Apoptotic cleavage of scaffold attachment factor A (SAF-A) by caspase-3 occurs at a noncanonical cleavage site.
Members of the caspase family of cysteine proteases play essential roles in the disintegration of cellular architecture during apoptosis. Caspases have been grouped into subfamilies according to their preferred cleavage sites, with the "apoptotic executioner" caspase-3 as the prototype of DEXD-dependent proteases. We show here that caspase-3 is more tolerant to variations of the cleavage site than previously anticipated and present an example of a noncanonical recognition site that is efficiently cleaved by caspase-3 in vitro and in vivo. The new cleavage site was identified in human scaffold attachment factor A, one of the major scaffold attachment region DNA-binding proteins of human cells thought to be involved in nuclear architecture by fastening chromatin loops to a proteinaceous nuclear skeleton, the so-called nuclear matrix or scaffold. Using an amino-terminal recombinant construct of scaffold attachment factor A and recombinant caspase-3, we have mapped the cleavage site by matrix-assisted laser desorption ionization/time of flight mass spectrometry and Edman sequencing. We find that cleavage occurs after Asp-100 in a sequence context (SALD) that does not conform to the hitherto accepted DEXD consensus sequence of caspase-3. A point mutation, D100A, abrogates cleavage by recombinant caspase-3 in vitro and during apoptosis in vivo, confirming SALD as a novel caspase-3 cleavage site.
Kipp
M
M
Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Schwab
B L
BL
Przybylski
M
M
Nicotera
P
P
Fackelmayer
F O
FO
D000595:Amino Acid Sequence; D017209:Apoptosis; D001483:Base Sequence; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D017931:DNA Primers; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D006868:Hydrolysis; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D012261:Ribonucleoproteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D017931:DNA Primers; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012261:Ribonucleoproteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases