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 NH 2 -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-B) and bound RICK, a CARDcontaining kinase that also induces NF-B activation. Nod1 mutants inhibited NF-B activity induced by RICK, but not that resulting from tumor necrosis factor-␣ stimulation. Thus, Nod1 is a leucine-rich repeatcontaining Apaf-1-like molecule that can regulate both apoptosis and NF-B activation pathways.
Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors. Cancer Res; 70(4); 1544-54. ©2010 AACR.
Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-X L , an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-X L was mediated through both its CED-4-like domain and the Cterminal domain containing WD-40 repeats. Expression of Bcl-X L inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-X L purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-X L failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-X L regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-X L associates with caspase-9 and Apaf-1, and show that Bcl-X L inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.
We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxylterminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with f luorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD͞MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-f luoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD͞MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.Apoptosis, a morphologically distinguished form of programmed cell death, is critical not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases, including cancer, autoimmune disease, viral infection, and neurodegenerative disorders (1-2). Whereas numerous genes that control apoptosis have been identified and partially characterized, the precise mechanisms by which these genes interact to execute the apoptotic program remains poorly understood. In mammals, a family of cysteine proteases (designated caspases) related to the Caenorhabditis elegans gene ced-3 appears to represent the effector arm of the apoptotic program (3-4). The first member of the family identified was interleukin 1-converting enzyme (5). To date, more than 10 caspases have been identified and partially characterized (3). Each caspase contains characteristic conserved sequences important for proteolytic activity and induction of apoptosis (3-4). Caspase activation is induced by a wide array of death signals and leads to precipitous cleavage of protein substrates and execution of the apoptotic program (1, 2).Engagement of the surface receptors, Fas͞APO-1͞CD95 and tumor necrosis factor receptor 1 (TNFR-1), leads to the activation of several caspases and apoptosis (6). Both CD95 and TNFR-1 use the adapter protein FADD͞MORT-1 to link cytoplasmic receptor sequences to the upstream FLICE͞ MACH͞Mch5 protease (caspase-8) and Mch4 (caspase-10) (7-14). Both FADD͞MORT-1 and caspase-8 interact through conserved death effector domains (DED) located in the N-terminal region of FADD͞MORT-1 and pro-domain of caspase-8 (7, 8, 11-13). Upon binding of the corresponding ligands, the CD95 re...
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