Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK's kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesiclebound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling.

The pathogen–host interaction database (PHI-base) is a web-accessible database that catalogues experimentally verified pathogenicity, virulence and effector genes from bacterial, fungal and Oomycete pathogens, which infect human, animal, plant, insect, fish and fungal hosts. Plant endophytes are also included. PHI-base is therefore an invaluable resource for the discovery of genes in medically and agronomically important pathogens, which may be potential targets for chemical intervention. The database is freely accessible to both academic and non-academic users. This publication describes recent additions to the database and both current and future applications. The number of fields that characterize PHI-base entries has almost doubled. Important additional fields deal with new experimental methods, strain information, pathogenicity islands and external references that link the database to external resources, for example, gene ontology terms and Locus IDs. Another important addition is the inclusion of anti-infectives and their target genes that makes it possible to predict the compounds, that may interact with newly identified virulence factors. In parallel, the curation process has been improved and now involves several external experts. On the technical side, several new search tools have been provided and the database is also now distributed in XML format. PHI-base is available at: http://www.phi-base.org/.

Highlights d The same enzyme, ZDHHC17, palmitoylates DLK and NMNAT2 d DLK palmitoylation by ZDHHC17 is essential for somal responses to axonal injury d Prolonged loss of ZDHHC17 causes NMNAT-dependent fragmentation of distal axons d Conserved motifs in NMNAT2 and DLK govern their ZDHHC17-dependent regulation

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes. Molecular & Cellular Proteomics 7:2475-2485, 2008.Synapses are dynamic structures that expand, retract, and remodel to accomplish activity-dependent modification of neuronal circuits. During peripheral inflammation, the synaptic contacts between primary sensory neurons and dorsal horn neurons are modified in a way that the responsiveness of the system to subsequent stimuli is increased, resulting in hypersensitivity to noxious stimuli (1, 2). These activity-dependent changes in synaptic morphology and strength are based on many different mechanisms including alterations in ion channel and receptor activities due to phosphorylation by protein kinases, the translocation of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1 receptors to the postsynaptic membrane (3, 4), transcription-and translation-dependent changes in protein expression (2, 5), and ubiquitylation-mediated protein degradation (6). Often these changes occur only at a few synapses, making the analysis by common proteomics techniques extremely difficult. Multiepitope ligand cartography (MELC) is a novel i...

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The E3 ubiquitin ligase MYCBP2 negatively regulates neuronal growth, synaptogenesis, and synaptic strength. More recently it was shown that MYCBP2 is also involved in receptor and ion channel internalization. We found that mice with a MYCBP2-deficiency in peripheral sensory neurons show prolonged thermal hyperalgesia. Loss of MYCBP2 constitutively activated p38 MAPK and increased expression of several proteins involved in receptor trafficking. Surprisingly, loss of MYCBP2 inhibited internalization of transient receptor potential vanilloid receptor 1 (TRPV1) and prevented desensitization of capsaicin-induced calcium increases. Lack of desensitization, TRPV internalization and prolonged hyperalgesia were reversed by inhibition of p38 MAPK. The effects were TRPVspecific, since neither mustard oil-induced desensitization nor behavioral responses to mechanical stimuli were affected. In summary, we show here for the first time that p38 MAPK activation can inhibit activity-induced ion channel internalization and that MYCBP2 regulates internalization of TRPV1 in peripheral sensory neurons as well as duration of thermal hyperalgesia through p38 MAPK.The E3-ubiquitin ligase MYCBP2 (Myc-binding protein 2; also known as protein associated with Myc (PAM)) 2 is an unusual large protein with a predicted size of 510 kDa. MYCBP2 orthologs have been described in mouse as Phr1, in zebrafish as Esrom, in drosophila as Highwire and in Caenorhabditis elegans as RPM-1. While MYCBP2 mRNA is found in nearly all human tissues, its expression is exceptionally high in peripheral and central neurons (1-3). MYCBP2 has been shown to act as negative regulators of synaptic growth, synaptogenesis, and neurite growth in C. elegans (4), Drosophila (5), zebrafish (6), and mice (7,8). In C. elegans and Drosophila MYCBP2-dependent growth inhibition is largely mediated by the p38 MAPK pathway (9, 10) whereas in mice the role of p38 MAPK in MYCBP2-regulated axonal growth is less clear.Whereas growth regulation of cortical axons by MYCBP2 does not involve p38 MAPK (8), MYCBP2-dependent axonal overgrowth of spinal cord motor neurons and sensory dorsal root ganglion (DRG) neurons was regulated by p38 MAPKmediated alterations in microtubule stability (11).Besides its role in the regulation of neuronal growth, also a function of MYCBP2 in neuronal transmission has been demonstrated. In C. elegans and drosophila loss-of-function mutations in the MYCBP2 orthologs decreased the number of synaptic vesicles at cholinergic and GABAergic synapses in a p38 MAPK-dependent manner (9) and reduced strength of synaptic transmission at neuromuscular junctions (5, 12, 13). More recently, it was shown that the MYCBP2 ortholog in C. elegans, RPM-1, prevents in central neurons activity-dependent internalization of AMPA receptors by inhibiting p38 MAPK signaling through ubiquitylation of MAPK kinase kinase 12 (MAPKKK12), (14). Loss of RPM-1 caused constitutive activation of p38MAPK leading to an increased internalization of the AMPA receptor ortholog GLR1.Interestingly, in ma...

After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can thus serve as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We optimized an HCS assay based on this readout, which showed highly robust performance in a 96-well format. Using this assay we screened a library of 1200 FDA-approved compounds and found that ketoconazole, the compound that most dramatically affected DLK localization in our primary screen, dose-dependently inhibited DLK palmitoylation in follow-up biochemical assays. Moreover, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to trophic deprivation, a well known model of DLK-dependent pro-degenerative signaling. Our HCS platform is thus capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.

Dual leucine-zipper kinase (DLK; a MAP3K) mediates neuronal responses to diverse injuries and insults through the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs). Here, we identified two ways through which DLK is coupled to the neural-specific isoform JNK3 to control prodegenerative signaling. JNK3 catalyzed positive feedback phosphorylation of DLK that further activated DLK and locked the DLK-JNK3 module in a highly active state. Neither homologous MAP3Ks nor a homologous MAPK could support this positive feedback loop. Unlike the related JNK1 isoform JNK2 and JNK3 promote prodegenerative axon-to-soma signaling and were endogenously palmitoylated. Moreover, palmitoylation targeted both DLK and JNK3 to the same axonal vesicles, and JNK3 palmitoylation was essential for axonal retrograde signaling in response to optic nerve crush injury in vivo. These findings provide previously unappreciated insights into DLK-JNK signaling relevant to neuropathological conditions and answer long-standing questions regarding the selective prodegenerative roles of JNK2 and JNK3.