Inhibition of CPP32 blocks surface IgM-mediated apoptosis and D4-GDI cleavage in human BL60 Burkitt lymphoma cells

Abstract

Apoptosis (programmed cell death) is instrumental in the process of controlling lymphocyte growth and selection. Negative selection, mediated by surface IgM (sIgM) signaling after encountering self antigen, eliminates autoreactive B cells. To identify proteins which are potentially involved in anti-IgM-mediated apoptosis, we used an anti-IgM-sensitive subclone of the human Burkitt lymphoma cell line BL60. After anti-IgM treatment and separation of apoptosis-committed cells, we performed high resolution two-dimensional gel electrophoresis (2-DE). Comparison of the 2-DE protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots. Subsequent analysis of these proteins was performed by mass spectrometry and Edman microsequencing. We report that one of these spots which disappears after sIgM cross-linking turned out to be D4-GDI. D4-GDI is an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPase. D4-GDI was rapidly truncated to a 23-kDa fragment in BL60 cells. By using a Rho-GDI-specific antiserum, which cross-reacts with D4-GDI, we observed the onset of cleavage after 8 h of stimulation with anti-IgM. Cleavage and apoptosis could be completely inhibited by z-DEVD-fmk, a selective irreversible inhibitor of CPP32 (caspase-3), whereas ac-YVAD-cmk, an inhibitor for interleukin-1beta-converting enzyme-like proteases, did not block cleavage of D4-GDI or apoptosis. Our results revealed the functional importance of caspases and a new target protein in the process of anti-IgM-mediated apoptosis.

Cleavage and nuclear translocation of the caspase 3 substrate Rho GDP-dissociation inhibitor, D4-GDI, during apoptosis

“…15 Previously D4-GDI has been reported to be cleaved at aspartate 19 by caspase 3 during apoptosis in Jurkat Tcells induced with anti-Fas antibody, overexpression of Bax, and staurosporine, 16,17 and in BL60 Burkitt lymphoma cells induced with anti-IgM. 18 We add in this report the observations that D4-GDI is cleaved in ML-1 cells and many other cell types induced to undergo apoptosis by incubation with etoposide, staurosporine, or anti-fas antibody. We also show that protease inhibitors, serine/ threonine phosphatase inhibitors and zinc can inhibit this cleavage.…”

“…The cleavage of D4-GDI by caspase 3-like activity has been detected during apoptosis induced by anti-FAS antibody, BAX, and staurosporine in Jurkat cells, 16,17 and anti IgM treatment of BL60 Burkitt lymphoma cells. 18 Inhibitors of caspases have been shown to inhibit this cleavage. Whether the cleavage of D4-GDI at the caspase 3 site activates Rho has yet to be determined.…”

Predominant Identification of RNA-binding Proteins in Fas-induced Apoptosis by Proteome Analysis

“…Among these proteins, ␣ NAC, hnRNP A0, hnRNP A2/B1, hnRNP A3, hnRNP K, hnRNP R, KH-type splicing regulatory protein, NS1-associated protein 1, p54 nrb , poly(A)-binding protein 4, and splicing factors ASF-2 and SRp30c were not known to be modified during apoptosis. However, we also identified proteins that were previously found to be modified during apoptosis by a proteome approach using the Burkitt lymphoma B cell line HL-60 and IgM as an inducer of apoptosis (16,40,41). These proteins included hnRNP A1, hnRNP C1/C2, FUSEbinding protein, nucleolin, Rho GDI 2, and the transcription factor BTF3.…”

Apoptosis-induced cleavage of keratin 15 and keratin 17 in a human breast epithelial cell line

“…31 Cells were lysed by boiling in 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1% SDS, and 30 ± 50 mg protein was separated by SDS ± PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Monoclonal anti-keratin 17 antibody was diluted 1 : 400.…”