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.     

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.     

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.     

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.     

MKBP, a Novel Member of the Small Heat Shock Protein Family, Binds and Activates the Myotonic Dystrophy Protein Kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as αB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by αB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.     

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.     

Coexpression of the CUG-binding protein reduces DM protein kinase expression in COS cells.

Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. Patients have a large CTG repeat expansion in the 3' untranslated region of the DMPK gene, which encodes DM protein kinase. RNA trans-dominant models, which hypothesize that the expanded CUG trinucleotide repeat on DMPK mRNA sequesters a factor or disrupts the RNA metabolism of the DMPK mRNA itself and other mRNAs in a trans dominant manner, have been proposed. A candidate for the sequestered factor, termed CUG-binding protein (CUG-BP), exists in several alternatively spliced isoforms. We found a human isoform with a twelve base insertion (deduced amino acids Leu-Tyr-Leu-Gln) and an isoform with a three base insertion (deduced amino acid Ala) insertion. In order to elucidate the effects of CUG-BP on DMPK expression, we introduced CUG-BP and DMPK cDNA transiently into COS-7 cells. Cotransfection of CUG-BP did not significantly affect the expression of either wild type or mutant DMPK at the mRNA level. On the other hand, cotransfection of CUG-BP significantly affected the expression of both the wild type and mutant DMPKs at the protein level. This reduction was remarkable when the mutant DMPK construct was used.     

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.     

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.     

Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive

RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence.     

Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro.

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.