Cloning and expression of the cDNA encoding rat caspase-2.
We have isolated cDNA clones for rat caspase-2 (also called Nedd2/Ich-1), that encodes a protein similar to interleukin-1beta-converting enzyme (ICE) and the product of the nematode Caenorhabditis elegans cell death gene ced-3 both of which play an important role in programmed cell death (PCD). The rat caspase-2 cDNA clones have an open reading frame (ORF) of 452 amino acids (aa). The predicted aa sequence of rat caspase-2 is highly similar to that of mouse Nedd2 (97.3%) and human Ich-1L (91.3%). The aa sequence QACRG containing the active Cys residue, that is necessary for the proteolytic activity of ICE/Ced-3 (caspase) family of proteases, is also conserved in rat caspase-2. Rat caspase-2 also has several Asp residues in the amino and carboxyl cleavage regions similar to other caspase family proteins. We have developed PC12 cells carrying an on/off switching cassette of caspase-2 (named PC-Nd cells), which contains the neo gene flanked by a pair of loxP sites, the Cre-specific recognition sequence of 34 nucleotides (nt), that lies between the promoter and the caspase-2 cDNA. This expression cassette was designed to express the neo gene initially and to turn on the expression of caspase-2 by site-specific recombinase Cre-mediated excisional deletion of the neo gene. After infection with Cre-producing recombinant adenovirus (re-Ad), the expression of caspase-2 was highly induced in PC-Nd cells and presumptive actively processed fragments of caspase-2 were also observed. This gene activation strategy of caspase-2 will be useful for the study of the biological effects of caspase family proteins in PCD.
Sato
N
N
Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Winston-Salem, NC 27157, USA. nsato@bgsm.edu
Milligan
C E
CE
Uchiyama
Y
Y
Oppenheim
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D053143:Caspase 2; D020169:Caspases; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016716:PC12 Cells; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011506:Proteins; D053143:Caspase 2; D020169:Caspases; D003546:Cysteine Endopeptidases