Zehava Levy

The p75 Neurotrophin Receptor Interacts with Multiple MAGE Proteins

The p75 neurotrophin receptor has been implicated in diverse aspects of neurotrophin signaling, but the mechanisms by which its effects are mediated are not well understood. Here we identify two MAGE proteins, necdin and MAGE-H1, as interactors for the intracellular domain of p75 and show that the interaction is enhanced by ligand stimulation. PC12 cells transfected with necdin or MAGE-H1 exhibit accelerated differentiation in response to nerve growth factor. Expression of these two MAGE proteins is predominantly cytoplasmic in PC12 cells, and necdin was found to be capable of homodimerization, suggesting that it may act as a cytoplasmic adaptor to recruit a signaling complex to p75. These findings indicate that diverse MAGE family members can interact with the p75 receptor and highlight type II MAGE proteins as a potential family of interactors for signaling proteins containing type II death domains.

CCM2 Mediates Death Signaling by the TrkA Receptor Tyrosine Kinase

The TrkA receptor tyrosine kinase is crucial for differentiation and survival of nerve-growth-factor-dependent neurons. Paradoxically, TrkA also induces cell death in pediatric tumor cells of neural origin, via an unknown mechanism. Here, we show that CCM2, a gene product associated with cerebral cavernous malformations, interacts with the juxtamembrane region of TrkA via its phosphotyrosine binding (PTB) domain and mediates TrkA-induced death in diverse cell types. Both the PTB and Karet domains of CCM2 are required for TrkA-dependent cell death, such that the PTB domain determines the specificity of the interaction, and the Karet domain links to death pathways. Downregulation of CCM2 in medulloblastoma or neuroblastoma cells attenuates TrkA-dependent death. Combined high expression levels of CCM2 and TrkA are correlated with long-term survival in a large cohort of human neuroblastoma patients. Thus, CCM2 is a key mediator of TrkA-dependent cell death in pediatric neuroblastic tumors.

A lyso‐platelet activating factor phospholipase C, originally suggested to be a neutral‐sphingomyelinase, is located in the endoplasmic reticulum

Recently a putative mammalian neutral‐sphingomyelinase was cloned [Tomiuk et al. (1998) Proc. Natl. Acad. Sci. USA 95, 3638–3643; GenBank accession number AJ222801]. We have overexpressed this enzyme in cultured cells and demonstrate, using four different tagged constructs, that it is localized at the endoplasmic reticulum and not at the plasma membrane. This localization precludes a role for enzyme AJ222801 in the sphingomyelin cycle. Furthermore, a recent publication demonstrated that this enzyme has lyso‐platelet activating factor (PAF) phospholipase C activity [Sawai et al. (1999) J. Biol. Chem. 274, 38131–38139]. Together, these data suggest a role for enzyme AJ222801 in the regulation of PAF metabolism.

Functional consequences of necdin nucleocytoplasmic localization.

Background Necdin, a MAGE family protein expressed primarily in the nervous system, has been shown to interact with both nuclear and cytoplasmic proteins, but the mechanism of its nucleocytoplasmic transport are unknown. Methodology/Principal Findings We carried out a large-scale interaction screen using necdin as a bait in the yeast RRS system, and found a wide range of potential interactors with different subcellular localizations, including over 60 new candidates for direct binding to necdin. Integration of these interactions into a comprehensive network revealed a number of coherent interaction modules, including a cytoplasmic module connecting to necdin through huntingtin-associated protein 1 (Hap1), dynactin and hip-1 protein interactor (Hippi); a nuclear P53 and Creb-binding-protein (Crebbp) module, connecting through Crebbp and WW domain-containing transcription regulator protein 1 (Wwtr1); and a nucleocytoplasmic transport module, connecting through transportins 1 and 2. We validated the necdin-transportin1 interaction and characterized a sequence motif in necdin that modulates karyopherin interaction. Surprisingly, a D234P necdin mutant showed enhanced binding to both transportin1 and importin β1. Finally, exclusion of necdin from the nucleus triggered extensive cell death. Conclusions/Significance These data suggest that necdin has multiple roles within protein complexes in different subcellular compartments, and indicate that it can utilize multiple karyopherin-dependent pathways to modulate its localization.

STK25 protein mediates TrkA and CCM2 protein-dependent death in pediatric tumor cells of neural origin

Background: The cytoplasmic adaptor protein CCM2 interacts with the TrkA receptor tyrosine kinase to induce pediatric tumor cell death. Results: STK25 interacts with CCM2, and its kinase activity is necessary for TrkA-dependent death of medulloblastoma cells. Conclusion: STK25 mediates TrkA-CCM2 death signaling in medulloblastoma cells. Significance: These findings identify a downstream catalytic effector for receptor tyrosine kinase death signaling. The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin.

A Genome Wide Screening Approach for Membrane-targeted Proteins

Membrane-associated proteins are critical for intra- and intercellular communication. Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue. Here we describe a modification of the yeast Ras recruitment system to this end and designate the modified approach the Ras membrane trap (RMT). A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome. 112 gene products were identified in the screen of which 79 lack assignable homologs in available data bases. Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested. Thus, genome annotation using currently available tools is likely to underpredict representation of membrane-associated gene products. The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes. Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks and indeed from representatives of all major invertebrate phyla.

A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor.

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.