Identification of Clusterin Domain Involved in NF-κB Pathway Regulation

Abdellatif Essabbani,Florence Margottin-Goguet,Gilles Chiocchia

doi:10.1074/jbc.c109.057133

Abstract

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-kappaB 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-kappaB regulatory activity. Sequence comparison analysis showed sequence motif identity between CLU and beta-transducin repeat-containing protein (beta-TrCP), a main E3 ubiquitin ligase involved in IkappaB-alpha degradation. These homologies were localized in the disulfide constraint region of CLU. We generated a specific molecular construct of this region, named DeltaCLU, and showed that it has the same NF-kappaB regulatory activity as CLU. Neither the alpha-chain nor the beta-chain of CLU had any NF-kappaB regulatory activity. Furthermore, we showed that following tumor necrosis factor-alpha stimulation of transfected cells, we could co-immunoprecipitate phospho-IkappaB-alpha with DeltaCLU. Moreover, we showed that DeltaCLU could localize both in the cytoplasm and in the nucleus. These results demonstrate the identification of a new CLU activity site involved in NF-kappaB pathway regulation.

Highlights

Clusterin (CLU)3 is a ubiquitous glycoprotein that shows a very broad pattern of tissue expression in mammals
The second CLU gene transcript lacks the endoplasmic reticulum-targeting sequence at exon 2, and its product is detected as a 49-kDa nonglycosylated precursor nuclear form of CLU (nCLU) protein in the cytosol and a 55-kDa glycosylated protein in the nucleus. pnCLU is stored in the cytoplasm; upon stress induced by ionizing radiation, pnCLU is activated and becomes the mature form nCLU, which locates to the nucleus
Because CLU was shown to be a negative regulator of NF-␬B and stabilizer of I␬B-␣, we performed sequence alignments between clusterin and proteins known to act in the NF-␬B pathway

Summary

Introduction

Clusterin (CLU)3 is a ubiquitous glycoprotein that shows a very broad pattern of tissue expression in mammals. We and others have demonstrated that CLU intracellular forms are involved in NF-␬B pathway regulation through I␬B-␣ stabilization [16, 17]. CLU Molecular Constructs—We generated a set of gene constructs corresponding to several regions of clusterin in an expression vector pFLAG to facilitate visualization and homogenize technical parameters for manipulation of the expressed gene.

Results

Conclusion