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 hav e been grouped into subfamilies according to their preferred cleavage sites , with the "apoptotic executioner" caspase-3 as the prototype of DEXD-depen dent 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-bindin g proteins of human cells thought to be involved in nuclear architecture by fastening chromatin loops to a proteinaceous nuclear skeleton, the so-call ed nuclear matrix or scaffold. Using an amino-terminal recombinant construc t of scaffold attachment factor A and recombinant caspase-3, we have mapped the cleavage site by matrix-assisted laser desorption ionization/time of f light mass spectrometry and Edman sequencing, We find that cleavage occurs after Asp-100 in a sequence context (SALD) that does not conform to the hit herto accepted DEXD consensus sequence of caspase-3. A point mutation, D100 A, abrogates cleavage by recombinant caspase-3 in vitro and during apoptosi s in vivo, confirming SALD as a novel caspase-3 cleavage site.