Identification of the primary caspase 3 cleavage site in alpha II-spectrin during apoptosis.
Alpha II-spectrin is one of the major proteins responsible for maintaining the cytoskeletal integrity of the cell. The caspase 3-mediated cleavage of alpha II-spectrin during apoptotic cell death may play an important role in altering membrane stability and the formation of apoptotic bodies. In this study, we identified the primary caspase 3 cleavage site in alpha II-spectrin. We found that the transcriptional inhibitor, actinomycin D, induced caspase 3 activation and that caspase 3 activation is coincident with the cleavage of alpha II-spectrin protein at a primary cleavage site. Deletion analysis and site directed mutagenesis identified the primary cleavage site in alpha II spectrin at amino acid 1185 (DETD). The primary caspase 3 cleavage site in alpha II spectrin is conserved in immature and mature B cells. Our results indicate that alpha II-spectrin is initially cleaved at a caspase 3 consensus site and this primary event likely alters the structural conformation of the protein exposing subsequent cleavage sites and altering cytoskeletal integrity. Identification of the primary cleavage site for caspase 3 may help to elucidate the role of alpha II-spectrin in membrane stability and apoptosis as well as provide new insights into alpha II-spectrin autoantibody formation associated with the autoimmune disease, Sjögren's syndrome.
Williams
S T
ST
Department of Physiology & Biophysics, Wright State University School of Medicine, Dayton, OH 45435, USA.
Smith
A N
AN
Cianci
C D
CD
Morrow
J S
JS
Brown
T L
TL
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D053938:DNA Fragmentation; D003609:Dactinomycin / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D016297:Mutagenesis, Site-Directed; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D012859:Sjogren's Syndrome; D013049:Spectrin / Q000378:metabolism*
D019384:Nucleic Acid Synthesis Inhibitors; D020033:Protein Isoforms; D013049:Spectrin; D003609:Dactinomycin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases