Journal of Biological Chemistry
Volume 274, Issue 43, 22 October 1999, Pages 30874-30881
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CELL BIOLOGY AND METABOLISM
The PEST Domain of IκBα Is Necessary and Sufficient forin Vitro Degradation by μ-Calpain*

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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.

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This work was supported by Grant R01CA81065 from National Institutes of Health, a Howard Hughes Medical Institute fund through the University of Wisconsin Medical School, the Shaw Scientist Award from the Milwaukee Foundation (to S. M.), and funding from National Institutes of Health training Grant T32GM07215 (to S. D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.