Activation of caspases-8 and -10 by FLIP<sub>L</sub>

Kelly Boatright

Cristina Deis

Jean Denault

Daniel Sutherlin

Guy Salvesen

*Program in Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.

†Graduate Program in Molecular Pathology, University of California San Diego, La Jolla, CA 92037, U.S.A.

‡Department of Medicinal Chemistry, Genentech Inc., 1DNA Way, South San Francisco, CA 94080, U.S.A.

^{To whom correspondence should be addressed (email}gro.mahnrub@nesevlasg).

Received 2004 May 14; Revised 2004 Jun 14; Accepted 2004 Jun 21.

Abstract

The first step in caspase activation is transition of the latent zymogen to an active form. For the initiator caspases, this occurs through dimerization of monomeric zymogens at an activating complex. Recent studies have suggested that FLIP_L [FLICE-like inhibitory protein, long form; FLICE is FADD (Fas-associated death domain protein)-like interleukin-1β-converting enzyme], previously thought to act solely as an inhibitor of caspase-8 activation, can under certain circumstances function to enhance caspase activation. Using an in vitro induced-proximity assay, we demonstrate that activation of caspases-8 and -10 occurs independently of cleavage of either the caspase or FLIP_L. FLIP_L activates caspase-8 by forming heterodimeric enzyme molecules with substrate specificity and catalytic activity indistinguishable from those of caspase-8 homodimers. Significantly, the barrier for heterodimer formation is lower than that for homodimer formation, suggesting that FLIP_L is a more potent activator of caspase-8 than is caspase-8 itself.

Keywords: apoptosis, caspase-8, FLIP_L (FLICE-like inhibitory protein), protease, zymogen

Abbreviations: Ac-IETD-AFC, acetyl-Ile-Glu-Thr-Asp-7-amido-4-fluoromethylcoumarin; DED, death effector domain; DISC, death-inducing signalling complex; FADD, Fas-associated death domain protein; FLICE, FADD-like interleukin-1β-converting enzyme; FLIP, FLICE-like inhibitory protein; FLIP_L, FLIP long form; FLIP_S, FLIP short form; MMP, matrix metalloprotease; MT-MMP, membrane-type matrix metalloprotease; NFκB, nuclear factor κB; TIMP, tissue inhibitor of metalloproteases; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone

Abstract

Acknowledgments

We thank Scott Snipas and Annamarie Price for expert technical assistance, Markus Peter and Stan Krajewski for supplying caspase-8 antisera, and Pablo Fuentes-Prior for helpful discussions. This work was supported by California Breast Cancer Research Program Fellowship 8GB-0137 (K.M.B.) and NIH grant CA69381.

Acknowledgments

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