The serologically defined colon cancer antigen-3 interacts with the protein tyrosine phosphatase PTPN13 and is involved in the regulation of cytokinesis.

Cytokinesis is the final step of cell division. Increasing evidence suggests failure of cytokinesis might contribute to the development of cancer. Here, we demonstrate that the serologically defined colon cancer antigen-3 (SDCCAG3) forms a complex with PTPN13, a protein tyrosine phosphatase known to be involved in the regulation of cytokinesis, carcinogenesis and tumor aggressiveness. We show that SDCCAG3 is a novel endosomal protein, primarily localized at the early/recycling endosomal compartment. SDCCAG3 undergoes dynamic localization during cell division with strong accumulation at the midbody during cytokinesis. Overexpression as well as downregulation correlates with the generation of multinucleate cells. Furthermore, we show interaction of SDCCAG3 with the Arf GTPase activating protein GIT1 (G protein-coupled receptor kinase interactor-1). Overexpression of an ArfGAP-negative version of GIT1 also results in an increased number of multinucleate cells suggesting regulation of Arf-mediated vesicular trafficking or signaling via SDCCAG3. Finally, we demonstrate that SDCCAG3 expression levels are elevated in colon cancers. In summary, we have established SDCCAG3 as a novel endosomal protein, which is involved in the regulation of cytokinesis.

Reduced number of functional glutamatergic synapses in hippocampal neurons overexpressing full-length TrkB receptors.

Brain-derived neurotrophic factor (BDNF) acutely modulates the efficacy of central glutamatergic synapses via activation of the receptor tyrosine kinase TrkB. On a longer time scale, recent evidence suggests an additional role of TrkB signaling in the formation of excitatory synaptic connections. Here, we have overexpressed full-length TrkB receptors (fl-TrkB) in hippocampal neurons, to investigate the contribution of BDNF signaling to the maturation of glutamatergic synapses. Using patch clamp recordings, we show a three-fold reduction in glutamatergic excitatory autaptic and synaptic current amplitudes in neurons overexpressing fl-TrkB, and application of saturating concentrations of BDNF and NT-4/5 completely reverses this effect. Compatible with these overexpression data, in untransfected neurons, scavenging of endogenous BDNF and NT-4/5 by TrkB-IgGs reduces excitatory autaptic current (EAC) amplitudes. By overexpression of truncated TrkB receptors (TrkB.T1, TrkB.T2) and a chimeric receptor containing only the intracellular domain of fl-TrkB, we show that intra- and extracellular domains of fl-TrkB are necessary to observe the EAC reduction. Labeling of presynaptic terminals with FM 4-64 revealed, that the reduced EAC amplitudes in fl-TrkB overexpressing neurons are accompanied by a two-fold reduction in synapse number. These results suggest, that ligand-independent signaling through fl-TrkB receptors can decrease glutamatergic synaptic strength, if sufficient amounts of BDNF or NT-4/5 are not available.

Semaphorin4F interacts with the synapse-associated protein SAP90/PSD-95.

Semaphorins are a family of secreted and membrane-associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.

The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif.

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.

The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain.

Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying five PDZ protein-protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (K(D)) of 8.1 x 10(-9) M. We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC protein. The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as beta-catenin playing a major role in the regulation of cell division, migration and cell adhesion.

Identification and characterization of the human orthologue of yeast Pex14p.

Pex14p is a central component of the peroxisomal protein import machinery, which has been suggested to provide the point of convergence for PTS1- and PTS2-dependent protein import in yeast cells. Here we describe the identification of a human peroxisome-associated protein (HsPex14p) which shows significant similarity to the yeast Pex14p. HsPex14p is a carbonate-resistant peroxisomal membrane protein with its C terminus exposed to the cytosol. The N terminus of the protein is not accessible to exogenously added antibodies or protease and thus might protrude into the peroxisomal lumen. HsPex14p overexpression leads to the decoration of tubular structures and mislocalization of peroxisomal catalase to the cytosol. HsPex14p binds the cytosolic receptor for the peroxisomal targeting signal 1 (PTS1), a result consistent with a function as a membrane receptor in peroxisomal protein import. Homo-oligomerization of HsPex14p or interaction of the protein with the PTS2-receptor or HsPex13p was not observed. This distinguishes the human Pex14p from its counterpart in yeast cells and thus supports recent data suggesting that not all aspects of peroxisomal protein import are conserved between yeasts and humans. The role of HsPex14p in mammalian peroxisome biogenesis makes HsPEX14 a candidate PBD gene for being responsible for an unrecognized complementation group of human peroxisome biogenesis disorders.

Ectopic expression of a chimeric colony-stimulating factor-1/TrkB-receptor promotes CSF-1-dependent survival of cultured sympathetic neurons.

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.

Cloning and sequence analysis of a cDNA encoding a novel truncated form of the chicken TrkB receptor.

A cDNA clone encoding a novel truncated form of the chicken TrkB receptor has been isolated and sequenced. Compared to two previously reported forms from mouse and rat, this clone contains an 141-bp insert (47 amino acids) in the cytoplasmic region.