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Published online 24 July 2000. doi:10.1083/jcb.150.2.321
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© The Rockefeller University Press, 0021-9525/2000//321 $5.00
The Journal of Cell Biology, Volume 150, Number 2, , 2000 321-334

Original Article

Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (Icad/Cad)



Delphine Lechardeura, Luke Drzymalaa, Manu Sharmaa,c, Danuta Zylkaa, Robert Kinacha, Joanna Paciaa, Christopher Hicksa, Nawaid Usmania, Johanna M. Rommensb,d, and Gergely L. Lukacsa,c

a Program in Cell and Lung Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8
b Program in Genetics and Genomics Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8
c Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1X8
d Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5G 1X8
Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8.(416) 813-5771(416) 813-5125

glukacs{at}sickkids.on.ca

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD–ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3–deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3–dependent regulation of CAD activity takes place.

Key Words: apoptosis • chromosomal DNA degradation • caspase-activated DNase • nuclear targeting • caspase-3

© 2000 The Rockefeller University Press

The online version of this article contains supplemental material.

Abbreviations used in this paper: CAD, caspase-3–activated DNase; DFF, DNA fragmentation factor; EGFP, enhanced green fluorescent protein; GST, glutathione-S-transferase; hICAD-L, full-length human ICAD; hICAD-S, alternatively spliced variant of hICAD; ICAD, inhibitor of caspase-activated DNase; NLS, nuclear localization signal.


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