Mass spectrometry analysis of complexes formed by myotonic dystrophy protein kinase (DMPK).
Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats at the 3'-UTR of the serine/threonine protein kinase DMPK. Expanded CTG repeats are toxic since they are transcribed into an RNA molecule which is then sequestered within the nucleus in the form of foci. RNA cytotoxicity is linked to the aberrant splicing of several developmentally regulated genes. DMPK transcripts undergo alternative splicing giving rise to many isoforms but do not seem to be involved in the splicing dysregulation of DM1. However, decreased levels of DMPK in DM1 patients and DMPK involvement in muscle weakness and cardiac dysfunction in animal models have been reported. The variability in phenotypic expression of DMPK together with its differential subcellular targeting, suggests that different splicing isoforms may be involved in different signalling pathways, possibly through DMPK-interacting proteins. To gain better insight into the DMPK function, we used mass spectrometry to identify proteins co-segregating with DMPK in soluble complexes isolated from high-speed supernatant of rat muscles. We carried out experiments with native DMPK to preserve the physiological stoichiometry with potential partners. DMPK-containing complexes were isolated and immuno-detected by non-denaturing electrophoresis, gel filtration, ionic-exchange chromatography and immunoprecipitation. DMPK peptides were identified by high-resolution mass spectrometry together with several putative DMPK-binding proteins, including several heat shock proteins such as HSP20/HSPB6, HSP60/CPN60, HSP70 and HSP90. We also obtained evidence of a direct interaction of DMPK with alphaB-crystallin/HSPB5 and HSP25/HSPB1.
Forner
Francesca
F
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany.
Furlan
Sandra
S
Salvatori
Sergio
S
D020413:3' Untranslated Regions / Q000235:genetics; D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045586:Intranuclear Inclusion Bodies / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000473:pathology; D009223:Myotonic Dystrophy / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D065636:Myotonin-Protein Kinase; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014158:Transcription, Genetic / Q000235:genetics; D019680:Trinucleotide Repeat Expansion / Q000235:genetics
D020413:3' Untranslated Regions; D006360:Heat-Shock Proteins; D007527:Isoenzymes; D009097:Multienzyme Complexes; D010455:Peptides; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases