DLK (Dual Leucine Zipper-Bearing Kinase)
DLK is a serine/threonine kinase that belongs to the mixed-lineage kinase (MLK) family of mitogen-activated protein kinase kinase kinases (MAPKKKs) (Gallo and Johnson 2002). It was discovered in 1994 as a protein differentially expressed during the retinoic acid-induced neuronal differentiation of human NT2 teratocarcinoma cells and originally denoted zipper protein kinase (ZPK) (Reddy and Pleasure 1994). Parallel and subsequent studies led to the identification and cloning of the mouse and rat homologs of ZPK, respectively, termed DLK (Holzman et al. 1994) and MAP kinase upstream kinase (MUK) (Hirai et al. 1996).
Structure, Expression, and Subcellular Localization
Northern blot analysis of human and mouse tissues revealed that the highest levels of DLK mRNA are observed in brain and kidney (Reddy and Pleasure 1994; Holzman et al. 1994). DLK mRNA was also detected by in situ hybridization in mouse skin, stomach, small intestine, liver, pancreas, and testis (Nadeau et al. 1997). In all these tissues, the expression of DLK mRNA increases with development and correlates with areas occupied by differentiated rather than proliferating cells (Nadeau et al. 1997). For example, in developing mouse skin, DLK mRNA expression was detected in the suprabasal cell layers of the epidermis but not in the innermost basal layer, which contains mitotically active cells.
Consistent with the expression results described above, immunostaining experiments on mouse embryos revealed that the DLK protein is predominantly detected in neural tissues, including brain, spinal cord, and dorsal root ganglion (Hirai et al. 2002). In the developing brain, DLK is most abundant in cells of the subventricular and intermediate zones, which are comprised primarily of immature neurons (Hirai et al. 2002). Examination of the subcellular localization of DLK in cultured embryonic neurons indicated that it is preferentially associated with the microtubules and the Golgi apparatus (Hirai et al. 2002). In pancreatic β-cells, DLK is also known to localize in the cytoplasm and translocate into the nucleus upon exposure to inflammatory cytokines (Wallbach et al. 2016).
Signaling Properties and Regulation
To date, much remains to be established about the molecular mechanisms regulating DLK activation and signal suppression. Some evidence suggests, however, that dimerization or oligomerization of DLK mediated by the leucine zipper motifs is a prerequisite for autophosphorylation, activation, and stimulation of the JNK pathway (Gallo and Johnson 2002). Work from a number of laboratories has also indicated that the regulation of DLK is achieved by heterologous interactions with various cellular proteins. The binding of DLK to these proteins, in particular the scaffold JNK-interacting protein (JIP)-1 and MUK-binding inhibitory protein (MBIP), plays an important role in DLK regulation by preventing its dimerization and activation (Fukuyama et al. 2000; Gallo and Johnson 2002). Another important mechanism of DLK regulation is phosphorylation, a process that modulates the stability and enzymatic activity of DLK. In this regard, it was shown that DLK undergoes JNK-dependent phosphorylation and stabilization in response to neuronal stress (Huntwork-Rodriguez et al. 2013). This phosphorylation at critical residues outside of the catalytic domain upregulates DLK protein abundance via reduction of DLK ubiquitination, which is mediated at least in part by the E3 ubiquitin ligase Phr1. More recently, DLK was also shown to have increased stability in cultured embryonic rat cortical neurons exposed to forskolin, an activator of adenylyl cyclase and protein kinase A (PKA). This effect of forskolin was blocked by pretreatment with the PKA inhibitor H89, indicating that the increase in DLK protein levels is PKA-dependent. In support of this, it was found that overexpression of the catalytic subunit of PKA alone could activate DLK and promote its stabilization, thus reinforcing the importance of phosphorylation in DLK regulation (Hao et al. 2016). Besides its stimulatory effect, phosphorylation is also implicated in the negative regulation of DLK activity and function. Indeed, it has been demonstrated that the kinase activity of DLK can be inhibited in mouse embryonic stem cells by Akt-mediated phosphorylation at two distinct sites (Wu et al. 2015). Such an inhibition was found to be required for maintaining the self-renewal capacity of these cells. Finally, in addition to phosphorylation, palmitoylation is another modification involved in DLK regulation. A recent study has reported that DLK is palmitoylated on a conserved cysteine residue adjacent to the kinase domain and that palmitoylation is required for DLK-dependent retrograde signaling in axons (Holland et al. 2016). Mechanistically, this was associated with the ability of palmitoylation to modulate DLK attachment to trafficking vesicles, interactions with protein partners and catalytic activity.
Wallenda and DLK-1, the respective Drosophila and C. elegans orthologues of DLK, are also known to be regulated by a ubiquitin-proteasomal degradation mechanism involving the E3 ligase Highwire/RPM-1 (Nakata et al. 2005; Collins et al. 2006). In the worm, this degradation is facilitated by PPM-2, a protein phosphatase that dephosphorylates DLK-1 at a serine residue critical for the binding of a shorter DLK-1 isoform (Baker et al. 2014). The primary function of this isoform is to inhibit DLK-1 function because its Ca2+-mediated dissociation switches DLK-1 activity from off to on state in neurons (Yan and Jin 2012). Furthermore, Wallenda and DLK-1 are both activated by conditions that affect cytoskeletal stability, such as mutations in the actin-microtubule cross-linking protein Short stop (Valakh et al. 2013) or tubulin (Chen et al. 2014). Consistent with these data are studies in mammalian sensory neurons showing that genetic ablation of DLK abolished almost completely phosphorylation of the JNK substrate c-Jun in response to cytoskeleton-disrupting drugs (Valakh et al. 2015). Taken together, these findings point to a role for DLK signaling in the detection of cytoskeletal perturbations.
In vitro studies with different types of cells have identified a role for DLK in the regulation of many physiological processes. For example, in rat pheochromocytoma PC12 cells and sympathetic neurons, ectopic expression of DLK induces apoptosis, whereas kinase-deficient DLK severely inhibits death caused by nerve growth factor deprivation (Xu et al. 2001). Consistent with a role in cell death, downregulation of DLK by RNA interference in mouse NIH 3 T3 fibroblasts and human MDA-MB-231 breast cancer epithelial cells blocked the apoptotic response induced by calphostin C (Robitaille et al. 2008). Based on its distribution during development, a role for DLK in cell differentiation has also been proposed (Nadeau et al. 1997). Accordingly, it was reported that DLK overexpression in poorly differentiated normal keratinocytes is sufficient to induce phenotypic changes associated with keratinocyte terminal differentiation, including upregulation of filaggrin expression, DNA fragmentation, activation of transglutaminases, and formation of corneocytes (Robitaille et al. 2005). Other evidence suggests that DLK may also play a role in adipocyte cell differentiation. Hence, in 3T3-L1 cells induced to undergo adipocyte differentiation, DLK expression is upregulated, and its knockdown by RNA interference completely blocks the accumulation of lipid droplets as well as the expression of the adipogenic markers adiponectin and fatty acid synthase. In agreement with this, cells lacking DLK show significantly less expression of the master regulators of adipogenesis, peroxisome proliferator-activated receptor (PPAR)-γ2, and the CCAAT enhancer-binding protein (C/EBP)α (Couture et al. 2009). Interestingly, DLK was also identified as a key regulator of axon growth in mammals. This is supported by the findings that DLK knockdown prevents neurite extension from cultured PC12 cells and mouse embryonic cortical neurons (Eto et al. 2010; Hirai et al. 2011). Finally, a recent work by Stahnke et al. (2014) has revealed a new role for DLK in regulating insulin gene expression through modulation of the transcriptional activity of MafA in islet beta cells. Collectively, these data indicate that DLK may fulfill different functions, depending on the stimuli and the cellular context.
Considerable progress has also been made within the last few years in understanding the in vivo biological functions of DLK. In mice, knockout of the DLK gene results in a lethal phenotype around birth (Hirai et al. 2006), suggesting that it might have a crucial role during embryogenesis and organogenesis. Embryos lacking DLK display abnormal brain development, characterized by defects in axon growth, neuron migration, apoptosis, and axon degeneration (Hirai et al. 2006, 2011; Bloom et al. 2007; Ghosh et al. 2011; Itoh et al. 2011). In addition, these mice have reduced JNK activity and reduced phosphorylation of JNK substrates, such as the microtubule-stabilizing proteins Doublecortin and MAP2c (Hirai et al. 2006). Besides its role during development, it has been reported that DLK plays a crucial role in both the degeneration and regeneration of mature neurons in response to injury. Support for this idea is provided by the observations that conditional deletion of DLK significantly attenuates the neuronal and axonal degeneration caused by mechanical injury and glutamate-induced excitotoxicity (Miller et al. 2009; Pozniak et al. 2013; Watkins et al. 2013; Welsbie et al. 2013). Additionally, the absence of DLK in mouse has been shown to impair axonal growth in optic and sciatic nerve crush injury models (Shin et al. 2012; Watkins et al. 2013). The exact mechanism of this response remains unclear, but these studies showed the involvement of DLK in retrograde axonal transport of injury signals as well as in transcriptional regulation of both pro-apoptotic and regeneration-associated genes. Interestingly, this dual function of DLK seems to be conserved throughout evolution, since a dramatic defect in axon degeneration or regeneration after injury has been noticed in Drosophila and C. elegans mutants defective in Wallenda and DLK-1 (Miller et al. 2009; Hammarlund et al. 2009). Thus, these findings demonstrate a key role for DLK in controlling neuronal development as well as degenerative and regenerative responses to axonal injury.
DLK is a serine/threonine kinase that functions as an upstream activator of the MAPK pathways. It is expressed in a tissue-specific manner and regulated by mechanisms that involve phosphorylation, palmitoylation, interactions with different protein partners, and ubiquitin-mediated degradation. The functions of DLK are diverse and include regulation of development, cell differentiation, and apoptosis. Studies of mouse, fly, and worm mutants defective in DLK show that this interesting kinase may also be required for axonal degeneration and regeneration in response to injury. Thus, DLK appears to be a pivotal signaling component for regulation of various fundamental biological processes, although the precise molecular mechanisms by which it is activated and by which it mediates such effects are still elusive. Further work is therefore required to address these issues and expand our understanding of DLK’s action.
We thank Dr. Alain Lavigueur for critical reading of the manuscript and the Natural Sciences and Engineering Research Council of Canada for its financial support. We also apologize to our colleagues whose work could not be cited due to space limitations.
- Hao Y, Frey E, Yoon C, Wong H, Nestorovski D, Holzman LB, Giger RJ, DiAntonio A, Collins C. An evolutionarily conserved mechanism for cAMP elicited axonal regeneration involves direct activation of the dual leucine zipper kinase DLK. Elife. 2016;5:e14048. doi: 10.7554/eLife.14048.PubMedPubMedCentralCrossRefGoogle Scholar
- Huntwork-Rodriguez S, Wang B, Watkins T, Ghosh AS, Pozniak CD, Bustos D, Newton K, Kirkpatrick DS, Lewcock JW. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis. J Cell Biol. 2013;202:747–63. doi: 10.1083/jcb.201303066.PubMedPubMedCentralCrossRefGoogle Scholar
- Watkins TA, Wang B, Huntwork-Rodriguez S, Yang J, Jiang Z, Eastham-Anderson J, et al. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury. Proc Natl Acad Sci USA. 2013;110:4039–44. doi: 10.1073/pnas.1211074110.PubMedPubMedCentralCrossRefGoogle Scholar