Nonmuscle Myosin Light Chain Research Articles

Analysis of cDNAs encoding Drosophila melanogaster myosin light chain kinase.

Myosin light chain kinase regulates the activity of myosin by phosphorylating the myosin regulatory light chain. Here we describe the cloning and characterization of cDNAs encoding Drosophila melanogaster myosin light chain kinase. We amplified a fragment of the Drosophila mlck gene using degenerate primers homologous to a highly conserved region in myosin light chain kinase proteins of vertebrate species. We used the gene fragment to isolate corresponding Drosophila mlck cDNAs. The deduced protein sequence of the cDNAs shows high homology to the catalytic and regulatory domains of vertebrate nonmuscle, smooth muscle and skeletal muscle myosin light chain kinase. Protein motifs I and II, which are present in vertebrate nonmuscle and smooth muscle myosin light chain kinase, but not in skeletal muscle myosin light chain kinase, are also present in Drosophila myosin light chain kinase. Transcript and cDNA analysis shows the gene encodes multiple messages and is expressed in nonmuscle and muscle cells, including the adult indirect flight muscle. Genomic Southern analysis and chromosome hybridization suggest mlck is a single copy gene which maps to chromosome band 52D, and is not haplo-insufficient for flight.

Identification and Characterization of Novel Genes Modulated in the Thyroid of Dogs Treated with Methimazole and Propylthiouracil

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific stimulation or repression of a large number of genes. To better understand differentiated epithelial cell growth regulation, we have initiated a study to identify genes which are regulated by the thyrotropin-dependent mitogenic pathway in dog thyroid cells. A thyroid cDNA library was prepared from a methimazole and propylthiouracil-treated dog and differentially screened with probes derived from control or stimulated thyroids. Among 19 clones isolated, 6 encode known proteins (inwardly rectifying potassium channel, nucleosome assembly protein, ribosomal protein L7, elongation factor 1alpha, non-muscle myosin light chain, and heat shock protein 90beta). The 13 others correspond to proteins whose function is unknown. Among them, 5 correspond to mRNAs whose expression was modulated by mitogenic stimulation of thyrocytes in primary culture. A preliminary characterization of two of these cDNAs is reported: clone 5, which might represent a novel, atypical protein kinase, and clone 3, which contains ankyrin-like repeats, suggesting that it might interact with other proteins.

Essential light chain of Drosophila nonmuscle myosin II

We have cloned and sequenced a cDNA encoding the essential (alkaline) light chain of nonmuscle myosin from Drosophila melanogaster. The protein predicted from the cDNA matches partial amino acid sequence derived from essential light chain protein that copurifies with native nonmuscle myosin heavy chain. This completes the sequence of the three myosin subunits, two of which have been shown genetically to be required for morphogenesis and cytokinesis (the heavy chain encoded by zipper and the regulatory light chain encoded by spaghetti squash). The essential light chain protein is 147 amino acids in length and is 53% identical to human smooth muscle essential light chain. The sequence is consistent with the presence of four helix-loop-helix domains seen in crystallographic structures of the striated muscle myosin light chains and their close relative, calmodulin. We identified the most conserved residues among essential light chain sequences from multiple phyla and present their locations on the crystallographic structure of striated muscle essential light chain. This highlights several conserved contacts among the myosin subunits that may be important for the structure and regulation of the myosin motor. The gene encoding Drosophila nonmuscle essential light chain (Mlc-c) localizes to cytological position 5A6 and we discuss prospects for genetic analysis in this region.

Drosophila nonmuscle myosin II is required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos

The X-linked Drosophila gene spaghetti squash (sqh) encodes the regulatory light chain of nonmuscle myosin II. To assess the requirement for myosin II in oogenesis and early embryogenesis, we induced homozygous germline clones of the hypomorphic mutation sqh1 in otherwise heterozygous mothers. Developing oocytes in such sqh1 germline clones often failed to attain full size due to a defect in 'dumping', the rapid phase of cytoplasmic transport from nurse cells. In contrast to other dumpless mutants described to date, sqh1 egg chambers showed no evidence of ring canal obstruction, and no obvious alteration in the actin network. However the distribution of myosin II was abnormal. We conclude that the molecular motor responsible for cytoplasmic dumping is supplied largely, if not exclusively, by nurse cell myosin II and we suggest that regulation of myosin activity is one means by which cytoplasmic transport may be controlled during oocyte development. The eggs resulting from sqh1 clones, though smaller than normal, began development but exhibited an early defect in axial migration of cleavage nuclei towards the posterior pole of the embryo, in a similar manner to that seen in early cleavage eggs in which the actin cytoskeleton is disrupted. Thus both nurse cell dumping and axial migration require a maternally supplied myosin II.

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or -2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized. Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of the myosin light chain, and in a residue tentatively identified as serine-1944 in the myosin heavy chain.

Non-muscle and smooth muscle myosin light chain kinases: no end in sight

Non-muscle and smooth muscle myosin light chain kinases: no end in sight

Sarcomeric myosin heavy chain expressed in nonmuscle cells forms thick filaments in the presence of substoichiometric amounts of light chains.

Central to the function of myosin is its ability to assemble into thick filaments which interact precisely and specifically with other myofibrillar proteins. We have established a novel experimental system for studying myofibrillogenesis using transient transfections of COS cells, a monkey kidney cell line. We have expressed both full-length rat alpha cardiac myosin heavy chain (MHC) and a truncated heavy meromyosin-like alpha MHC (sHMM) and shown that immunoreactive MHC proteins of the expected sizes were detected in lysates of transfected cells. Surprisingly, the full-length MHC formed large spindle-shaped structures throughout the cytoplasm of transfected cells as determined by immunofluorescence microscopy. The structures were not found in cells expressing the sHMM construct, indicating that their formation required an MHC rod. The spindle-shaped structures ranged in length from approximately 1 micron to over 20 microns in length and were birefringent suggesting that they are ordered arrays of thick filaments. This was confirmed by electron microscopic analysis of the transfected cells which revealed arrays of filamentous structures approximately 12 nm in diameter at their widest point. In addition, the vast majority of transfected MHC did not associate with the endogenous nonmuscle myosin light chains, demonstrating that myosin thick filaments can form in the absence of stoichiometric amounts of myosin light chains.

Differential gene expression in wild-type and X-ray-sensitive mutants of Chinese hamster ovary cell lines

Complementary DNA cloning, differential screening and Northern hybridization techniques were used to study differential gene expression in the wild-type Chinese hamster ovary (CHO) K1 cell line and its two X-ray sensitive mutants, xrs-5 and xrs-6. 11 species of mRNAs were found underexpressed in the two independently isolated mutants. The steady-state levels of those mRNAs are 3–26-fold less in the two mutants, depending on the particular species. 6 of the underexpressed mRNAs have been identified by comparing the sequences of the cloned cDNAs to the known sequences in GenBank. 4 of them code for the structural proteins of ferritin heavy chain, nonmuscle myosin light chain 3nm, ribosomal protein S17 and L7, respectively. The other two have strong homology with mouse B2 or retroviral sequences. The remaining 5 mRNAs did not show significant homology with any of the known sequences and apparently represent newly isolated species. The effect of 137Cs γ-rays on the expression of the 11 mRNAs has been studied. Radiation inhibited the expression of the B2-like gene in the mutants but not in the wild-type CHO cells. The levels of the other 10 mRNAs were not affected by radiation. The underexpression of this group of genes in both xrs-5 and xrs-6 mutants seems to be related to their radiation-sensitive phenotype, although the specific gene responsible has not been identified. Two models are proposed to explain the mechanism of underexpression. It is suggested that a cellular factor or/and chromosome structural changes are involved.

The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila

Abstract Two independent approaches to understanding the molecular mechanism of cytokinesis have converged on the gene spaghetti-squash (sqh) . A genetic screen for mitotic mutants identified sqh 1 , a mutation that disrupts cytokinesis, which was then cloned by transposon tagging. Independently, the gene that encodes the regulatory light chain of the biochemically defined nonmuscle myosin (MRLC-C) was also cloned. We show here that sqh encodes MRLC-C and that in sqh 1 mutants, the level of stable light chain transcript is greatly reduced. Reversion by transposon excision or transformation with a wild-type copy of the sqh transcription unit rescues cytokinesis fallure and other defects in sqh 1 . Vertebrate homologs of MRLC-C are phosphorylatable and regulate myosin activity in vitro. These studies provide genetic proof that MRLC-C is required for cytokinesis, suggest a role for the protein in regulating contractile ring function, and establish a genetic system to evaluate its function.

Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II

Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (i.e., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase II (CaMPK-II), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:1 stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops II and III decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS20F and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-81 while RS20CK induced a blue-shift. The quenching rate of Trp-81 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures.

The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila

Two independent approaches to understanding the molecular mechanism of cytokinesis have converged on the gene spaghetti-squash (sqh). A genetic screen for mitotic mutants identified sqh 1 , a mutation that disrupts cytokinesis, which was then cloned by transposon tagging. Independently, the gene that encodes the regulatory light chain of the biochemically defined nonmuscle myosin (MRLC-C) was also cloned. We show here that sqh encodes MRLC-C and that in sqh 1 mutants, the level of stable light chain transcript is greatly reduced. Reversion by transposon excision or transformation with a wild-type copy of the sqh transcription unit rescues cytokinesis fallure and other defects in sqh 1 . Vertebrate homologs of MRLC-C are phosphorylatable and regulate myosin activity in vitro. These studies provide genetic proof that MRLC-C is required for cytokinesis, suggest a role for the protein in regulating contractile ring function, and establish a genetic system to evaluate its function.

