The PEST domain of IκBα is necessary and sufficient for in vitro degradation by μ-calpain

Polypeptide sequences enriched in proline (P), glutamate (E), serine (S), and threonine (T), dubbed PEST domains, are proposed to expedite the degradation of proteins. The proteolysis of one PEST-containing protein, I kappa B alpha, is prerequisite to the activation of the transcription factor NF-kappa B, Two mechanisms of I kappa B alpha degradation in vivo have been described, one well characterized through the ubiquitin-proteasome pathway, and another less characterized through calpain. In this report, a mutational analysis was done to identify any regions of I kappa B alpha that facilitate its recognition and proteolysis by calpain in, vitro. These studies revealed that the PEST sequence of I kappa B alpha is critical for its calpain-dependent degradation. Furthermore, the I kappa B alpha-PEST domain binds to the calmodulin-like domain of the large subunit of mu-calpain (mu CaMLD). Transfer of the I kappa B alpha-PEST domain to a protein incapable of either binding to or being degraded by mu-calpain allowed for the interaction of the chimeric protein with mu CaMLD and resulted in its susceptibility to calpain proteolysis. Moreover, the mu CaMLD of calpain acts as a competitive inhibitor of calpain-dependent I kappa B alpha degradation. Our data demonstrate that the I kappa B alpha-PEST sequence acts as a modular domain to promote the physical association with and subsequent degradation by mu-calpain and suggest a functional role for PEST sequences in other proteins as potential calpain-targeting units.