Cleavage of transcription factor SP1 by caspases during anti-IgM-induced B-cell apoptosis.
Apoptosis is instrumental in the processes generating the diversity of the B-cell repertoire. Autoreactive B-cells are eliminated by anti-IgM crosslinking after encountering self-antigens, but precise mechanisms leading to B-cell apoptosis are still not well understood. We report here the cleavage of the transcription factor SP1 in the human Burkitt lymphoma cell line BL60 during anti-IgM-induced apoptosis. Western blot analysis revealed two cleavage products of approximately 68 kDa and 45 kDa after induction of apoptosis. Cleavage could be completely inhibited by zDEVD-fmk, an inhibitor specific for caspase 3-like proteases. In-vitro cleavage of recombinant SP1 by recombinant caspase 3 (CPP32) or caspase 7 (Mch 3) results in similar cleavage products as those observed in vivo. Recombinant caspase 6 (Mch 2) primarily generates a 68-kDa cleavage product, as observed after calcium ionophore (CaI) induced B-cell apoptosis. In contrast, caspase 1 (ICE) did not cleave SP1 in vitro. The time course of SP1 cleavage during anti-IgM-induced apoptosis is paralleled by an increase of caspase activity measured by DEVD-p-nitroanilide (DEVD-pNA) cleavage. DNA band-shift assays revealed a decrease in the intensity of the full length SP1/DNA complex and an increase in the intensity of a smaller complex due to the binding of one SP1 cleavage product. By Edman sequencing we could identify a caspase 3 cleavage site after Asp584 (D584AQPQAGR), generating a 22-kDa C-terminal SP1 protein fragment which still contains the DNA binding site. Our results show the cleavage of the human transcription factor SP1 in vivo and in vitro, underlining the central role of caspase 3-like proteases during the process of anti-IgM-induced apoptosis.
Rickers
A
A
Medizinische Onkologie und Tumorimmunologie, Max Delbück Center for Molecular Medicine, Berlin-Buch, Germany.
Peters
N
N
Badock
V
V
Beyaert
R
R
Vandenabeele
P
P
Dörken
B
B
Bommert
K
K
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic; D017209:Apoptosis / Q000276:immunology / Q000502:physiology*; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D006801:Humans; D007075:Immunoglobulin M; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000888:Antibodies, Anti-Idiotypic; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases