The effect of ICAD-S on the formation and intracellular distribution of a nucleolytically active caspase-activated DNase.
We show here that co-expression of murine CAD with either ICAD-L or ICAD-S in Escherichia coli as well as mammalian cells leads to a functional DFF complex, which after caspase-3 activation releases a nucleolytically active DNase. The chaperone activity of ICAD-S is between one and two orders of magnitude less effective than that of ICAD-L, as deduced from cleavage experiments with different activated recombinant DFF complexes produced in E.coli. With nucleolytically active EGFP fusion proteins of CAD it is demonstrated that co-expression of ICAD-S, which lacks the C-terminal domain of ICAD-L, including the NLS, leads to a homogeneous intracellular distribution of the DNase in transfected cells, whereas co-expression of human or murine ICAD-L variants lacking the NLS leads to exclusion of EGFP-CAD from the nuclei in approximately 50% of cells. These results attribute a particular importance of the NLS in the long isoform of the inhibitor of CAD for nuclear accumulation of the DFF complex in living cells. It is concluded that ICAD-L and ICAD-S in vivo might function as tissue-specific modulators in the regulation of apoptotic DNA degradation by controlling not only the enzymatic activity but also the amount of CAD available in the nuclei of mammalian cells.
Institut für Biochemie (FB 08), Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Sebastian Richard
SR
Scholz
Christian
C
Korn
Oleg
O
Gimadutdinow
Michael
M
Knoblauch
Alfred
A
Pingoud
Gregor
G
Meiss
D016475:3T3 Cells; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009711:Nucleotides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C109664:caspase-activated DNase inhibitor; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease