CDK14 Promotes Axon Regeneration by Regulating the Noncanonical Wnt Signaling Pathway in a Kinase-Independent Manner

The postinjury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the nonmuscle myosin light chain-4 (MLC-4) phosphorylation signaling pathway. In this study, we have identified svh-16/cdk-14, a mammalian CDK14 homolog, as a positive regulator of axon regeneration in motor neurons. We then isolated the CDK-14-binding protein MIG-5/Disheveled (Dsh) and found that EGL-20/Wnt and the MIG-1/Frizzled receptor (Fz) are required for efficient axon regeneration. Further, we demonstrate that CDK-14 activates EPHX-1, the C. elegans homolog of the mammalian ephexin Rho-type GTPase guanine nucleotide exchange factor (GEF), in a kinase-independent manner. EPHX-1 functions as a GEF for the CDC-42 GTPase, inhibiting myosin phosphatase, which maintains MLC-4 phosphorylation. These results suggest that CDK14 activates the RhoGEF–CDC42–MLC phosphorylation axis in a noncanonical Wnt signaling pathway that promotes axon regeneration. SIGNIFICANCE STATEMENT Noncanonical Wnt signaling is mediated by Frizzled receptor (Fz), Disheveled (Dsh), Rho-type GTPase, and nonmuscle myosin light chain (MLC) phosphorylation. This study identified svh-16/cdk-14, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that CDK-14 binds to MIG-5/Dsh, and that EGL-20/Wnt, MIG-1/Fz, and EPHX-1/RhoGEF are required for axon regeneration. The phosphorylation-mimetic MLC-4 suppressed axon regeneration defects in mig-1, cdk-14, and ephx-1 mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a guanine nucleotide exchange factor for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the noncanonical Wnt signaling pathway controls axon regeneration via the CDK-14–EPHX-1–CDC-42–MLC phosphorylation axis.


Introduction
The ability of axons to regenerate after an injury is a fundamental and conserved property of neurons, governed by interactions between the intrinsic growth state and the extracellular microenvironment (Kaplan et al., 2015). Neurons in the mammalian peripheral nervous system, like neurons in most invertebrates, retain the ability to regenerate. In contrast, neurons in the mammalian CNS have a limited regenerative capacity (Case and Tessier-Lavigne, 2005). This difference in regenerative potential reflects alterations in the intrinsic capacity for axon growth. Accordingly, understanding the intrinsic mechanisms that regulate axon regeneration provides insights into possible neurologic injuries and disease treatments. However, these intrinsic signaling mechanisms operating in the adult nervous system are not yet fully understood.
Axon regeneration of injured neurons involves reorganization of the cytoskeleton (Bradke et al., 2012). After injury, neurons transform the damaged axon ends into new growth cone-like structures, driven by a dynamic reorganization of the actin network (Coles and Bradke, 2015). The Rho-type GTPase family is central to the regulation of cytoskeletal dynamics, including actin polymerization, F-actin stabilization, and actomyosin assembly in neuronal and non-neuronal cells (Hall, 1998). Rho GTPase functions as a bimolecular switch, with inactive GDP-bound and active GTP-bound forms. Activated Rho GTPases interact with effector proteins that trigger various cellular responses (Bishop and Hall, 2000). Among the Rho GTPase effectors, Rho-associated coiled-coil kinase (ROCK) plays an essential role in actin organization via myosin activation (Amano et al., 1996). Several Rho GTPases, including Rho, Cdc42, and Rac, have been demonstrated to function in the noncanonical Wnt pathway (Schlessinger et al., 2009). This pathway uses universal Wnt signaling components, such as Frizzled receptor (Fz) and Disheveled (Dsh). Although several Wnts have been shown to function in the nervous system, including neuron migration, polarity, neurite extension, and neurite pruning Lu et al., 2004;Montcouquiol et al., 2006;Hayashi et al., 2009), the Wnt pathway contribution to axon regeneration is poorly understood.
Recent studies on axon regeneration in genetic model organisms, such as Caenorhabditis elegans, have identified common biological pathways that use conserved molecules to control regeneration (Byrne and Hammarlund, 2017). Axon regeneration in C. elegans is regulated by the p38 MAP kinase (MAPK), JNK MAPK, and Rho GTPase signaling pathways (Hammarlund and Jin, 2014;Hisamoto and Matsumoto, 2017;Shimizu et al., 2018). The JNK pathway consists of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 JNK; it is inactivated at the KGB-1 activation step by VHP-1, which is a member of the MAPK phosphatase family (Mizuno et al., 2004). The vhp-1 loss-of-function mutation causes hyperactivation of the JNK pathway, resulting in developmental arrest at an early larval stage. We have previously undertaken a genome-wide RNAi screen for suppressors of vhp-1 lethality and isolated 92 svh genes (Li et al., 2012;Shimizu et al., 2021). Analysis of these svh genes was able to shed new light on the regulation of axon regeneration. Moreover, we demonstrated that in C. elegans, the RHO-1/RhoA-LET-502/ROCK signaling pathway promotes axon regeneration via phosphorylation of the regulatory nonmuscle myosin light chain [MLC (MLC-4); Shimizu et al., 2018]. We have also found that the svh-15 gene was previously identified as brc-2, which encodes a homolog of the mammalian BRCA2 tumor suppressor (Martin et al., 2005;Shimizu et al., 2018). We have demonstrated that SVH-15/BRC-2 is required for the activation of LET-502 by GTPbound RHO-1. MLC phosphorylation activates the Mg ATPase activity of nonmuscle myosin II and stimulates the interaction between actin and myosin (Somlyo and Somlyo, 2003). A growth cone contains a specific actin structure, which generates a mechanical force at the leading edge of the lamellipodium (Bradke et al., 2012). A previous study has revealed that myosin II functions in actin-bundle turnover in the growth cones of cultured neurons (Medeiros et al., 2006). These results suggest that MLC-4 phosphorylation regulates the dynamics of regenerating growth cones via the alteration of actin structures. Indeed, in C. elegans, MLC-4 is phosphorylated at the ends of the injured axons (Shimizu et al., 2018). The altered phosphorylation state of MLC is attributed to direct ROCK-mediated phosphorylation and inhibition of the myosin-binding subunit of myosin phosphatase (MYPT), which is a part of the MLC phosphatase complex (Totsukawa et al., 2000). Therefore, MYPT is expected to be involved in regulating C. elegans axon regeneration.

Materials and Methods
C. elegans strains. The C. elegans strains used in this study are listed in Table 1. All strains were maintained on nematode growth media and fed with OP50, as described previously (Brenner, 1974).
Microscopy. Fluorescence imaging of transgenic animals was performed under a 100Â objective lens on a fluorescent microscope (model ECLIPSE E800, Nikon) and photographed using a Zyla CCD camera (Oxford Instruments).
Axotomy. Axotomies were performed as described previously (Li et al., 2012). Animals were subjected to axotomy at the young adult stage. Commissures that displayed growth cones or small branches on the proximal fragment were counted as "regenerated." Proximal fragments that showed no change after 24 h were counted as "no regeneration." At least 20 individuals with one to three axotomized commissures were observed for most experiments.
Measurements of regenerating axons. The length of regenerating axons for D-type motor neurons was measured using the segmented line tool of ImageJ. Measurements were made from the site of injury to the tip of the longest branch of the regenerating axon. Axons that did not regenerate were excluded. Data were plotted using R (version 4.0.1) and R studio (version 1.3.959).
Biochemical experiments using mammalian cells. For immunoprecipitation, transfected COS-7 cells were lysed in RIPA buffer [50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM  (K89) is required for kinase activity. The region deleted in the tm4238 allele is indicated by the black bar. B, Representative D-type motor neurons in wild-type and cdk-14 mutant animals 24 h after laser surgery. In wild-type animals, severed axons exhibited regenerating growth cones (yellow arrowhead). In cdk-14 mutants, the proximal ends of axons failed to regenerate (white arrowhead). Scale bars, 10 mm. C, Percentage of axons that initiated regeneration 24 h after laser surgery in young adults. The number of axons examined is shown. Error bars indicate 95% confidence intervals. *p , 0.05, **p , 0.01, as determined by Fisher's exact test. NS, Not significant. D, Length of regenerating axons 24 h after laser surgery. Data are presented as a boxplot representing median (thick line within the box) and interquartile range (edge of box) with individual data points. The number (n) of axons examined is shown. Statistical significance was determined by the Wilcoxon rank-sum test.
Experimental design and statistical analyses. All experiments were not randomized, and the investigators were not blinded to the group allocation during experiments and outcome assessment. No statistical methods were used to predetermine sample size. Data visualization was performed using Microsoft Excel 2016, R (version 4.0.1), and R studio (version 1.3.959). Statistical analysis was conducted as described previously (Li et al., 2012). Briefly, 95% confidence intervals were calculated using the modified Wald method, and the two-tailed p values were calculated using Fisher's exact test on GraphPad QuickCalcs (http://www. graphpad.com/quickcalcs/contingency1/). The Wilcoxon rank-sum test (two tailed) was performed using R (version 4.0.1), R studio (version 1.3.959), and the R exactRankTests package.
Homology search, phylogenetic analysis, identification of domains, and alignments of amino acids. Homology search, identification of conserved domains, and alignments of amino acids were executed using BLAST [Basic Local Alignment Search Tool; National Center for Biotechnology Information (NCBI)], CD-search (NCBI), and Genetyx-Mac programs.

SVH-16/CDK-14 is involved in axon regeneration
So far, we have isolated 92 svh genes involved in axon regeneration (Li et al., 2012;Shimizu et al., 2018Shimizu et al., , 2021. The svh-16 gene corresponds to Wormbase ORF ZC123.4, which encodes a protein with high homology to mammalian CDK14, a member of the CDK family ( Fig. 1A; Malumbres, 2014). We, therefore, renamed svh-16 as cdk-14. To clarify the role of cdk-14 in axon regeneration, we performed laser axotomy and assessed the regeneration of GABA-releasing D-type motor neurons ( Fig. 1B;  Yanik et al., 2004). In young adult wild-type animals, ;65% of severed axons started to regenerate within 24 h after axonal injury, whereas the frequency was reduced in cdk-14(tm4238) mutants ( Fig. 1B,C, Table 2). We investigated the effects of cdk-14 on growth cone behavior and found that, in cdk-14(tm4238) mutants, the length of regenerated axons or axon guidance remains unaffected (Fig. 1D). Thus, CDK-14 is explicitly required for initiating axon regeneration. Notably, the effect of cdk-14 is specific to injuryinduced regeneration, as the cdk-14(tm4238) mutation did not affect nerve development per se In Wormbase, the genomic sequence of the cdk-14 gene is predicted to encode three isoforms, CDK-14a, CDK-14b, and CDK-14c ( Fig. 2A,B). To confirm that the cdk-14 mutation is responsible for the axon regeneration defect, we generated the transgene Punc-25::cdk-14c, which contains the cdk-14c cDNA and D-type motor neuron-specific unc-25 promoter. Expression of cdk-14c in D-type neurons rescued the defect in axon regeneration observed in cdk-14(tm4238) mutants (Fig. 1C, Table 2). Therefore, we will hereafter refer to cdk-14c as cdk-14. Our results indicate that CDK-14 regulates axon regeneration of injured D-type motor neurons after laser axotomy in a cell-autonomous manner.
Mammalian CDK14, also referred to as PFTK1, is activated by a specific interaction with cyclin Y (Jiang et al., 2009). However, the C. elegans cyclin Y homolog cyy-1 is not involved in axon regeneration (Nix et al., 2014). Thus, we hypothesized that the kinase activity of CDK-14 is not required for axon regeneration. To test this possibility, we used CRISPR-Cas9 mutagenesis to generate a catalytically inactive cdk-14(K89R) mutant at the endogenous cdk-14 locus with the invariant Lys-89 mutation in subdomain II, which is critical for ATP binding (Fig. 2C; Shu et al., 2007). The cdk-14(K89R) mutation had no effect on axon regeneration (Fig. 1C, Table 2), suggesting that CDK-14 regulates axon regeneration independent of its kinase activity.
As Dsh proteins link Fz receptors to the Wnt signaling pathway (Cadigan and Liu, 2006), we examined the possibility that Wnt regulates axon regeneration. In this pathway, a Wnt ligand binds to the Fz receptor and activates Dsh (Fig. 3A). To determine whether the Wnt pathway regulates axon regeneration, we further examined whether there are svh genes encoding components that function in the Wnt pathway. We then identified svh-21, which is identical to mig-1, encoding an Fz family member of C. elegans ( Fig. 4A; Pan et al., 2006). Next, we inquired whether the mig-1 gene was also involved in axon regeneration, wherein we found that the mig-1(e1787) mutation (Fig. 4A) significantly reduced axon regeneration after the laser injury (Fig. 4B, Table  2). Like cdk-14 mutants, the mig-1(e1787) mutation did not affect axon guidance or nerve development.

EPHX-1/RhoGEF functions between CDK-14 and CDC-42
GTPase in the axon regeneration pathway Activation of Rho GTPase depends on GEF family members that catalyze the GDP-GTP exchange reaction (Rossman et al., 2005). Therefore, a GEF for the CDC-42 GTPase should function in the noncanonical Wnt pathway to regulate axon regeneration. Human weak-similarity GEF (WGEF) directly interacts with Dsh and functions within the noncanonical Wnt pathway ( Fig.  6A; Tanegashima et al., 2008). WGEF belongs to the Dbl family of RhoGEF proteins that usually possess tandem Dbl homology (DH), pleckstrin homology (PH), and Src homology 3 (SH3) domains ( Fig. 6A; Tanegashima et al., 2008). The C. elegans ephx-1 gene encodes a Dbl family protein homologous to WGEF and ephexin (Fig. 6A). We, therefore, examined whether EPHX-1 acts upstream of CDC-42 to regulate axonal regeneration. We found that the ephx-1(ok494) mutant (Fig. 6B) was impaired in axon regeneration (Fig. 6C, Table  2). Like the cdk-14 mutant, the ephx-1(ok494) mutant did not affect the length of the regenerated axons (Fig. 6D), axon guidance, or nerve development. Furthermore, the ephx-1 phenotype defective in axon regeneration was suppressed by the expression of GTP-bound CDC-42(G12V), but not by that of LET-502DC (Fig. 6E, Table 2). These results support the hypothesis that EPHX-1 functions as a GEF for the CDC-42 GTPase in the noncanonical Wnt pathway to regulate axon regeneration.

CDK-14 interacts with EPHX-1
The GEF potential of Dbl family members is autoinhibited by an intramolecular interaction between the N-terminal poly-proline region and the C-terminal SH3 domain ( Fig. 7A; Schiller et al., 2006). Indeed, the SH3-containing domain of EPHX-1 was associated with the N-terminal domain in the yeast two-hybrid assay (Fig. 7B,C), and the EPHX-1 N-terminus contains poly-proline regions (Fig. 7B). Since CDK-14 regulation of axon regeneration is kinase independent (Fig.  1C), we hypothesized that CDK-14 interacts with EPHX-1 to activate EPHX-1 activity by inhibiting intramolecular binding. To investigate this possibility, we asked whether CDK-14 physically interacts with EPHX-1. Yeast two-hybrid analysis revealed that CDK-14 is associated with the SH3-containing (amino acids 1033À1159) domain of EPHX-1 (Fig. 7D). These results suggest that the interaction between CDK-14 and the SH3 domain of EPHX-1 disrupts intramolecular binding and activates EPHX-1 in axon regeneration.

EPHX-1 N-terminal domain inhibits interaction with CDC-42
Since the SH3 domain of EPHX-1 interacted with the N-terminal region (Fig. 7C), we expected the N-terminal region of EPHX-1 to exert GEF autoinhibitory activity. The ok494 allele of the ephx-1 gene encodes a protein with a frameshift deletion between amino acids 719 and 977 of the DH domain, essentially a truncation at amino acid 723 (Fig. 6B). The tm11319 allele has a deletion of the N-terminal domain, resulting in a truncated EPHX-1 (EPHX-1d), with the N-terminal 467 aa deleted (Fig. 6B). We observed a reduced frequency of axon regeneration in ephx-1 (ok494) mutants (Fig. 6C, Table 2), whereas the tm11319 mutation had no effect on axon regeneration (Fig. 8A, Table 2). These results indicate that the EPHX-1 N-terminal domain is unnecessary for axon regeneration. If the N-terminal region of EPHX-1 inhibits GEF activity, then deletion of the N terminus would be expected to increase EPHX-1 activity. Consistent with this prediction, we found that the ephx-1(tm11319) mutation was able to suppress the regeneration defect of cdk-14(tm4238) mutants (Fig.  8A, Table 2). Therefore, truncation of the N-terminal domain of EPHX-1 can relieve the autoinhibition of GEF activity.   Increased activity of the N-terminal EPHX-1 deletion mutant could be explained by a conformational change in the remaining part of the molecule, affecting its binding to CDC-42. We tested this possibility by analyzing the binding activity of EPHX-1 (full-length) and EPHX-1DN(1À467) with CDC-42. EPHX-1DN(1À467) corresponds to the EPHX-1d isoform (Figs. 6B, 8B). We cotransfected FLAGtagged GDP-bound CDC-42(T17N) and T7-tagged EPHX-1 into mammalian COS-7 cells. Coimmunoprecipitation experiments revealed that EPHX-1DN(1À467) had significantly higher CDC-42-binding activity than full-length EPHX-1 (Fig. 8B), suggesting that the N-terminal domain of EPHX-1 has an autoinhibitory function.

