Fusing DEDD with ubiquitin changes its intracellular localization and apoptotic potential.
DEDD, a highly conserved and ubiquitous death effector domain containing protein, exists in non, mono, and diubiquitinated forms. We previously reported that endogenous unmodified DEDD is only found in nucleoli and that mono- and diubiquitinated DEDD associate with caspase-3 in the cytosol suggesting that ubiquitination may be important to the apoptosis regulating functions of DEDD in the cytosol. We now demonstrate that many of its 16 lysine residues can serve as alternative acceptors for ubiquitination to maintain the monoubiquitination status of DEDD. A central region in DEDD (amino acids 109-305) outside the death effector domain was found to be essential for ubiquitination and/or the docking of the ubiquitination machinery. Fusion of ubiquitin to the C-terminus of DEDD to mimic monoubiquitinated DEDD relocated DEDD from nucleoli to the cytosol. This fusion protein also demonstrated a greater apoptosis potential than unmodified DEDD. Finally, we show that both mono- and polyubiquitination of DEDD can be achieved by the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP-1/2). In addition, the cotransfection of DEDD with cIAP-1 or cIAP-2 results in the relocalization of the IAPs to the nucleoli. Our data suggest that monoubiquitination of DEDD regulates both its cytoplasmic localization and its proapoptotic potential and that IAP proteins can regulate DEDD's ubiquitination status.
Lee
J C
JC
The Ben May Institute for Cancer Research, University of Chicago, 924 E. 57th Street, Chicago, IL 60637, USA.
Wang
G X
GX
Schickling
O
O
Peter
M E
ME
D017209:Apoptosis; D002466:Cell Nucleolus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism*; D008239:Lysine; D050505:Mutant Proteins; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
D053418:Death Domain Receptor Signaling Adaptor Proteins; D051034:Inhibitor of Apoptosis Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D008239:Lysine