Toshihiko Hanada

Human Homologue of the Drosophila Discs Large Tumor Suppressor Binds to p56 lck Tyrosine Kinase and Shaker Type Kv1.3 Potassium Channel in T Lymphocytes

Human homologue of the Drosophiladiscs large tumor suppressor protein (hDlg) belongs to a newly discovered family of proteins termed MAGUKs that appear to have structural as well as signaling functions. Consistent with the multi-domain organization of MAGUKs, hDlg consists of three copies of the PDZ (PSD-95/Discs large/zO-1) domain, an SH3 motif, and a guanylate kinase-like domain. In addition, the hDlg contains an amino-terminal proline-rich domain that is absent in other MAGUKs. To explore the role of hDlg in cell signaling pathways, we used human T lymphocytes as a model system to investigate interaction of hDlg with known tyrosine kinases. In human T lymphocyte cell lines, binding properties of hDlg were studied by immunoprecipitation, immunoblotting, and immune complex kinase assays. Our results show that protein tyrosine kinase activity is associated with the immunoprecipitates of hDlg. Immunoblotting experiments revealed that the immunoprecipitates of hDlg contain p56 lck , a member of the Src family of tyrosine kinases. The specificity of the interaction is demonstrated by the lack of p59 fyn tyrosine kinase and phosphotidylinositol 3-kinase in the hDlg immunoprecipitates. Direct interaction between hDlg and p56 lck is demonstrated using glutathione S-transferase fusion proteins of hDlg and recombinant p56 lck expressed in the baculovirus-infected Sf9 cells. The p56 lck binding site was localized within the amino-terminal segment of hDlg containing proline-rich domain. In addition, we show in vivo association of hDlg with Kv1.3 channel, which was expressed in T lymphocytes as an epitope-tagged protein using a vaccinia virus expression system. Taken together, these results provide the first evidence of a direct interaction between hDlg and p56 lck tyrosine kinase and suggest a novel function of hDlg in coupling tyrosine kinase and voltage-gated potassium channel in T lymphocytes.

Transport of PIP3 by GAKIN, a kinesin-3 family protein, regulates neuronal cell polarity

Phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), a product of phosphatidylinositol 3-kinase, is an important second messenger implicated in signal transduction and membrane transport. In hippocampal neurons, the accumulation of PIP3 at the tip of neurite initiates the axon specification and neuronal polarity formation. We show that guanylate kinase–associated kinesin (GAKIN), a kinesin-like motor protein, directly interacts with a PIP3-interacting protein, PIP3BP, and mediates the transport of PIP3-containing vesicles. Recombinant GAKIN and PIP3BP form a complex on synthetic liposomes containing PIP3 and support the motility of the liposomes along microtubules in vitro. In PC12 cells and cultured hippocampal neurons, transport activity of GAKIN contributes to the accumulation of PIP3 at the tip of neurites. In hippocampal neurons, altered accumulation of PIP3 by overexpression of GAKIN constructs led to the loss of the axonally differentiated neurites. Together, these results suggest that, in neurons, the GAKIN–PIP3BP complex transports PIP3 to the neurite ends and regulates neuronal polarity formation.

Dlg1, Sec8, and Mtmr2 Regulate Membrane Homeostasis in Schwann Cell Myelination

How membrane biosynthesis and homeostasis is achieved in myelinating glia is mostly unknown. We previously reported that loss of myotubularin-related protein 2 (MTMR2) provokes autosomal recessive demyelinating Charcot–Marie–Tooth type 4B1 neuropathy, characterized by excessive redundant myelin, also known as myelin outfoldings. We generated a Mtmr2-null mouse that models the human neuropathy. We also found that, in Schwann cells, Mtmr2 interacts with Discs large 1 (Dlg1), a scaffold involved in polarized trafficking and membrane addition, whose localization in Mtmr2-null nerves is altered. We here report that, in Schwann cells, Dlg1 also interacts with kinesin 13B (kif13B) and Sec8, which are involved in vesicle transport and membrane tethering in polarized cells, respectively. Taking advantage of the Mtmr2-null mouse as a model of impaired membrane formation, we provide here the first evidence for a machinery that titrates membrane formation during myelination. We established Schwann cell/DRG neuron cocultures from Mtmr2-null mice, in which myelin outfoldings were reproduced and almost completely rescued by Mtmr2 replacement. By exploiting this in vitro model, we propose a mechanism whereby kif13B kinesin transports Dlg1 to sites of membrane remodeling where it coordinates a homeostatic control of myelination. The interaction of Dlg1 with the Sec8 exocyst component promotes membrane addition, whereas with Mtmr2, negatively regulates membrane formation. Myelin outfoldings thus arise as a consequence of the loss of negative control on the amount of membrane, which is produced during myelination.

Dematin and Adducin Provide a Novel Link between the Spectrin Cytoskeleton and Human Erythrocyte Membrane by Directly Interacting with Glucose Transporter-1

Dematin and adducin are actin-binding proteins located at the spectrin-actin junctions, also called the junctional complex, in the erythrocyte membrane. Here we propose a new model whereby dematin and adducin link the junctional complex to human erythrocyte plasma membrane. Using a combination of surface labeling, immunoprecipitation, and vesicle proteomics approaches, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the junctional complex to the lipid bilayer. Because homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

Centaurin-α1 interacts directly with kinesin motor protein KIF13B

Centaurin-α1 is a phosphatidylinositol 3,4,5-trisphosphate binding protein as well as a GTPase activating protein (GAP) for the ADP-ribosylation factor (ARF) family of small GTPases. To further understand its cellular function, we screened a rat brain cDNA library using centaurin-α1 as bait to identify centaurin-α1 interacting proteins. The yeast two-hybrid screen identified a novel kinesin motor protein as a centaurin-α1 binding partner. The motor protein, termed KIF13B, encoded by a single ∼9.5-kb transcript, is widely expressed with high levels observed in brain and kidney. Yeast two-hybrid and GST pull-down assays showed that the interaction between centaurin-α1 and KIF13B is direct and mediated by the GAP domain of centaurin-α1 and the stalk domain of KIF13B. Centaurin-α1 and KIF13B form a complex in vivo and the KIF13B interaction appears to be specific to centaurin-α1 as other members of the ARF GAP family did not show any binding activity. We also show that KIF13B and centaurin-α1 colocalize at the leading edges of the cell periphery whereas a deletion mutant of centaurin-α1 that lacks the KIF13B binding site, failed to colocalize with KIF13B in vivo. Finally, we demonstrate that KIF13B binding suppresses the ARF6 GAP activity of centaurin-α1 in intact cells. Together, our data suggest a mechanism where direct binding between centaurin-α1 and KIF13B could concentrate centaurin-α1 at the leading edges of cells, thus modulating ARF6 function.

Human Dlg protein binds to the envelope glycoproteins of human T-cell leukemia virus type 1 and regulates envelope mediated cell-cell fusion in T lymphocytes

Human homologue of the Drosophila Dlg tumor suppressor (hDlg) is a widely expressed scaffold protein implicated in the organization of multi-protein complexes at cell adhesion sites such as the neuronal synapse. hDlg contains three PDZ domains that mediate its binding to the consensus motifs present at the C-termini of various cell surface proteins, thus inducing their clustering and/or stabilization at the plasma membrane. Using a yeast two-hybrid screen, we identified hDlg as a cellular binding partner of a viral membrane integral protein, the envelope glycoprotein (Env) of human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 is a human retrovirus that infects CD4+ T lymphocytes and is preferentially transmitted via direct contacts between infected and target cells, through a structure referred to as the virological synapse. Here, we demonstrate that hDlg interacts with a classical PDZ domain-binding motif present at the C-terminus of the cytoplasmic domain of HTLV-1 Env and conserved in the related HTLV-2 virus. We further document that, in HTLV-1 infected primary T cells, hDlg and Env are concentrated in restricted areas of the plasma membrane, enriched in molecules involved in T-cell contacts. The presence of Gag proteins responsible for viral assembly and budding in these areas indicated that they constitute platforms for viral assembly and transmission. Finally, a mutant virus unable to bind hDlg exhibited a decreased ability to trigger Env mediated cell fusion between T lymphocytes. We thus propose that hDlg stabilizes HTLV-1 envelope glycoproteins at the virological synapse formed between infected and target cells, hence assisting the cell-to-cell transmission of the virus.

Protein 4.1-mediated Membrane Targeting of Human Discs Large in Epithelial Cells

Human discs large (hDlg) protein binds to protein 4.1R via a motif encoded by an alternatively spliced exon located between the SH3 and the C-terminal guanylate kinase-like domains. To evaluate the functional significance of protein 4.1R binding for subcellular localization of hDlg in vivo, we expressed full-length recombinant constructs of two naturally occurring isoforms of hDlg termed hDlg-I2 and hDlg-I3. The hDlg-I3 but not the hDlg-I2 isoform binds to the FERM (Four.1-Ezrin-Radixin-Moesin) domain of protein 4.1R in vitro. Upon transient transfection into subconfluent Madine-Darby canine kidney (MDCK) epithelial cells, the hDlg-I3 fused with the green fluorescent protein accumulated predominantly at the plasma membrane of cell-cell contact sites, whereas the hDlg-I2 fusion protein distributed in the cytoplasm. In contrast, in stably transfected confluent MDCK cells, both hDlg-I2 and -I3 isoforms localized efficiently to the lateral membrane, consistent with the previous notion that the N-terminal domain of hDlg mediates its membrane targeting in polarized epithelial cells. We introduced a double mutation (I38A/I40A) into the N-terminal domain of hDlg, which disrupted its interaction with DLG2, a key event in the membrane targeting of hDlg. Interestingly, the hDlg-I2 isoform harboring the I38A/I40A mutation mislocalized from the membrane into cytoplasm. Importantly, the hDlg-I3 isoform with the same mutation localized efficiently to the membrane of confluent MDCK cells. Together, our results demonstrate that in addition to the N-terminal targeting domain, the alternatively spliced I3 insertion plays a critical role in recruiting hDlg to the lateral membrane in epithelial cells via its interaction with protein 4.1R.

VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain.

The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.

Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells

Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA- band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs.

Glutamate receptors localize postsynaptically at neuromuscular junctions in mice.

Dlg (Discs Large) is a multidomain protein that interacts with glutamate receptors and potassium channels at Drosophila neuromuscular junctions (NMJs) and at mammalian central nervous system synapses. Dlg also localizes postsynaptically at cholinergic mammalian NMJs. We show here that α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionate (AMPA) receptor subunits, together with glutamate, are present at the mammalian NMJ. Both AMPA and NMDA (N‐methyl‐D‐aspartate) glutamate receptor subunits display overlapping postsynaptic localization patterns with Dlg at all NMJs examined in normal mice. Kir2 potassium channels also localize with Dlg and glutamate receptors at this synapse. Localization of the components of a glutamatergic system suggests novel mechanisms at mammalian neuromuscular synapses. Muscle Nerve 39: 343–349, 2009