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.

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.

Differential regulation of cardiac angiotensin converting enzyme binding sites and AT1 receptor density in the failing human heart.

BACKGROUND: The regulation and interaction of ACE and the angiotensin II (Ang II) type I (AT1) receptor in the failing human heart are not understood. METHODS AND RESULTS: Radioligand binding with 3H-ramiprilat was used to measure ACE protein in membrane preparations of hearts obtained from 36 subjects with idiopathic dilated cardiomyopathy (IDC), 8 subjects with primary pulmonary hypertension (PPH), and 32 organ donors with normal cardiac function (NF hearts). 125I-Ang II formation was measured in a subset of hearts. Saralasin (125I-(Sar1,Ile8)-Ang II) was used to measure total Ang II receptor density. AT1 and AT2 receptor binding were determined with the AT1 receptor antagonist losartan. Maximal ACE binding (Bmax) was 578+/-47 fmol/mg in IDC left ventricle (LV), 713+/-97 fmol/mg in PPH LV, and 325+/-27 fmol/mg in NF LV (P<0.001, IDC or PPH versus NF). In IDC, PPH, and NF right ventricles (RV), ACE Bmax was 737+/-78, 638+/-137, and 422+/-49 fmol/mg, respectively (P=0.02, IDC versus NF; P=0.08, PPH versus NF). 125I-Ang II formation correlated with ACE binding sites (r=0.60, P=0.00005). There was selective downregulation of the AT1 receptor subtype in failing PPH ventricles: 6.41+/-1.23 fmol/mg in PPH LV, 2.37+/-0.50 fmol/mg in PPH RV, 5.38+/-0.53 fmol/mg in NF LV, and 7.30+/-1.10 fmol/mg in NF RV (P=0.01, PPH RV versus PPH LV; P=0.0006, PPH RV versus NF RV). CONCLUSIONS: ACE binding sites are increased in both failing IDC and nonfailing PPH ventricles. In PPH hearts, the AT1 receptor is downregulated only in the failing RV.

Selective downregulation of the angiotensin II AT1-receptor subtype in failing human ventricular myocardium.

BACKGROUND: The regulation of angiotensin II receptors and the two major subtypes (AT1 and AT2) in chronically failing human ventricular myocardium has not been previously examined. METHODS AND RESULTS: Angiotensin II receptors were measured by saturation binding of 125I-[Sar1,Ile8]angiotensin II in crude membranes from nonfailing (n = 19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n = 31) or ischemic cardiomyopathy (ISC; n = 21) and membranes from a limited number of right ventricles in each category. The AT1 and AT2 fractions were determined by use of an AT1-selective antagonist, losartan. beta-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the beta 1 and beta 2 fractions determined by use of a beta 1-selective antagonist, CGP20712A, AT1 but not AT2 density was significantly decreased in the combined (IDC + ISC) failing left ventricles (nonfailing: AT1 4.66 +/- 0.48, AT2 2.73 +/- 0.39; failing: AT1 3.20 +/- 0.29, AT2 2.70 +/- 0.33 fmol/mg protein; mean +/- SE). The decrease in AT1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73 +/- 0.40, P < .01; ISC: 3.89 +/- 0.39 fmol/mg protein, P = NS versus nonfailing). beta 1 but not beta 2 density was decreased in the failing left ventricles. AT1 density was correlated with beta 1 density in all left ventricles (r = .43). AT1 density was also decreased in IDC right ventricles. In situ reverse transcription-polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT1 mRNA was present in both myocytes and nonmyocytes. CONCLUSIONS: AT1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of beta 1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.

Overexpression of myotonic dystrophy kinase in BC3H1 cells induces the skeletal muscle phenotype.

Myotonic muscular dystrophy is an autosomal dominant defect that produces muscle wasting, myotonia, and cardiac conduction abnormalities. The myotonic dystrophy locus codes for a putative serine-threonine protein kinase of unknown function. We report that overexpression of human myotonic dystrophy protein kinase induces the expression of skeletal muscle-specific genes in undifferentiated BC3H1 muscle cells. BC3H1 clones expressing myotonic dystrophy kinase appear equivalent to differentiated cells with respect to expression of myogenin, retinoblastoma tumor supressor gene, M creatine kinase, beta-tropomyosin, and vimentin. In addition, differential display analysis demonstrates that the pattern of gene expression exhibited by myotonic dystrophy kinase-expressing cells is essentially identical to that of differentiated BC3H1 muscle cells. These observations suggest that myotonic dystrophy kinase may function in the myogenic pathway.

Identification, tissue-specific expression, and subcellular localization of the 80- and 71-kDa forms of myotonic dystrophy kinase protein.

The protein product of the myotonic dystrophy (DM) gene is a putative serine-threonine protein kinase (DM kinase). Previous reports have characterized the DM gene product as various 50-62-kDa proteins. The predicted protein size from DM cDNA sequence is 69 kDa. We therefore expressed a full-length recombinant human DM kinase protein and compared its size and expression to heart, cardiac Purkinje fibers, and skeletal muscle from normal and DM subjects. Recombinantly expressed DM kinase and endogenous DM kinase in human heart, displayed two immunoreactive DM kinase proteins with apparent molecular sizes of 71 and 80 kDa, suggesting that these prior reports are incorrect. In cardiac Purkinje fibers the 71-kDa protein was the major form, and in skeletal muscle the 80-kDa protein was the major form. Immunostaining showed DM kinase localized to neuromuscular junctions in skeletal muscle and intercalated discs in heart and Purkinje fibers. DM subjects showed low abundance of DM kinase in heart and skeletal muscle, suggesting haplotype insufficiency as a potential mechanism for disease expression. These studies describe differential expression of two protein forms of DM kinase, which are localized to specialized cellular structures associated with impulse transmission.

Angiotensin-converting enzyme DD genotype in patients with ischaemic or idiopathic dilated cardiomyopathy.

Polymorphism in the angiotensin-converting enzyme (ACE) gene has been shown to correlate with circulating ACE concentrations, and also to be an independent risk factor for the development of myocardial infarction, particularly in men thought to be at low risk by standard criteria. We determined the genotypes of individuals with end-stage heart failure due to either ischaemic dilated cardiomyopathy (102) or idiopathic dilated cardiomyopathy (112) and compared these to organ donors with normally functioning hearts (79). Genotypes were determined by the polymerase chain reaction with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene to amplify template DNA isolated from patients. Compared with the DD frequency in the control population, the frequency of the ACE DD genotype was 48% higher in individuals with idiopathic dilated cardiomyopathy (p = 0.008) and 63% higher in subjects with ischaemic cardiomyopathy (p = 0.008), suggesting that an ACE gene variant may contribute to the pathogenesis of both types of cardiomyopathy.

Transcriptional-translational regulation of muscle-specific protein synthesis and its relationship to chondrogenic stimuli.

Demineralized bone (bone matrix) has a well-characterized ability to evoke the re-differentiation of cells derived from skeletal muscle into chondrocytes. Recent investigations in this laboratory have shown that muscle-specific (alpha) actin synthesis continues throughout redifferentiation. Conversely, expression of the cartilage phenotype is associated with repression of muscle-specific enzyme synthesis. The present experiments were undertaken to determine the mode of genomic regulation responsible for control of these muscle-specific syntheses. As part of these experiments, we investigated the ability of embryonic and adult RNA to direct translation in vitro. The results indicate that unfractionated (total) RNA is capable of directing the efficient synthesis of actin, but not myosin heavy or light chains. Decreased abundance of polyadenylated mRNA cannot account for lack of myosin synthesis. Polyadenylated mRNA, however, directed synthesis of actin and myosin with an efficiency greater than that of total RNA. This data suggested that embryonic total RNA was subject to translational control. Dot blot hybridization against cDNA probes for alpha-actin, myosin heavy chain, and fast light chains demonstrated that myogenic cells were subject to a pattern of mixed transcriptional and translational control. It is hypothesized that full expression of the muscle phenotype involves sequential release of transcriptional, and subsequently, the translational controls. We have also observed that cultures of skeletal muscle on bone matrix contain mRNA for muscle-specific proteins, even through the period normally characterized by chondrogenesis. In the absence of concurrent enzyme protein synthesis, it appears that one action of bone matrix is to continue genomic controls that in the source skeletal muscle maintain the genome in an embryonic (translationally repressed) state.