Discussion
The Wnt signaling pathway plays multiple roles in the nervous system, including axon guidance, polarity establishment, axon regeneration, and synaptic specificity (He et al., 2018). The importance of Wnt signaling is demonstrated by the conservation of its molecular components across organisms from C. elegans to humans. The Wnt pathway is classified into canonical or noncanonical pathways. In the canonical Wnt signaling pathway, Wnt binds to the Fz receptor to activate the transcription of target genes through the regulation of b -catenin and T-cell factor (TCF). The noncanonical Wnt pathway is independent of b -catenin and TCF. This pathway uses universal Wnt signaling components such as Fz and Dsh, but, unlike the canonical Wnt pathway, Dsh functions through Rho family GTPases, subsequently activating downstream JNK to induce cytoskeletal rearrangements (Montcouquiol et al., 2006). Recent studies in mammalian and fish model systems have demonstrated that Wnt signaling promotes axon regeneration after injury in the adult optic nerve and spinal cord (Osakada et al., 2007;Herman et al., 2018), increasing the potential therapeutic value of Wnt. However, our understanding of its downstream signaling pathways remains limited. Our genetic analysis identified a set of Wnt components and demonstrated that EGL-20/Wnt functions in axon regeneration via the Fz receptor MIG-1. Our study reveals that the noncanonical Wnt pathway triggers downstream events that eventually act on the cytoskeleton to initiate axon regeneration (Fig. 9).
How is Wnt signaling transduced from Dsh to Rho GTPase activation in axon regeneration? Our results reveal a mechanistic link between Wnt-Dsh signaling and CDK14. Mammalian CDK14, also called PFTK1, is a member of the CDKs and is activated by a specific interaction with cyclin Y (Jiang et al., 2009). The CDK14/PFTK1-cyclin Y complex activates the noncanonical Wnt pathway, which subsequently activates Rho GTPases, such as Rho, Cdc42, and Rac, resulting in actin polymerization that underlies cell motility and migration (Sun et al., 2014). Analogous to mammalian CDK14/PFTK1, we showed that C. elegans CDK-14 functions as an essential regulator of the noncanonical Wnt pathway via activation of CDC-42 in axon regeneration. However, in contrast to mammalian CDK14, the C. elegans cyclin Y homolog is not involved in axon regeneration (Nix et al., 2014). Therefore, the kinase activity of CDK-14 is not required for axon regeneration. Consistent with this, a kinasenegative cdk-14(K89R) mutation had no effect on axon regeneration, indicating that CDK-14 regulation of axon regeneration is kinase independent.
How does CDK-14 control axon regeneration in a kinase-independent manner? Several studies have shown that the noncanonical Wnt pathway depends on the activation of Rho GTPases (Strutt et al., 1997;Choi and Han, 2002;Habas et al., 2003), which is mediated by RhoGEFs that catalyze the GDP-GTP exchange reaction (Piekny et al., 2000). Indeed, WGEF has been identified in Xenopus as a link between Dsh and Rho activation in the noncanonical Wnt signaling pathway (Tanegashima et al., 2008). We also found that C. elegans EPHX-1/ ephexin RhoGEF mediates noncanonical Wnt signaling in axon regeneration and demonstrated that CDK-14 promotes axon regeneration by activating EPHX-1, which acts on CDC-42. We propose that EPHX-1 is a necessary component in the pathway linking CDK-14 to CDC-42 activation (Fig. 9).
