Palmitoylation-Dependent Regulation of the DLK/JNK/cJun and the GP130/JAK/STAT Retrograde Signaling Pathways

Abstract

Palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to protein cysteine residues. This process is best known for its roles in targeting proteins to lipid membranes, including both the plasma membrane and vesicles. Palmitoylation occurs in all eukaryotic cells, but appears to be particularly important in neurons, because genetic mutation or loss of several palmitoyl acyltransferases (PATs, the enzymes that catalyze palmitoylation), leads to predominantly neuropathological defects. In addition, a growing number of recent studies have revealed key roles for palmitoylation of specific proteins in neuronal regulation. However, most of these studies have focused on how palmitoylation regulates postsynaptic protein targeting. In contrast, it is far less clear how palmitoylation might regulate the specialized subcellular processes that are important in axons. One particularly important process in axons is retrograde signaling, in which information is conveyed from distal locations back to the cell body. Following injury to axons of the peripheral nervous system (PNS), retrograde signals are critical to activate transcription of pro-regenerative genes. Key retrograde signaling pathways include the DLK/JNK/c-Jun (Dual Leucine Zipper Kinase/c-Jun N-terminal Kinase/c-Jun) signaling pathway and the GP130/JAK/STAT (Glycoprotein 130/Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway, both of which are activated following nerve injury and are vital to promote regeneration. Though both of these pathways are critical for conveying distal information from the periphery to the cell body, many of their component proteins are predicted to be soluble and diffusible. This raises the question of how these proteins can directionally signal over the long distances that axons extend. Interestingly, bio-informatic and proteomic studies suggested that DLK, GP130, JAK and STAT may be palmitoylated. We hypothesized that palmitoylation could be important for the roles of these proteins in retrograde signaling. Because retrograde signals are initiated in distal axons, a considerable distance from the cell body, we further hypothesized axonally localized PATs might play key roles in the control of retrograde signaling. We find that the retrograde signaling protein DLK is palmitoylated at a highly conserved cysteine residue. This modification is necessary for its localization to motile vesicles and for its interaction with the retrograde signaling protein JIP3. Notably, we also describe a novel role for palmitoylation in regulating DLK’s kinase activity. In addition, our study identifies the first axonally enriched PATs in sensory neurons; DHHC5 and DHHC8. shRNA knockdown experiments in sensory neurons reveal that these axonal PATs control both palmitoylation and surface expression of GP130 and are essential for GP130/JAK/STAT3-dependent retrograde signaling. These findings reveal a novel role for palmitoylation in the control of axonal retrograde signaling, provide key insights into the molecular roles of this modification and identify new potential targets for therapy to improve nerve regeneration post-injury.