Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies.
There is considerable confusion in the literature about the size of the myotonic dystrophy protein kinase (DMPK) and its localization within tissues. We have used a new panel of monoclonal antibodies (mAbs) to begin to resolve these issues, which are important for understanding the possible role of DMPK in myotonic dystrophy. Antisera raised against the catalytic and coil domains of DMPK recognized a major 55 kDa protein and a minor 72-80 kDa doublet on western blots of human skeletal muscle. Ten mAbs, five against the catalytic domain and five against the coil region, recognized only the 72-80 kDa doublet. The 72 kDa protein was present in all tissues tested, whereas the 80 kDa component was variably expressed, mainly in skeletal and cardiac muscles. The 72 kDa protein was absent in a DMPK knockout mouse and was greatly increased in a transgenic mouse overexpressing human DMPK, confirming its identity as authentic DMPK. Two mAbs against the catalytic domain recognized only the more abundant 55 kDa protein, which was found only in skeletal muscle. Nine out of 10 mAbs located DMPK to intercalated discs in human heart, an affected tissue in myotonic dystrophy. However, co-localization of DMPK with acetylcholine receptors at neuromuscular junctions was not observed with any of the mAbs. Subcellular fractionation and sedimentation analysis suggest that a major proportion of the DMPK in skeletal muscle and brain is cytosolic.
Pham
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YC
MRIC Biochemistry Group, NE Wales Institute, Mold Road, Wrexham LL11 2AW, UK.
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Lam
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Morris
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008099:Liver / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D009223:Myotonic Dystrophy / Q000201:enzymology*; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D012584:Sciatic Nerve / Q000201:enzymology; D012967:Sodium Dodecyl Sulfate; D013116:Spinal Cord / Q000201:enzymology; D013154:Spleen / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; C585517:DMPK protein, human; C585516:DMPK protein, mouse; D012967:Sodium Dodecyl Sulfate; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases