Isoform-specific palmitoylation of JNK regulates axonal development

Abstract

The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1-3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3's distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching. The c-jun N-terminal kinase (JNK) family consists of JNK1, JNK2 and JNK3 subgroups, which participate in diverse biological and pathological processes. 1,2 At least 10 JNK isoforms of varied sizes, with most between 46 and 54 kDa, are produced from the Jnk1-3 genes, giving rise to JNK isoforms. 1 In the nervous system, JNKs (particularly isoform JNK3) have been extensively studied as the key players in apoptosis and neurodegeneration. [3][4][5][6] However, accumulating evidence supports a physiological role of JNK in regulating neurite formation and morphogenesis. 7-12 Pharmacological inhibition of JNK activity blocks axogenesis in hippocampal neurons, arguing for an essential role of JNK in neurite development. 13 Growth factors and signalling molecules, including secreted proteins of the Wnt family, have also been found to activate JNK for remodelling dendrites and axons, a process that relies on cytoskeletal rearrangement. 11,[14][15][16] This has led to the identification of several actin-or microtubule-associated proteins as JNK substrates that modulate different aspects of cytoskeletal activity. 17 However, all the 10 JNK isoforms share the same kinase domain and activation mechanism. 17 The differential roles of JNK isoforms in neurite development and the mechanisms underlying isoform-specific regulation are poorly understood.Analysis of animals null for particular JNK isoforms provides the first evidence to support isoform-specific roles of JNKs in the brain. Mice with Jnk1 À/À show abnormalities in neurite development, 7 whereas mice null for Jnk1 and Jnk2 show embryonic lethality due to severe neuro...

Slippery signaling: Palmitoylation-dependent control of neuronal kinase localization and activity

“…The importance of palmitoylation in DLK-JNK signaling is reinforced by a report that another member of this pathway, the neurally-enriched JNK3, is also palmitoylated (Yang et al, 2012). In peripheral neurons JNK3 plays key roles in retrograde responses to axonal injury and was previously reported to colocalize with JIP3 on axonal vesicles (Keramaris et al, 2005, Cavalli et al, 2005).…”

“…These studies reported an impressive number of palmitoyl-proteins, but did not identify several palmitoylated neuronal proteins, including kinases, that have been identified in targeted studies e.g. (Yang et al, 2012, Holland et al, 2016, George et al, 2015, Levy et al, 2011). This raises the strong possibility that additional studies from different neuronal preparations may identify additional palmitoyl-kinases.…”

“…If so, then palmitoylation would play a key role in modifying multiple DLK-JNK pathway components to ensure the specificity and transport of neuronal injury signals. It is important to note, though, that in CNS neurons, the palmitoylated form of JNK3 was reported to regulate secretory trafficking (Yang et al, 2013) and axonal development (Yang et al, 2012). These different roles of JNK3 may reflect CNS-PNS differences, but multiple functions for JNK3 palmitoylation should perhaps be expected, given that this kinase can play both physiological and pathological roles in neurons (Waetzig and Herdegen, 2003).…”

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…Furthermore, the long (p54) isoforms of JNK1 and JNK2 harbor a caspase cleavage site and their C termini can be processed during the onset of apoptosis, with unclear functional consequences (171). Lastly, the N-and Cterminal-lengthened JNK3␣2(L) isoform of JNK3 was palmitoylated on its C terminus by the DHHC family palmitoyltransferase ZDHHC15, which regulates JNK3␣2(L) subcellular localization and activity; in contrast, the almost-identical C terminus of JNK1␣2 was not palmitoylated (172). Taken together, these studies reinforce the differences in the potential regulatory mechanisms for the different JNK isoforms.…”

Regulation of protein trafficking: JNK3 at the Golgi complex

“…First, JNK3 in neurons undergoes isoform-specific palmitoylation, a lipid modification that generally enhances the hydrophobicity of proteins and regulates their association with membrane structures. Palmitoylation of JNK3 impedes axon growth, a process that relies on active Golgi functions 4 . Second, palmitoylation directs JNK3 to the Golgi complex.…”