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

Abstract

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, IκBα, is prerequisite to the activation of the transcription factor NF-κB. Two mechanisms of IκBα 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 IκBα that facilitate its recognition and proteolysis by calpain in vitro. These studies revealed that the PEST sequence of IκBα is critical for its calpain-dependent degradation. Furthermore, the IκBα-PEST domain binds to the calmodulin-like domain of the large subunit of μ-calpain (μCaMLD). Transfer of the IκBα-PEST domain to a protein incapable of either binding to or being degraded by μ-calpain allowed for the interaction of the chimeric protein with μCaMLD and resulted in its susceptibility to calpain proteolysis. Moreover, the μCaMLD of calpain acts as a competitive inhibitor of calpain-dependent IκBα degradation. Our data demonstrate that the IκBα-PEST sequence acts as a modular domain to promote the physical association with and subsequent degradation by μ-calpain and suggest a functional role for PEST sequences in other proteins as potential calpain-targeting units.