RRC ID 28600
Author Ono Y, Ojima K, Torii F, Takaya E, Doi N, Nakagawa K, Hata S, Abe K, Sorimachi H.
Title Skeletal muscle-specific calpain is an intracellular Na+-dependent protease.
Journal J Biol Chem
Abstract Because intracellular [Na(+)] is kept low by Na(+)/K(+)-ATPase, Na(+) dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca(2+)-activated intracellular "modulator proteases" that is responsible for a limb-girdle muscular dystrophy ("calpainopathy"), underwent Na(+)-dependent, but not Cs(+)-dependent, autolysis in the absence of Ca(2+). Furthermore, Na(+) and Ca(2+) complementarily activated autolysis of p94 at physiological concentrations. By blocking Na(+)/K(+)-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na(+)/Ca(2+)-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca(2+)-binding sequence in the protease acted as a Na(+) sensor. Proteomic analyses using Cs(+)/Mg(2+) and p94CS-KI mice as negative controls revealed that Na(+) and Ca(2+) direct p94 to proteolyze different substrates. We propose three roles for Na(+) dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca(2+)], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na(+)-dependent enzyme.
Volume 285(30)
Pages 22986-98
Published 2010-7-23
DOI 10.1074/jbc.M110.126946
PII S0021-9258(20)60341-6
PMID 20460380
PMC PMC2906292
MeSH Amino Acid Sequence Animals Autolysis Binding Sites Calcium / metabolism Calcium / pharmacology Calpain / chemistry Calpain / genetics Calpain / metabolism* Enzyme Activation / drug effects Humans Intracellular Space / enzymology* Mice Molecular Sequence Data Muscle, Skeletal / enzymology* Mutation Organ Specificity Protein Structure, Tertiary Rats Sodium / metabolism* Sodium / pharmacology Substrate Specificity
IF 4.238
Times Cited 33
DNA material pcDNA3.1-N-FlagC-humanCAPN3(p94) (RDB08250) pBluescript-human-CAPN3(p94) (RDB08249)