title: Identification of glycogen phosphorylase and creatine kinase as calpain substrates in skeletal muscle.
abstract: The goal of this study was to identify calpain substrates in muscle cells. Our hypothesis was that the yeast two-hybrid method could be used to identify novel calpain substrates. To accomplish this, native mu- and m-calpains, as well as a variety of calpain DNA fragments, were expressed in yeast cells and used to screen for binding proteins in a human skeletal muscle cDNA library. Calpain constructs that were used in the screening process included native mu- and m-calpains, a dominant negative (DN) m-calpain (i.e. active site modified), N-terminal truncated DN m-calpain (i.e. autolyzed DN-m-calpain) and, finally, an N- and C-terminal truncated m-calpain (i.e. autolyzed DN-m-calpain lacking a calcium-binding domain). Yeast cells were transformed using yeast two-hybrid expression vectors containing the different calpain constructs as "baits". Beta-galactosidase activity was assayed as an index of interaction between calpain and its potential target proteins. From this analysis, four clones (Ca2+-ATPase, novel nebulin-related protein (N-RAP), creatine kinase and glycogen phosphorylase) were recovered. Two of these, creatine kinase and glycogen phosphorylase, were selected for further study. In in-vitro assays, calpain was able to partially digest both proteins, suggesting that both creatine kinase and glycogen phosphorylase are natural calpain substrates.
authors.affiliation: Department of Animal Sciences, Oregon State University, Corvallis, OR 97331-6702, USA.
authors.forename: J
authors.initials: J
authors.lastname: Purintrapiban
authors.forename: M
authors.initials: M
authors.lastname: Wang
authors.forename: N E
authors.initials: NE
authors.lastname: Forsberg
mesh_terms: D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D003402:Creatine Kinase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D006005:Phosphorylases / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D006005:Phosphorylases; D003402:Creatine Kinase; D002154:Calpain