The isoenzyme-diagnostic regions of muscle-type creatine kinase, the M-260 and M-300 box, are not responsible for its binding to the myofibrillar M-band.
Muscle-type creatine kinase is known for its unique interaction with the myofibrillar M-band, but the molecular origin for this structural relationship is not well understood. A systematic sequence comparison between the highly homologous cytosolic isoforms, muscle-type and brain-type creatine kinase, yielded two isoenzyme-specific regions in the muscle-type creatine kinases, the M-260 box (residues 258-270) and the M-300 box (residues 300-315). These particular regions were conspicuous for the specific interaction of this CK isoenzyme, but not of brain-type creatine kinase, with the sarcomeric M-band. In situ diffusion assays with fluorescently labeled native, as well as mutated muscle-type creatine kinase variants, were used to study by laser confocal microscopy their association with the M-band of chemically skinned muscle fibers. Neither a set of charge mutants of the M-260 box and/or the M-300 box nor a hybrid construct of both isoforms with the entire C-terminal region derived from the brain-type isoform showed any significant alteration in the in situ M-band-binding properties when compared to the wild-type form of muscle-type creatine kinase. This indicates that in the intact protein of muscle type creatine kinase, these C-terminal isoenzyme-specific regions are not important for M-band interaction and that the actual M-band interaction domain(s) lay mostly within the N-terminal half of the molecule. The highly conserved motives (M-260 box and M-300 box) may serve an isoenzyme-specific purpose yet to be identified.
Stolz
M
M
Swiss Federal Institute of Technology (ETH), Institute of Cell Biology, Zürich.
Kraft
T
T
Wallimann
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003402:Creatine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D014644:Genetic Variation; D007525:Isoelectric Focusing; D007527:Isoenzymes; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D009210:Myofibrils / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D003402:Creatine Kinase