Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody: possible role of the protein in cytoskeleton rearrangements during differentiation.
Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.
Beffy
Pascale
P
Institute of Clinical Physiology, C.N.R., Research Area, 56100 Pisa, Italy.
Barsanti
Cristina
C
Del Carratore
Renata
R
Simi
Silvana
S
Benedetti
Pier Alberto
PA
Benzi
Luca
L
Prelle
Alessandro
A
Ciscato
Patrizia
P
Simili
Marcella
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D020134:Catalytic Domain / Q000276:immunology; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000032:analysis; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology*; D009206:Myocardium / Q000378:metabolism; D065636:Myotonin-Protein Kinase; D009469:Neuromuscular Junction / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; C585517:DMPK protein, human; C585516:DMPK protein, mouse; D007527:Isoenzymes; D011994:Recombinant Proteins; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases