Myotonic dystrophy protein kinase domains mediate localization, oligomerization, novel catalytic activity, and autoinhibition

Human myotonic dystrophy protein kinase (DMPK) is a member of a novel class of multidomain protein kinases that regulate cell size and shape in a variety of organisms. However, little is currently known about the general properties of DMPK including domain function, substrate specificity, and potential mechanisms of regulation. Two forms of the kinase are expressed in muscle, DMPK-1 and DMPK-2. We demonstrate that the larger DMPK-1 form (the primary translation product) is proteolytically cleaved near the carboxy terminus to generate the smaller DMPK-2 form. We further demonstrate that the coiled-coil domain is required for DMPK oligomerization; coiled-coil mediated oligomerization also correlated with enhanced catalytic activity. DMPK was found to exhibit a novel catalytic activity similar to, but distinct from, related protein kinases such as protein kinase C and A, and the Rho kinases. We observed that recombinant DMPK-1 exhibits low activity, whereas the activity of carboxy-terminally truncated DMPK is increased approximately 3-fold. The inhibitory activity of the full-length kinase was mapped to what appears to be a pseudosubstrate autoinhibitory domain at the extreme carboxy terminus of DMPK. To date, endogenous activators of DMPK are unknown; however, we observed that DMPK purified from cells exposed to the G protein activator GTP-gamma-S exhibited an approximately 2-fold increase in activity. These results suggest a general model of DMPK regulation with two main regulatory branches: short-term activation of the kinase in response to G protein second messengers and long-term activation as a result of proteolysis.     

Homodimerization through coiled-coil regions enhances activity of the myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) is the protein product of the human DM-1 locus on chromosome 19q13.1 and has been implicated in the cardiac and behavioral dysfunctions of the disorder. DMPK contains four distinct regions: a leucine-rich repeat (L), a serine-threonine protein kinase catalytic domain (PK), an alpha-helical coiled-coil region (H), and a putative transmembrane-spanning tail (T). Multiple protein kinases that participate in cytoskeletal and cell cycle functions share homology with DMPK in the PK and H regions. Here we show that the LPKH and PKH subfragments of DMPK formed dimers of 140000 molecular weight, whereas the LPK subfragment remained a monomer of 62000 apparent molecular weight. The H domain thus appeared to be required for dimerization of DMPK subfragments. Caspase 1 cleaved LPKH between the PK and H regions. After cleavage, LPKH dimers became LPK-like monomers, consistent with the H domain mediating dimerization. The V(max) and k(cat)/K(m) of LPKH with a synthetic peptide kinase substrate were over 10-fold greater than either LPK or caspase-cleaved LPKH. The K(m) of dimeric LPKH was over three-fold greater than those of the monomeric proteins. Dimerization appeared to significantly affect the catalytic efficiency and substrate binding of DMPK. These interactions are likely to be functionally significant in other members of the myotonic dystrophy family of protein kinases with extensive coiled-coil domains.     

Phospholemman is a substrate for myotonic dystrophy protein kinase

The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I(Cl(PLM))) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I(Cl(PLM)) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I(Cl(PLM)). This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(-) PLM mutant in which all potential phosphorylation had been disabled by Ser --> Ala substitution. The biophysical characteristics of I(Cl(PLM)) were not changed by DMPK or by the phos(-) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I(Cl(PLM)) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.     

Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway

The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4^DCAF1. Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin''s auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin''s binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin''s auto-inhibitory tail does not obviously alter Merlin''s conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.     

The tumor suppressor Lats2 is pivotal in Aurora A and Aurora B signaling during mitosis

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.     

N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor.

The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH2-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH2 (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C11H23CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited the phosphorylation of poly E4Y by Src with an apparent Ki of 3 microm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 microm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 microm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E4Y. The non-acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing the affinity or characteristics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the peptide.     

Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases

Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer.     

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.     

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.     

The cyclin-dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism

cdk2.cyclin E and cdk5.p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2.GST-cyclin E and cdk5.GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K(i) value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K(i) value of 2 microm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2.GST-cyclin E the kinetic parameters were determined to be K(m, ATP) = 3.6 +/- 1.0 microm, K(m, peptide) = 4.6 +/- 1.4 microm, and the anticooperativity factor, alpha = 130 +/- 44. For cdk5.GST-p25, the K(m, ATP) = 3.2 +/- 0.7 microm, K(m, peptide) = 1.6 +/- 0.3 microm, and alpha = 7.2 +/- 1.8.     

Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.     

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.     

Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity.

We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.     

Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.     

A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.

A peptide derived from p34cdc2, cdc2(6-20)NH2 with the amino acid sequence of KVEKIGEGTYGVVYK-amide, was found to be a specific and efficient substrate for a pp60c-src-related protein tyrosine kinase from bovine spleen (STK). Glu-12 and Thr-14 were identified to be substrate specificity determinants in this peptide (Cheng, H.-C., Litwin, C. M. E., Hwang, D. M., and Wang, J. H. (1991) J. Biol. Chem. 266, 17919-17925). In this study, we demonstrated the presence of cdc2(6-20)NH2 peptide tyrosine kinase activity in the membrane fractions of bovine brain, spleen, thymus, lung, liver, and kidney. Hydroxylapatite column chromatography of thymus membrane extract revealed four protein tyrosine kinases, TK-I, TK-II, TK-III, and TK-IV, with different relative activities toward cdc2(6-20)NH2 and a general tyrosine kinase substrate, poly(Glu/Tyr). Only TK-I and TK-II showed significant activity toward cdc2(6-20)NH2, they were suggested as belonging to the src-family by virtue of their cross-reactivity with an antibody against a synthetic peptide corresponding to a conserved sequence of src-family kinases. Further immunological characterization using antibodies specific to individual src-related protein tyrosine kinases suggested that TK-I, TK-II, and STK are bovine homologs of p56lck, p55fyn, and p56lyn, respectively. Substrate specificity and kinetic characterization of src-family tyrosine kinases including human platelet pp60c-src, bovine p56lyn, p56lck, and p55fyn, as well as several non-src-related tyrosine kinases including epidermal growth factor receptor, p43v-abl, TK-III, and TK-IV showed that all the src-family tyrosine kinases but none of the other kinases displayed efficient cdc2(6-20)NH2 phosphorylation. In all cases, the high efficiency of cdc2(6-20)NH2 peptide phosphorylation could be markedly attenuated when Glu-12 and Thr-14 of the peptide were substituted, respectively, by valine and serine.     

Properties of several protein kinases that copurify with rat spinal cord neurofilaments

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.     

Abnormal actomyosin assembly in proliferating and differentiating myoblasts upon expression of a cytosolic DMPK isoform

DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells.