The PEST domain of IkappaBalpha 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, IkappaBalpha, is prerequisite to the activation of the transcription factor NF-kappaB. Two mechanisms of IkappaBalpha 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 IkappaBalpha that facilitate its recognition and proteolysis by calpain in vitro. These studies revealed that the PEST sequence of IkappaBalpha is critical for its calpain-dependent degradation. Furthermore, the IkappaBalpha-PEST domain binds to the calmodulin-like domain of the large subunit of mu-calpain (muCaMLD). Transfer of the IkappaBalpha-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 muCaMLD and resulted in its susceptibility to calpain proteolysis. Moreover, the muCaMLD of calpain acts as a competitive inhibitor of calpain-dependent IkappaBalpha degradation. Our data demonstrate that the IkappaBalpha-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.
Shumway
S D
SD
Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53792, USA.
Maki
M
M
Miyamoto
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002154:Calpain / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D020672:I-kappa B Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D046911:Macromolecular Substances; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D011994:Recombinant Proteins; D000072000:NF-KappaB Inhibitor alpha; D002154:Calpain