Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B-cells 1. Targeted re-sequencing studies have revealed mutations in various genes in the NFkB pathway 2 , 3 that contribute to the activated B-cell Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Protein phosphorylation and protein ubiquitination regulate most aspects of cell life, and defects in these control mechanisms cause cancer and many other diseases. In the past decade, protein kinases have become one of the most important classes of drug targets for the pharmaceutical industry. In contrast, drug discovery programs that target components of the ubiquitin system have lagged behind. In this Perspective, we discuss the reasons for the delay in this pipeline, the drugs targeting the ubiquitin system that have been developed, and new approaches that may popularize this area of drug discovery in the future.

The nerve growth factor (NGF) family of neurotrophins binds two classes of cell-surface receptors, trk receptor tyrosine kinases and the shared p75 receptor. Rapid internalization and retrograde trafficking of neurotrophin-trk complexes have been demonstrated in a number of systems and are thought to transmit trophic signals from terminals to neuronal cell bodies. In contrast, the internalization and trafficking of neurotrophin-p75 complexes are not well understood. In this study, we used biotinylated NGF and a fluorescent-labeled anti-p75 antibody to follow the kinetics and route of ligand-induced internalization of the p75 receptor in cycling and differentiated PC12 cells. Binding of neurotrophins to p75 induced internalization at a rate approximately three times slower than that of transferrin and NGF-TrkA complexes in the same cells. The ligand-p75 complex was internalized via clathrin-coated pits into early endosomes and eventually accumulated in recycling endosomes in the cell body and vesicles colabeled by the cholera toxin B-subunit in the growth cones. Both internalized ligand and p75 were protected from proteolytic degradation and accumulated in vesicles that did not undergo acidification. Finally, NGF induced endosomal association of p75 and its MAGE interactors, necdin and NRAGE. These data suggest that signaling endosomes containing activated p75 are involved in neurotrophin signaling, and that such endosomes may be temporally and spatially distinct from those containing trk receptors.

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.Neuronal responses to activation of the p75 neurotrophin receptor range from enhanced outgrowth to increased cell death, and p75 null mice exhibit a plethora of defects in both neuronal and non-neuronal systems (1, 2). However, the primary interactors and signaling mechanisms activated by p75 are not well understood (3). Although p75 belongs to the TNF receptor superfamily, its intracellular type II death domain does not self-aggregate (4) and does not interact with the major binding partners of type I death domains. Identification of specific signaling partners for p75 has been frustrating, as this particular receptor is a difficult bait in conventional yeast two-hybrid. Nonetheless seven candidate interactors have been reported so far for p75 (3,5). Most of these candidate interactors lack known catalytic domains, suggesting that they must recruit additional binding partners to form a functional signaling complex. There are no general sequence or functional homologies between the described p75 interactors, and the physiological significance of most of them remains to be established.To identify biologically relevant interactors for p75, we used the Ras rescue system (RRS) 1 for protein interaction trapping in yeast (6). RRS selects for protein interactions in the membrane proximal region of the cytoplasm, and there is no requirement for nuclear or other translocations of the binding partners. The system thus provides an appropriate screening method for the membrane-proximal binding of a receptor intracellular domain with its primary interactors. Our RRS screens identified two MAGE family proteins as novel p75 interactors and highlight type II MAGE proteins as a potential interactor family for type II death domain proteins. ) regions of p75-ICD were generated by PCR from rat p75. Synthetic oligonucleotides were designed to encode the nine-residue (SES-TATSPV) tail domain of p75-ICD. A pADH death domain bait was used to screen a mouse embryonic head cDNA library in pMyr. Alignments of MAGE sequences and phylogenetic trees were constructed using ClustalX. MATERIALS AND METHODS RRSExpression Analysis-Rat tissues for We...

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75 exonIII؊/؊ mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase ؊/؊ mice were as susceptible to NGF-induced death as wild type neurons. The p75 neurotrophin receptor (p75) 1 is the shared receptor for all four mammalian neurotrophins (1) as well as other unrelated ligands (2-6). It is expressed in a wide range of neuronal and non-neuronal cells (7,8), with a corresponding diversity of roles attributed throughout development and in the adult (9 -11). In addition to enhancing responsiveness of cells co-expressing p75 and Trk receptors (12), p75 has in recent years been established as a signaling receptor in its own right (13,14). Independent signaling of p75 has been reported to modulate many aspects of neuronal physiology including sensory functions (15), axon outgrowth (16 -18), and survival or apoptotic effects of neurotrophins (19 -24). Typically, the diverse effects observed after p75 activation are explained by "cell context," which may involve the differential activation of a number of intracellular signaling pathways, including NFB translocation (25), Jun kinase phosphorylation (26), and ceramide generation (27).Ceramide is a lipid second messenger implicated in diverse intracellular pathways, most prominently those regulating cell death in assorted cell types (28 -30). A plethora of studies have looked at the effects of exogenously added ceramide analogues on cultured cells, and in cultured neurons both outgrowth and survival/death effects have been reported (31-33). However, less attention has been paid to endogenous ceramide generation in neurons. Endogenous ceramide can be generated by hydrolysis of sphingomyelin (SM) or by de novo synthesis, processes that occur at different intracellular locations, and by different modes of regulation (29). Ceramide generated by SM hydrolysis can be produced by either neutral or acid sphingomyelinases (N-SMase or A-SMase, respectively) (30), which may be differentially distributed between the cell body and the axon (34).A prominent example of an endogenous signaling system that generates ceramide in the nervous system is p75 (27, 35). Although an early report (26) suggested a role for ceramide in p75-mediated cell death of oligodendrocytes, subsequent s...