Michael Fainzilber

Ceramide Signaling Downstream of the p75 Neurotrophin Receptor Mediates the Effects of Nerve Growth Factor on Outgrowth of Cultured Hippocampal Neurons

The p75 neurotrophin receptor (p75NTR) binds all known neurotrophins and has been suggested to either function as a coreceptor for the trk receptor tyrosine kinases or be involved in independent signaling leading to cell death. We have analyzed the effects of nerve growth factor (NGF) on the growth of cultured hippocampal pyramidal neurons and examined the possibility that the effects of NGF are mediated via generation of ceramide produced by neutral sphingomyelinase (N-SMase). During the initial hour of culture, the only detectable NGF receptor is p75NTR, which by comparative Western blot is expressed at 50- to 100-fold lower levels than on PC12 cells. At this early stage of culture, NGF accelerates neurite formation and outgrowth and induces ceramide formation in a dose-dependent manner. An NGF mutant that is deficient in p75NTR binding has no effect on neuronal morphology or ceramide formation. Furthermore, two anti-p75NTR antibodies (REX and 9651), which are known to compete with NGF for binding to p75NTR, mimic the effects of NGF, whereas a monoclonal antibody (MC192) targeted against a different epitope does not. Finally, scyphostatin, a specific N-SMase inhibitor, blocks the effects of NGF. We propose that a neurotrophin–p75NTR–ceramide signaling pathway influences outgrowth of hippocampal neurons. This signaling role of p75NTR may be distinct from other signaling pathways that lead to apoptosis.

O-Sulfonation of Serine and Threonine Mass Spectrometric Detection and Characterization of a New Posttranslational Modification in Diverse Proteins Throughout the Eukaryotes

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

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.