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 act ivated by calcium. They display a relatively strict specificity for their s ubstrates which they usually cleave at only a limited number of sites. Moti fs 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 vit ro. They thus provide favourable protein contexts for studying the structur al requirements for recognition and degradation by these proteases. Using i n vitro degradation assays and site-directed mutagenesis, we report here th at susceptibility to calpains is primarily determined by conformational det erminants 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 d ivided into at least two classes of sensitivity, the most sensitive ones be ing 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 recogni zed independently and their recognition is dependent on the local conformat ion of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.