D4-GDI, a Substrate of CPP32, Is Proteolyzed during Fas-induced Apoptosis

doi:10.1074/jbc.271.19.11209

D4-GDI, a Substrate of CPP32, Is Proteolyzed during Fas-induced Apoptosis (*)

From the (1)Department of Molecular Sciences, Central Research Division, Pfizer Inc., Groton, Connecticut 06340 and the
(2)Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037

^¶ To whom correspondence may be addressed:
Central Research, Pfizer, Inc., Groton, CT 06345.
Tel.: 860-441-5334; Fax: 860-441-5719.
**To whom correspondence may be addressed:
Dept. of Immunology, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037.
Tel.: 619-554-8217; Fax: 619-554-8218.

Abstract

Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis. ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 μM DEVD-CHO, a reported selective inhibitor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 μM. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp and Ser of the poly(ADP-ribose) polymerase-like cleavage sequence DELDS. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein.

Footnotes

↵^§ Supported by a fellowship from the National Arthritis Foundation.
↵* This work was supported by U.S. Public Health Service Grants GM39434 and GM44428 (to G. M. B.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ICE

interleukin-1β converting enzyme

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
↵²S. J. Martin, G. P. Amarante-Mendes, M. Tewari, L. Shi, T. H. Chuang, C. Casciano, P. Fitzgerald, E. M. Tan, G. M. Bokoch, V. M. Dixit, A. H. Greenberg, and D. R. Green, submitted for publication.
↵³Ammirati, M. J., Mansour, M. N., La Liberte, R., Carty, T. J., Daumy, G. O., Robinson, R., and Danley, D. E.(1992) Poster S178 presented at the Sixth Symposium of the Protein Society, San Diego, CA, 1992
↵⁴G. M. Bokoch, unpublished observations.
- Received January 19, 1996.
- Revision received February 16, 1996.