The sensitivity of c-Jun and c-Fos proteins to calpains depends on conformational determinants of the monomers and not on formation of dimers.
Milli- and micro-calpains are ubiquitous cytoplasmic cysteine proteases activated by calcium. They display a relatively strict specificity for their substrates which they usually cleave at only a limited number of sites. Motifs responsible for recognition by calpains have not been characterized yet, and recently a role for PEST motifs in this process has been ruled out. c-Fos and c-Jun transcription factors are highly sensitive to calpains in vitro. They thus provide favourable protein contexts for studying the structural requirements for recognition and degradation by these proteases. Using in vitro degradation assays and site-directed mutagenesis, we report here that susceptibility to calpains is primarily determined by conformational determinants of the monomers and not by the quaternary structure of c-Fos and c-Jun proteins. The multiple cleavage sites borne by both proteins can be divided into at least two classes of sensitivity, the most sensitive ones being easily visualized in the presence of rate-limiting amounts of calpains. One site located at position 90-91 in c-Fos protein is extremely sensitive. However, efficient proteolysis did not have any strict dependence on the nature of the amino acids on either side of the scissile bond in the region extending from P2 to P'2. The structural integrity of the monomers is not crucial for recognition by calpains. Rather, sensitive sites can be recognized independently and their recognition is dependent on the local conformation of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.
Pariat
M
M
Institut de Génétique Moléculaire de Montpellier, CNRS, UMR 5535, Route de Mende, 34293 Montpellier Cedex 05, France.
Salvat
C
C
Bébien
M
M
Brockly
F
F
Altieri
E
E
Carillo
S
S
Jariel-Encontre
I
I
Piechaczyk
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D019281:Dimerization; D006801:Humans; D019169:Jurkat Cells; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D002154:Calpain