Events in Apoptosis

doi:10.1074/jbc.271.27.16260

Events in Apoptosis

ACIDIFICATION IS DOWNSTREAM OF PROTEASE ACTIVATION AND BCL-2 PROTECTION*

From the ^‡ Scripps Research Institute, La Jolla, California 92037, the
^§ La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, and the
^∥ Veterans Affairs Medical Center, San Diego, California 92161

^” To whom correspondence should be addressed:
Dept. of Molecular and Experimental Medicine, The Scripps Research Inst. NX7, 10666 N. Torrey Pines Rd., La Jolla, CA 92037.
Tel.: 619-784-7929; Fax: 619-784-7981; E-mail: robbieg{at}scripps.edu

Abstract

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems. However, its relation to other events in the process of apoptosis is not yet characterized. In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling. Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1β converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation. These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.

Footnotes

↵^¶ Recipient of Wellcome Trust Fellowship 041080.
↵* This work was supported in part by United States Public Health Service Grants AI01345 and AG13501 (to R. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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

ZVAD

carbobenzoxy-Val-Ala-Asp-fluoromethylketone

SNARF-1

seminaphthorhodafluor-1.
↵² S. J. Martin, manuscript in preparation.
- Received March 25, 1996.