most tissues except for peripheral blood lymphocytes, but it is inactivated in 44% of NSCLC and 30-60% of various cancers, including liver, pancreatic, and prostate cancers, especially in those with invasion or metastasis. Inactivation occurs by two hits: through promoter methylation, and through loss of heterozygosity at the gene locus. TSLC1/IGSF4 encodes an immunoglobulin superfamily cell adhesion molecule and associates with an actin-binding protein, DAL-1/4.1B, and members of the membrane-associated guanylate kinase homologue (MAGuK) group, providing a novel tumor suppressor cascade that is inactivated in more than 80% of NSCLC. TSLC1/IGSF4 appears to be involved in the formation of an epithelial cell structure with DAL-1/4.1B and MAGuK. Furthermore, TSLC1/IGSF4 may act as a tumor antigen recognized by activated NK or CD8+ T cells. These two distinct mechanisms based on homophilic and heterophilic interactions would be responsible for tumor suppression by TSLC1/IGSF4. TSLC1/IGSF4 is ectopically expressed in adult T-cell leukemia (ATL) cells, providing not only a diagnostic marker for ATL, but also a possible therapeutic target against its invasion. The distinct roles of TSLC1/IGSF4 in the oncogenesis of carcinomas and ATL could be due to tissue-specific differences in the downstream cascades, and is a novel concept with respect to cell adhesion in human oncogenesis.

The LFA-1-associated Molecule PTA-1 (CD226) on T Cells Forms a Dynamic Molecular Complex with Protein 4.1G and Human Discs Large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

MAGUKs in synapse assembly and function: an emerging view.

Neuronal morphogenesis, synaptogenesis and synaptic plasticity are fundamental aspects of nervous system development. Much of our current understanding of how each of these processes contributes to the establishment and maintenance of neural circuitry has come from a molecular description of specific classes of key molecules. With regard to synapse assembly and function, a family of membrane-associated guanylate kinase homologs (MAGUKs) have emerged as central organizers of multicomponent protein signaling complexes. In particular MAGUKs appear to play fundamental roles in the transport, anchoring and signaling of specific subclasses of synaptic receptors and ion channels. In this review, we will focus on the role that subfamilies of MAGUKs play during the formation, maintenance and plasticity of the vertebrate central nervous system glutamatergic synapse.

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

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 k(cat) = 45 s(-1), K(0.5 (MT)) = 0.1 microm. 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.

The Tight Junction Protein ZO-2 Localizes to the Nucleus and Interacts with the Heterogeneous Nuclear Ribonucleoprotein Scaffold Attachment Factor-B

Zonula occludens proteins (ZOPs), currently comprising ZO-1, ZO-2, and ZO-3, belong to the family of membrane-associated guanylate kinase homologue (MAGUK) proteins that are involved in the organization of epithelial and endothelial intercellular junctions. ZOPs bind to the cytoplasmic C termini of junctional transmembrane proteins linking them to the actin cytoskeleton. They are characterized by several conserved modules, including three PDZ domains, one SH3 domain, and a guanylate kinase-like domain, elements indicating that ZOPs may serve multiple purposes. Interestingly, ZOPs contain some unique motifs not shared by other MAGUK family members, including nuclear localization and nuclear export signals and a leucine zipper-like sequence. Their potential involvement in cell growth and proliferation has been suggested earlier based on the observation that the N-terminal half of ZOPs displays significant similarity to the product of the Drosophila tumor suppressor gene lethal(1)discs-large (dlg). The nuclear targeting of ZOPs in subconfluent epithelial cell cultures is well documented, although the action of the junctional MAGUKs in the nucleus has remained elusive. Here we show for the first time that nuclear ZO-2 directly interacts with the DNA-binding protein scaffold attachment factor-B (SAF-B). Our results from two-hybrid assays and in vivo co-immunoprecipitation studies provide evidence to suggest that ZO-2 associates with the C-terminal portion of SAF-B via its PDZ-1 domain. We further demonstrate that enhanced green fluorescent protein (EGFP)- and DsRed-tagged ZO-2 and SAF-B fusion proteins partially co-localize in nuclei of transfected epithelial cells. As shown by laser confocal microscopy and epifluorescent analysis, nuclear ZO-2 is present in epithelial and endothelial cells, particularly in response to environmental stress conditions. Interestingly, no association of SAF-B with ZO-1 was found, which supports the notion that junctional MAGUKs serve nonredundant functions.