Products/Services Used | Details | Operation |
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Mutagenesis Services> | The casp9 constitutively two-chain (C9 CT) expression construct consists of a synthetic gene (Genscript, Piscataway NJ) in pET21b that encodes E. coli codon-optimized human casp-9 that is built for separate expression of the casp-9 large subunit (amino acids 1- 315) independently from the small subunit (amino acids 316-416 + His6x) which was under the control of a second ribosome binding site. Casp-9 variants encoding amino acid substitutions were generated using the QuikChange site-directed mutagenesis method (Stratagene/Agilent, Santa Clara CA). The casp-3 wild type expression construct consists of the full-length, C-terminal His6x-tagged human casp-3 gene (amino acids 1-279 plus six terminal His) in pET23b [63] (gift of Guy Salvesen). | Get A Quote |
Caspases, the cysteine proteases which facilitate the faithful execution of apoptosis, are tightly regulated by a number of mechanisms including phosphorylation. In response to cAMP, PKA phosphorylates caspase-9 at three sites preventing caspase-9 activation, and suppressing apoptosis progression. Phosphorylation of caspase-9 by PKA at the functionally relevant site Ser-183 acts as an upstream block of the apoptotic cascade, directly inactivating caspase-9 by a two-stage mechanism. First, Ser-183 phosphorylation prevents caspase-9 self-processing and directly blocks substrate binding. In addition, Ser-183 phosphorylation breaks the fundamental interactions within the caspase-9 core, promoting di... More