Inhibition of CPP32 blocks surface IgM-mediated apoptosis and D4-GDI cleavage in human BL60 Burkitt lymphoma cells.
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.
Rickers
A
A
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Brockstedt
E
E
Mapara
M Y
MY
Otto
A
A
Dörken
B
B
Bommert
K
K
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002051:Burkitt Lymphoma / Q000473:pathology*; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000502:physiology*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology*; D018189:Immunomagnetic Separation; D009842:Oligopeptides / Q000494:pharmacology; D011947:Receptors, Antigen, B-Cell / Q000276:immunology*; D017634:Self Tolerance / Q000276:immunology*; D014407:Tumor Cells, Cultured; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569658:ARHGDIB protein, human; D000590:Amino Acid Chloromethyl Ketones; D000888:Antibodies, Anti-Idiotypic; D015853:Cysteine Proteinase Inhibitors; D020730:Guanine Nucleotide Dissociation Inhibitors; D007075:Immunoglobulin M; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D009842:Oligopeptides; D011947:Receptors, Antigen, B-Cell; C044203:anti-IgM; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D019204:GTP-Binding Proteins