CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis

CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis

Departments of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109

Edited by David V. Goeddel, Tularik, Inc., South San Francisco, CA, and approved July 30, 1997 (received for review June 2, 1997)

Abstract

We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.

Footnotes

↵ * To whom reprint requests should be addressed. e-mail: Gabriel.Nunez{at}umich.edu.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: CLARP, caspase-like apoptosis-regulatory protein; AMC, amino-4-methylcoumarin; DED, death effector domain; TNFR-1, tumor necrosis factor receptor 1; EST, expressed sequence tag; AU1-caspase-8-mt, AU1-tagged Ser-377 mutant of caspase-8; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone; DEVD-AMC, acetyl-Asp-Glu-Val-Asp-AMC.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF005774).