Identification of Clusterin Domain Involved in NF-��B Pathway Regulation

Clusterin (CLU) is a ubiquitous protein that has been implicated in tumorigenesis, apoptosis, inflammation, and cell proliferation. We and others have previously shown that CLU is an inhibitor of the NF-κB pathway. However, the exact form of CLU and the region(s) of CLU involved in this effect were unknown. Using newly generated molecular constructs encoding for CLU and various regions of the molecule, we demonstrated that the presecretory form of CLU (psCLU) form bears the NF-κB regulatory activity. Sequence comparison analysis showed sequence motif identity between CLU and β-transducin repeat-containing protein (β-TrCP), a main E3 ubiquitin ligase involved in IκB-α degradation. These homologies were localized in the disulfide constraint region of CLU. We generated a specific molecular construct of this region, named ΔCLU, and showed that it has the same NF-κB regulatory activity as CLU. Neither the α-chain nor the β-chain of CLU had any NF-κB regulatory activity. Furthermore, we showed that following tumor necrosis factor-α stimulation of transfected cells, we could co-immunoprecipitate phospho-IκB-α with ΔCLU. Moreover, we showed that ΔCLU could localize both in the cytoplasm and in the nucleus. These results demonstrate the identification of a new CLU activity site involved in NF-κB pathway regulation.