Myotonic dystrophy and myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) was designated as a gene responsible for myotonic dystrophy (DM) on chromosome 19, because the gene product has extensive homology to protein kinase catalytic domains. DM is the most common disease with multisystem disorders among muscular dystrophies. The genetic basis of DM is now known to include mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'-untranslated region (UTR) of DMPK. Full-length DMPK was detected and various isoforms of DMPK have been reported in skeletal and cardiac muscles, central nervous tissues, etc. DMPK is localized predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in skeletal muscle. In cardiac muscle it is localized in intercalated dises and Purkinje fibers. Electron microscopically it is detected in the terminal cisternae of SR in skeletal muscle and the junctional and corbular SR in cardia muscle. In central nervous system, it is located in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rough endoplasmic reticulum. The functional role of DMPK is not fully understood, however, it may play an important role in Ca2+ homeostasis and signal transduction system. Diseased amount of DMPK may play an important role in the degeneration of skeletal muscle in adult type DM. However, other molecular pathogenetical mechanisms such as dysfunction of surrounding genes by structural change of the chromosome by long trinucleotide repeats, and the trans-gain of function of CUG-binding proteins might be responsible to induce multisystemic disorders of DM such as myotonia, endocrine dysfunction, etc.     

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: 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 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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

High-fat diet induced adiposity and insulin resistance in mice lacking the myotonic dystrophy protein kinase

Myotonic dystrophy 1 (MD1) is caused by a CTG expansion in the 3′-unstranslated region of the myotonic dystrophy protein kinase (DMPK) gene. MD1 patients frequently present insulin resistance and increased visceral adiposity. We examined whether DMPK deficiency is a genetic risk factor for high-fat diet-induced adiposity and insulin resistance using the DMPK knockout mouse model. We found that high-fat fed DMPK knockout mice had significantly increased body weights, hypertrophic adipocytes and whole-body insulin resistance compared with wild-type mice. This nutrient-genome interaction should be considered by physicians given the cardiometabolic risks and sedentary lifestyle associated with MD1 patients.