Alternative Splicing Controls Myotonic Dystrophy Protein Kinase Structure, Enzymatic Activity, and Subcellular Localization

Abstract

Transcripts of the myotonic dystrophy protein kinase (DMPK) gene, a member of the Rho kinase family, are subject to cell-type specific alternative splicing. An imbalance in the splice isoform profile of DMPK may play a role in the pathogenesis of DM1, a severe multisystemic disorder. Here, we report how structural subdomains determine biochemical properties and subcellular distribution of DMPK isoforms. A newly developed kinase assay revealed that DMPK is a Lys/Arg-directed kinase. Individual DMPK isoforms displayed comparable transphosphorylation activity and sequence preference for peptide substrates. However, DMPK autophosphorylation and phosphorylation of MYPT1 (as putative in vivo target of DMPK), were dependent on presence of an alternatively spliced VSGGG motif and the nature of the C terminus. In-gel effects of the VSGGG motif on the migration behavior of full-length kinase provide evidence for a model in which this motif mediates 3-D-conformational changes in DMPK isoforms. Finally, different C termini endow DMPK with the ability to bind to either endoplasmic reticulum or mitochondria or to adopt a cytosolic location. Our results suggest that DMPK isoforms have cell-type and location dependent substrate specificities with a role in organellar and cytoarchitectural dynamics.Myotonic dystrophy (DM1) is the most common form of muscular dystrophy in adults (23), caused by amplification of an unstable (CTG) n repeat in the 3Ј untranslated region (3Ј-UTR) of the DM1 protein kinase (DMPK) gene (10,20,29). The severity of the disease is correlated to the length of this repeat expansion, whereas there is an inverse correlation to the age of onset. The favored explanation for the DM1 phenotype is a gain-of-function at the RNA level (reviewed in references 41 and 53), whereby long (CUG) n repeat tracts in DMPK transcripts cause a global perturbation of RNA processing events in the nucleus. Reports of cis-effects on the accumulation of (CUG) n repeat containing DMPK transcripts in the nucleus and trans-effects on alternative splicing of transcripts encoding other muscle or brain proteins support this hypothesis (15,16,30,40,43,45).It is widely accepted, nonetheless, that the highly variable and complex DM1 phenotype is not caused solely by detrimental effects of (CUG) n expansion at the RNA level, but that also direct effects on DMPK gene products and local gene effects are involved in specific disease features. Various studies have shown that expansion of the (CTG) n repeat results in reduced appearance of DMPK in the cytoplasm (53). Studies in knockout mouse and myocyte cell models indicated that lack of DMPK protein may be associated with typical DM1 symptoms like myopathy and heart conduction defects, perhaps via effects on Ca 2ϩ or Na ϩ ion homeostasis (5,6,25,35,42).DMPK is a member of the AGC group of serine/threonine kinases (31) and is most homologous to the p21-activated kinases MRCK (28) and ROCK/rho-kinase/ROK (4). Other mammalian homologues are NDR1 (32), warts/lats (26, 55), and citron kinase (17). DM...

Myotonic Dystrophy Protein Kinase Phosphorylates Phospholamban and Regulates Calcium Uptake in Cardiomyocyte Sarcoplasmic Reticulum

“…The screening of a library of synthetic peptides has shown that the optimal DMPK substrate sequences should consist of three to four arginines (or lysines) at distinct positions Nterminal to the phosphoacceptor (8). Remarkably, the PLN sequence surrounding Ser-16 is similar to that of the DMPKpreferred Ser substrate (R/K)XXXRRf(S)Xf (where X is any amino acid, preferably not P or E; f is a hydrophobic residue; and boldface type indicate the phosphoacceptor).…”

“…2M) and colocalizes with phospholamban in these cells (Fig. 2, A-L), and the phospholamban sequence contains several putative DMPK phosphorylation sites (7,8), we hypothesized that PLN could be a substrate for DMPK. We generated a hDMPK mutated at the ATP-binding site (K110A), which is a kinase-deficient mutant as observed by using myelin basic protein as a substrate (data not shown).…”

“…This kinase exists both as a membrane-associated and as a soluble form in human left ventricular samples (7). The different C termini of DMPK that arise from alternative splicing determine its localization to the endoplasmic reticulum, mitochondria, or cytosol in transfected COS-1 cells (8). Among the substrates for DMPK proposed by in vitro studies are phospholemman, the dihydropyridine receptor, and the myosin phosphatase targeting subunit (9 -11).…”

Myotonic Dystrophy Protein Kinase: Structure, Function and Its Possible Role in the Pathogenesis of Myotonic Dystrophy Type 1

“…The presence of both tail 1 and tail 2 confers to the protein the capacity to anchor into membranes of endoplasmic reticulum and mitochondria. In contrast, isoforms with tail 3 as C terminus (isoforms E and F) adopt a cytosolic localization (van Herpen et al, 2005;Wansink et al, 2003). Interestingly, a tissue-specific expression pattern of DMPK isoforms have been showed in brain and smooth, cardiac and skeletal muscle.…”

“…This transcript encodes a ~ 69-kDa protein with a unique C-terminal tail (tail 4). Because of the absence of the (CUG) n repeat, DMPK G transcripts can freely exit the nucleus, which might result in efficient DMPK G expression in DM1 cells (Tiscornia & Mahadevan, 2000;Wansink et al, 2003).…”