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.

Characterization of human myosin light chains 1sa and 3nm: implications for isoform evolution and function.

We have isolated a cDNA clone for the human slow-twitch muscle isoform myosin light-chain 1slow-a (MLC1sa) from a skeletal muscle library and for the human nonmuscle isoform myosin light-chain 3nonmuscle (MLC3nm) from a fibroblast library. The nucleotide sequence of both isoforms was determined, and isoform-specific probes were constructed. In addition, MLC1sa was subsequently isolated from the fibroblast library. MLC1sa and MLC3nm were found to be very closely related to each other and distant from all other myosin light-chain isoforms so far described. We concluded that MLC1sa arose by duplication of MLC3nm rather than from any other isoform. A comparison was made between all human myosin light chains described to date and a model proposed for the evolution of this multigene family. A comparison between human and chicken myosin light-chain isoforms showed that human isoforms are more similar to their chicken counterparts than to human MLC1sa. The expression of MLC1sa and MLC3nm was studied in humans, rabbits, mice, and rats. MLC1sa was detected at the onset of both human and murine myogenesis in vitro. With development, MLC1sa may be replaced by the other slow-twitch muscle isoform, 1sb, in slow-twitch skeletal muscle, but the proportion of MLC1sa to 1sb expression varies between different species. MLC1sa was detected in nonmuscle cells in humans, mice, and rats. MLC3nm was the major nonmuscle alkaline myosin light chain in all species tested, but its pattern of expression in nonmuscle tissues was not identical to that of beta- or gamma-actin. We have shown that in the human, as in the chicken, one exon is spliced out of the MLC3nm transcript in smooth muscle to give an alternative product. We concluded that all alkali myosin light-chain isoforms may be functionally different.

Characterization of Human Myosin Light Chains 1sa and 3nm: Implications for Isoform Evolution and Function

We have isolated a cDNA clone for the human slow-twitch muscle isoform myosin light-chain 1slow-a (MLC1sa) from a skeletal muscle library and for the human nonmuscle isoform myosin light-chain 3nonmuscle (MLC3nm) from a fibroblast library. The nucleotide sequence of both isoforms was determined, and isoform-specific probes were constructed. In addition, MLC1sa was subsequently isolated from the fibroblast library. MLC1sa and MLC3nm were found to be very closely related to each other and distant from all other myosin light-chain isoforms so far described. We concluded that MLC1sa arose by duplication of MLC3nm rather than from any other isoform. A comparison was made between all human myosin light chains described to date and a model proposed for the evolution of this multigene family. A comparison between human and chicken myosin light-chain isoforms showed that human isoforms are more similar to their chicken counterparts than to human MLC1sa. The expression of MLC1sa and MLC3nm was studied in humans, rabbits, mice, and rats. MLC1sa was detected at the onset of both human and murine myogenesis in vitro. With development, MLC1sa may be replaced by the other slow-twitch muscle isoform, 1sb, in slow-twitch skeletal muscle, but the proportion of MLC1sa to 1sb expression varies between different species. MLC1sa was detected in nonmuscle cells in humans, mice, and rats. MLC3nm was the major nonmuscle alkaline myosin light chain in all species tested, but its pattern of expression in nonmuscle tissues was not identical to that of beta- or gamma-actin. We have shown that in the human, as in the chicken, one exon is spliced out of the MLC3nm transcript in smooth muscle to give an alternative product. We concluded that all alkali myosin light-chain isoforms may be functionally different.

Expression of nonmuscle myosin heavy and light chains in smooth muscle

We have compiled evidence that nonmuscle isoforms of both myosin heavy chain (NM MHC) and myosin regulatory light chain (NM LC20) are present in fully differentiated smooth muscles (SM). In swine carotid media sodium dodecyl sulfate-gel electrophoresis separated three MHC bands. The upper two bands were identified by immunoblotting as SM-specific isoforms. The lowest MHC band amounted to 14 +/- 2% of the total MHC and was electrophoretically and antigenically similar to platelet MHC. Two-dimensional gel electrophoresis of swine carotid media extracts resolved multiple LC20 species, including phosphorylated and "satellite" forms. Mass spectrometric analysis of tryptic peptides from blots of these gels demonstrated two LC20 isoforms. The measured peptide masses correspond with two published cDNA sequences proposed to represent SM and NM LC20 isoforms. These sequences readily explain the electrophoretic behavior of the isoforms. The minor isoform's abundance (16 +/- 3%, corresponding to NM MHC), antigenic properties, and pattern of expression in tissue culture all confirm that this is a NM LC20 isoform. The localization and functional significance of NM myosin in smooth muscle is unknown.

