Identification of glycogen phosphorylase and creatine kinase as calpain substrates in skeletal muscle.
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.
Purintrapiban
J
J
Department of Animal Sciences, Oregon State University, Corvallis, OR 97331-6702, USA.
Wang
M
M
Forsberg
N E
NE
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
D011993:Recombinant Fusion Proteins; D006005:Phosphorylases; D003402:Creatine Kinase; D002154:Calpain