Takeyoshi Koseki

Nod1, an Apaf-1-like Activator of Caspase-9 and Nuclear Factor-κB

Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-κB) and bound RICK, a CARD-containing kinase that also induces NF-κB activation. Nod1 mutants inhibited NF-κB activity induced by RICK, but not that resulting from tumor necrosis factor-α stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-κB activation pathways.

CIDE, A NOVEL FAMILY OF CELL DEATH ACTIVATORS WITH HOMOLOGY TO THE 45 KDA SUBUNIT OF THE DNA FRAGMENTATION FACTOR

DFF45 is a subunit of the DNA fragmentation factor (DFF) that is cleaved by caspase‐3 during apoptosis. However, the mechanism by which DFF45 regulates apoptotic cell death remains poorly understood. Here we report the identification and characterization of two mammalian genes, CIDE‐A and CIDE‐B, encoding highly related proteins with homology to the N‐terminal region of DFF45. CIDE‐A and CIDE‐B were found to activate apoptosis in mammalian cells, which was inhibited by DFF45 but not by caspase inhibitors. Expression of CIDE‐A induced DNA fragmentation in 293T cells, which was inhibited by DFF45, further suggesting that DFF45 inhibits the apoptotic activities of CIDEs. In addition to mammalian CIDE‐A and CIDE‐B, we identified DREP‐1, a Drosophila melanogaster homolog of DFF45 that could inhibit CIDE‐A‐mediated apoptosis. Mutant analysis revealed that the C‐terminal region of CIDE‐A was necessary and sufficient for killing whereas the region with homology to DFF45 located in the N‐terminus was required for DFF45 to inhibit CIDE‐A‐induced apoptosis. CD95/Fas‐mediated apoptosis was enhanced by CIDEs but inhibited by DFF45. These studies suggest that DFF45 is evolutionarily conserved and implicate CIDEs as DFF45‐inhibitable effectors that promote cell death and DNA fragmentation.

RICK, a novel protein kinase containing a caspase recruitment domain, interacts with CLARP and regulates CD95-mediated apoptosis.

Signaling through the CD95/Fas/APO-1 death receptor plays a critical role in the homeostasis of the immune system. RICK, a novel protein kinase that regulates CD95-mediated apoptosis was identified and characterized. RICK is composed of an N-terminal serine-threonine kinase catalytic domain and a C-terminal region containing a caspase-recruitment domain. RICK physically interacts with CLARP, a caspase-like molecule known to bind to Fas-associated protein with death domain (FADD) and caspase-8. Expression of RICK promoted the activation of caspase-8 and potentiated apoptosis induced by Fas ligand, FADD, CLARP, and caspase-8. Deletion mutant analysis revealed that both the kinase domain and caspase-recruitment domain were required for RICK to promote apoptosis. Significantly, expression of a RICK mutant in which the lysine of the putative ATP-binding site at position 38 was replaced by a methionine functioned as an inhibitor of CD95-mediated apoptosis. Thus, RICK represents a novel kinase that may regulate apoptosis induced by the CD95/Fas receptor pathway.

CIPER, a novel NF κB-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10

We have identified and characterized CIPER, a novel protein containing a caspase recruitment domain (CARD) in its N terminus and a C-terminal region rich in serine and threonine residues. The CARD of CIPER showed striking similarity to E10, a product of the equine herpesvirus-2. CIPER formed homodimers via its CARD and interacted with viral E10 but not with several apoptosis regulators containing CARDs including ARC, RAIDD, RICK, caspase-2, caspase-9, or Apaf-1. Expression of CIPER induced NF-κB activation, which was inhibited by dominant-negative NIK and a nonphosphorylable IκB-α mutant but not by dominant-negative RIP. Mutational analysis revealed that the N-terminal region of CIPER containing the CARD was sufficient and necessary for NF-κB-inducing activity. Point mutations in highly conserved residues in the CARD of CIPER disrupted the ability of CIPER to activate NF-κB and to form homodimers, indicating that the CARD is essential for NF-κB activation and dimerization. We propose that CIPER acts in a NIK-dependent pathway of NF-κB activation.

Identification of Regulatory and Catalytic Domains in the Apoptosis Nuclease DFF40/CAD

The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290–345) abrogated the caspase-3-induced nuclease activity of DFF40/CAD but not its ability to interact with DFF45/ICAD. Conversely, removal of the regulatory domain (residues 1–83) yielded a constitutively active DFF40/CAD nuclease that neither bound to its inhibitor nor required caspase-3 for activation. Amino acid alignment revealed that the regulatory domain of DFF40/CAD has homology to the N-terminal region of mammalian andDrosophila DFF45/ICAD and CIDE-N, a regulatory domain previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus, CIDE-N represents a new type of domain that is associated with the regulation of the apoptosis/DNA fragmentation pathway.