The Alkali Light Chains of Human Smooth and Nonmuscle Myosins Are Encoded by a Single Gene: Tissue-specific Expression by Alternative Splicing Pathways

Human smooth muscle and nonmuscle cells express closely related myosin alkali light chains which are different from the isoforms present in striated muscle tissues. To date no information on the amino acid sequence of these mammalian nonstriated muscle isoforms has been available. We have isolated full-length cDNA clones encoding the nonmuscle (lym4) and smooth muscle (GT6) myosin light chains (MLCs) from cultured human lymphoblasts and heart aorta smooth muscle cells, respectively. Here we present the complete nucleotide sequences for both cDNA clones, together with the deduced amino acid sequences for the peptides. Both cDNAs contain the same open reading frame for 151 amino acids with 5 amino acid differences located in the C terminus. These differences are encoded by a block of 44 nucleotides which is present only in the smooth muscle (SM) mRNA. To identify the human gene coding for the two MLC isoforms, we have isolated and sequenced the nonmuscle (NM)/SM MLC gene, together with several intronless pseudogenes. A single functional gene was found containing 7 exons which are utilized for the coding information of the SM MLC mRNA. In contrast, the NM MLC mRNA does not contain sequences encoded by exon 6 which corresponds to the 44 nucleotides expressed in SM mRNA. This genomic configuration suggests that both the smooth muscle and nonmuscle MLCs in man are generated from the identical primary transcript by alternative splicing pathways taking place in a tissue-dependent manner.

Synthetic peptides derived from the nonmuscle myosin light chains are highly specific substrates for protein kinase C.

The phosphorylation of synthetic peptides derived from the NH2-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1-13) (MLC(1-13)). Kinetic analysis of MLC(1-13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1-13) had a V/K of 2.4 min-1.mg-1, whereas the V/K of MLC(3-13) was 3.0 min-1.mg-1. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent Vmax.Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent Km increased 7-fold and the Vmax decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain peptides can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry.

Nonmuscle and smooth muscle myosin light chain mRNAs are generated from a single gene by the tissue-specific alternative RNA splicing.

We have isolated two cDNA clones for myosin alkali light chain (MLC) mRNA from two respective cDNA libraries of chick gizzard and fibroblast cells by cross-hybridization to the previously isolated cDNA of skeletal muscle MLC. Sequence analysis of the two cloned cDNAs revealed that both of them are homologous to but distinct from the cDNA sequence used as the probe so that they may be classified into members of the MLC family, that they are identical with each other in the 3' and 5' untranslated sequence as well as in the coding sequence with a notable exception of a 39-nucleotide insertion in the fibroblast cDNA, 26 nucleotides of which are used for encoding the C-terminal amino acid sequence, and, therefore, that they encode the identical 142-amino acid sequence with different C-terminals of nine amino acids, each specific for fibroblast and gizzard smooth muscle MLC. The position of the inserted block corresponds exactly to one of the exon-intron junctions in the other MLC genes whose structures have so far been elucidated. DNA blot analysis suggested that the two MLC mRNAs of gizzard (smooth muscle) and fibroblast cells (nonmuscle) are generated from a single gene, probably through alternative RNA splicing mechanisms. RNA blot analysis and S1 nuclease mapping analysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or very scantily if at all, in the gizzard, whereas the reverse is true for the gizzard smooth muscle MLC mRNA.

Surgery of the Tongue

The post-injury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In <i>Caenorhabditis elegans</i>, the initiation of axon regeneration is regulated by the non-muscle myosin light chain (MLC-4) phosphorylation signaling pathway. In this study, we have identified <i>svh-16</i>/<i>cdk-14</i>, 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 <i>C. elegans</i> 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 non-canonical Wnt signaling pathway that promotes axon regeneration. <b>Significance Statement:</b> Non-canonical Wnt signaling is mediated by Fz, Dsh, Rho-type GTPase, and non-muscle MLC phosphorylation. This study identified <i>svh-16</i>/<i>cdk-14</i>, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in <i>Caenorhabditis elegans</i> 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 <i>mig-1</i>, <i>cdk-14</i>, and <i>ephx-1</i> mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a GEF for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the non-canonical Wnt signaling pathway controls axon regeneration via the CDK-14–EPHX-1–CDC-42–MLC phosphorylation axis.