EPHX-1 belongs to the Dbl family of GEFs for Rho GTPases, whose domain structure consists of the DH-PH cassette followed by the SH3 domain (Rossman et al., 2005). The GEF potential of Dbl family members, such as WGEF and ephexin, is autoinhibited by intramolecular and intermolecular interactions (Schiller et al., 2006). The C-terminal SH3 domain binds to the N-terminal region, stabilizing the autoinhibitory helix on the DH domain. This interaction tightly suppresses Rho GTPase access to the DH domain required for GDP-GTP exchange. Indeed, truncation of the N-terminal region of Dbl results in its activation (Eva and Aaronson, 1985). We also showed that the N-terminal region of EPHX-1 inhibits binding to CDC-42. Based on these results, we propose the following mechanism by which CDK-14 activates the GEF activity of EPHX-1. EPHX-1 exchange potential is autoinhibited by an intramolecular interaction between the SH3 domain and the N-terminal region. The binding of CDK-14 to the SH3 domain of EPHX-1 removes the SH3 domain from the Nterminal region, activating the exchange potential of EPHX-1 (Fig. 9).
EPHX-1 functions as a GEF for RHO-1 in axon regeneration regulated by integrin signaling (Sakai et al., 2021b). This indicates that EPHX-1 can act on both RHO-1 and CDC-42 GTPases. How is the specificity of EPHX-1 RhoGEF for cognate GTPases determined? Mammalian ephexin is tyrosine phosphorylated in an N-terminal motif, which then alters the specificity Complex formation was detected by immunoprecipitation (IP) with anti-T7 antibody, followed by immunoblotting (IB) with anti-FLAG and anti-T7 antibodies. Total lysates were immunoblotted with anti-FLAG antibody. Schematic diagrams of EPHX-1 (full length) and EPHX-1DN(1À467) are shown above. Figure 9. Schematic model for the regulation of axon regeneration by the noncanonical Wnt signaling pathway. The EGL-20/Wnt-MIG-1/Fz signaling promotes axon regeneration by activating the noncanonical Wnt pathway consisting of the CDK-14-EPHX-1-CDC-42-MLC-4 phosphorylation axis. The exchange potential of EPHX-1 is autoinhibited by an intramolecular interaction between the SH3 domain and the N-terminal region. CDK-14 binding to the SH3 domain of EPHX-1 removes the SH3 domain from the N-terminal region, thereby activating the exchange potential of EPHX-1 toward CDC-42. Finally, GTP-bound CDC-42 causes the inactivation of MEL-11, leading to MLC-4 phosphorylation, which promotes axon regeneration.
for cognate GTPases. When ephexin is not tyrosine phosphorylated, it activates RhoA, Cdc42, and Rac1; however, it activates RhoA exclusively when the ephexin is phosphorylated (Sahin et al., 2005). Similarly, in C. elegans, integrin activation directs the exchange activity of EPHX-1 toward RHO-1 via the SRC-1mediated tyrosine phosphorylation of the EPHX-1 N-terminal region. These results suggest that tyrosine phosphorylation of EPHX-1 determines the specificity of its GEF activity for RHO-1. Consistent with this, activated LET-502DC suppressed the axon regeneration defect caused by the nonphosphorylated ephx-1 (Y568F) mutation, but not by the ephx-1 deletion mutation. In the noncanonical Wnt signaling pathway, however, the CDK-14 interaction determines the specificity of EPHX-1 GEF for CDC-42. In this case, the GTP-bound form of CDC-42 suppressed the ephx-1 deletion defect. One explanation for this result is that the protein kinase activated by active CDC-42 may phosphorylate both MLC-4 and MEL-11. MRCK and PAK are possible kinases that function downstream of CDC-42 (Wilkinson et al., 2005;Gally et al., 2009), but independent mutations in mrck-1 or pak-1 did not affect axon regeneration (Chen et al., 2011;Nix et al., 2014). Therefore, MRCK-1 and PAK-1 appear to be redundant kinases that induce MLC-4 phosphorylation during axon regeneration. Although this is the simplest model, we cannot exclude the possibility that a third kinase distinct from MRCK-1 and PAK-1 may work in the axon regeneration pathway. Thus, this calls for further studies to focus on identifying kinases that regulate axon regeneration downstream of CDC-42 in the noncanonical Wnt pathway.