The PEST Domain of I Kappa B alpha Is Necessary and Sufficient for in Vitro Degradation by mu -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.