Myotonic dystrophy protein kinase is critical for nuclear envelope integrity

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.     

Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody: possible role of the protein in cytoskeleton rearrangements during differentiation

Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.     

Myotonic dystrophy: molecular windows on a complex etiology.

Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy, with an incidence of approximately 1 in 8500 adults. DM is caused by an expanded number of trinucleotide repeats in the 3'-untranslated region (UTR) of a cAMP-dependent protein kinase (DM protein kinase, DMPK). Although a large number of transgenic animals have been generated with different gene constructions and knock-outs, none of them faithfully recapitulates the multisystemic and often severe phenotype seen in human patients. The transgenic data suggest that myotonic dystrophy is not caused simply by a biochemical deficiency or abnormality in the DM kinase gene product. Emerging studies suggest that two novel pathogenetic mechanisms may play a role in the disease: the expanded repeats appear to cause haploinsufficiency of a neighboring homeobox gene and also abnormal DMPK RNA appears to have a detrimental effect on RNA homeostasis. The complex, multisystemic phenotype may reflect an underlying multifaceted molecular pathophysiology: the facial dysmorphology may be due to pattern defects caused by haploinsufficiency of the homeobox gene, while the muscle disease and endocrine abnormalities may be due to both altered RNA metabolism and deficiency of the cAMP DMPK protein.     

Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions.

Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.     

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.     

Myotonic dystrophy protein kinase phosphorylates phospholamban and regulates calcium uptake in cardiomyocyte sarcoplasmic reticulum

Myotonic dystrophy (DM) is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Cardiovascular disease is one of the most prevalent causes of death in DM patients. Electrophysiological studies in cardiac muscles from DM patients and from DMPK(-/-) mice suggested that DMPK is critical to the modulation of cardiac contractility and to the maintenance of proper cardiac conduction activity. However, there are no data regarding the molecular signaling pathways involved in DM heart failure. Here we show that DMPK expression in cardiac myocytes is highly enriched in the sarcoplasmic reticulum (SR) where it colocalizes with the ryanodine receptor and phospholamban (PLN), a muscle-specific SR Ca(2+)-ATPase (SERCA2a) inhibitor. Coimmunoprecipitation studies showed that DMPK and PLN can physically associate. Furthermore, purified wild-type DMPK, but not a kinase-deficient mutant (K110A DMPK), phosphorylates PLN in vitro. Subsequent studies using the DMPK(-/-) mice demonstrated that PLN is hypo-phosphorylated in SR vesicles from DMPK(-/-) mice compared with wild-type mice both in vitro and in vivo. Finally, we show that Ca(2+) uptake in SR is impaired in ventricular homogenates from DMPK(-/-) mice. Together, our data suggest the existence of a novel regulatory DMPK pathway for cardiac contractility and provide a molecular mechanism for DM heart pathology.     

Myotonic dystrophy protein kinase (DMPK) and its role in the pathogenesis of myotonic dystrophy 1

Myotonic dystrophy 1 (DM1) is an autosomal, dominant inherited, neuromuscular disorder. The DM1 mutation consists in the expansion of an unstable CTG-repeat in the 3'-untranslated region of a gene encoding DMPK (myotonic dystrophy protein kinase). Clinical expression of DM1 is variable, presenting a progressive muscular dystrophy that affects distal muscles more than proximal and is associated with the inability to relax muscles appropriately (myotonia), cataracts, cardiac arrhythmia, testicular atrophy and insulin resistance. DMPK is a Ser/Thr protein kinase homologous to the p21-activated kinases MRCK and ROCK/rho-kinase/ROK. The most abundant isoform of DMPK is an 80 kDa protein mainly expressed in smooth, skeletal and cardiac muscles. Decreased DMPK protein levels may contribute to the pathology of DM1, as revealed by gene target studies. Here we review current understanding of the structural, functional and pathophysiological characteristics of DMPK.     

Expanding complexity in myotonic dystrophy

Myotonic dystrophy (DM) is a highly variable multisystemic disease belonging to the rather special class of trinucleotide expansion disorders. DM results from dynamic expansion of a perfect (CTG)_n repeat situated in a gene-dense region on chromosome 19q. Based on findings in patient materials or cellular and animal models, many mechanisms for the causes and consequences of repeat expansion have been proposed; however, none of them has enjoyed prolonged support. There is now circumstantial evidence that long (CTG)_n repeats may affect the expression of any of at least three genes, myotonic dystrophy protein kinase (DMPK), DMR-N9 (gene 59), and a DM-associated homeodomain protein (DMAHP). Furthermore, the new findings suggest that DM is not a simple gene-dosage or gain-or-loss-of-function disorder but that entirely new pathological pathways at the DNA, RNA, or protein level may play a role in its manifestation. BioEssays 20: 901–912, 1998. © 1998 John Wiley & Sons, Inc.     

Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.     

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.     

De novo myotonic dystrophy mutation in a Nigerian kindred.

An expansion of an unstable (CTG)n trinucleotide repeat in the 3' UTR of a gene encoding a putative serine/threonine protein kinase (DMPK) on human chromosome 19q13.3 has been shown to be specific for the myotonic dystrophy (DM) disease phenotype. In addition, a single haplotype composed of nine alleles within and flanking DMPK over a physical distance of 30 kb has been shown to be in complete linkage disequilibrium with DM. This has led to two hypotheses: (1) predisposition for (CTG)n instability results from a founder effect that occurred only once or a few times in human evolution; and (2) elements within the disease haplotype may predispose the (CTG)n repeat to instability. A detailed haplotype analysis of the DM region was conducted on a Nigerian (Yoruba) DM family, the only indigenous sub-Saharan DM case reported to date. Each affected member of this family had an expanded (CTG)n repeat in one of his or her DMPK alleles. However, unlike all other DM populations studied thus far, disassociation of the (CTG)n repeat expansion from other alleles of the putative predisposing haplotype was found. We conclude that the expanded (CTG)n repeat in this family is the result of an independent mutational event. Consequently, the origin of DM is unlikely to be a single mutational event, and the hypothesis that a single ancestral haplotype predisposes to repeat expansion is not compelling.     

Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface

The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.     

Assessment of a creatine kinase isoform M defect as a cause of myotonic dystrophy and the characterization of two novel CKMM polymorphisms

Summary Recent studies have shown the gene encoding creatine kinase isoform M (CKMM) to be very closely linked to the myotonic dystrophy (DM) locus on the long arm of chromosome 19. Given this close linkage to DM and the postulated role of CKMM in skeletal muscle contraction, the possibility of a defect in CKMM causing DM was investigated. CKMM cDNA was isolated from the skeletal muscle of an individual with DM. Sequencing of the CKMM cDNA from the DM chromosome 19 revealed two novel polymorphisms but no translationally significant mutation. This work rules out a defect in the coding segment of CKMM as a cause of DM in this family and, in light of genetic homogeneity shown to date for DM, probably in all cases of DM.     

Role of myotonic dystrophy protein kinase (DMPK) in glucose homeostasis and muscle insulin action

Myotonic dystrophy 1 (DM1) is caused by a CTG expansion in the 3'-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk-/-) mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk-/- mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk-/- mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.     

CTG repeats distribution and Alu insertion polymorphism at myotonic dystrophy (DM) gene in Amhara and Oromo populations of Ethiopia

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disease, highly variable and multisystemic, which is caused by the expansion of a CTG repeat located in the 3′ untranslated region of the DMPK gene. Normal alleles show a copy number of 5–37 repeats on normal chromosomes, amplified to 50–3000 copies on DM chromosomes. The trinucleotide repeat shows a trimodal allele distribution in the majority of the examined population. The first class includes alleles carrying (CTG)₅, the second class, alleles in the range 7–18 repeats, and the third class, alleles (CTG)_{≥ 19}. The frequency of this third class is directly related to the prevalence of DM in different populations, suggesting that normal large-sized alleles predispose toward DM. We studied CTG repeat allele distribution and Alu insertion and/or deletion polymorphism at the myotonic dystrophy locus in two major Ethiopian populations, the Amhara and Oromo. CTG allele distribution and haplotype analysis on a total of 224 normal chromosomes showed significant differences between the two ethnic groups. These differences have a bearing on the out-of-Africa hypothesis for the origin of the DM mutation. In addition, (CTG)_{≥ 19} alleles were exclusively detected in the Amhara population, confirming the predisposing role of these alleles compared with the DM expansion-mutation. Electronic Publication