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 crosslin king after encountering self-antigens, but precise mechanisms leading to B- cell apoptosis are still not well understood. We report here the cleavage o f the transcription factor SP1 in the human Burkitt lymphoma cell Line BL60 during anti-IgM-induced apoptosis. Western blot analysis revealed two clea vage products of approximately 68 kDa and 45 kDa after induction of apoptos is. Cleavage could be completely inhibited by zDEVD-fmk, an inhibitor speci fic for caspase 3-like proteases. In-vitro cleavage of recombinant SP1 by r ecombinant caspase 3 (CPP32) or caspase 7 (Mch 3) results in similar cleava ge products as those observed in vivo. Recombinant caspase 6 (Mch 2) primar ily generates a 68-kDa cleavage product, as observed after calcium ionophor e (CaI) induced B-cell apoptosis. In contrast, caspase 1 (ICE) did not clea ve SP1 in vitro. The time course of SP1 cleavage during anti-IgM-induced ap optosis is paralleled by an increase of caspase activity measured by DEVD-p -nitroanilide (DEVD-pNA) cleavage. DNA band-shift assays revealed a decreas e in the intensity of the full length SP1/DNA complex and an increase in th e intensity of a smaller complex due to the binding of one SP1 cleavage pro duct. By Edman sequencing we could identify a caspase 3 cleavage site after Asp584 (D(584)AQPQAGR), generating a 22-kDa C-terminal SP1 protein fragmen t 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-in duced apoptosis.