The cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2) proteins have been implicated in the activation of NF-B by TNF␣; however, genetic deletion of either cIAP1 or 2 did not support a physiologically relevant role, perhaps because of functional redundancy. To address this, we used combined genetic and siRNA knockdown approaches and report that cIAP1 and 2 are indeed critical, yet redundant, regulators of NF-B activation upon TNF␣ treatment. Whereas NF-B was properly activated by TNF␣ in cultured and primary cells deficient in either cIAP1 or 2, removal of both cIAPs severely blunted its activation. After treatment with TNF␣, cIAP1 and 2 were rapidly recruited to the TNF receptor 1, along with the adapter protein TNF receptor associated factor 2. Importantly, either cIAP1 or 2 was required for proper TNF receptor 1 signalosome function. In their combined absence, polyubiquitination of receptor interacting protein 1, an upstream event necessary for NF-B signaling, was attenuated. As a result, phosphorylation of the inhibitor of B kinase ␤ was diminished, and signal transduction was severely blunted. Consequently, cells missing both cIAP1 and 2 were sensitized to TNF␣-mediated apoptosis.Collectively, these data demonstrate that either cIAP1 or 2 is required for proper Rip1 polyubiquitination and NF-B activation upon TNF␣ treatment.apoptosis ͉ Receptor Interacting Protein (RIP1)

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a putative tumor suppressor in which expression is significantly reduced in human cancer cell lines and primary tumors. The proapoptotic effects of XAF1 have been attributed to both caspase-dependent and -independent means. In particular, XAF1 reverses the anti-caspase activity of XIAP, a physiological inhibitor of apoptosis. We further investigated the function of XAF1 by examining its relationship with other IAPs. Immunoprecipitation studies indicate that XAF1 binds to XIAP, cIAP1, cIAP2, Livin, TsIAP, and NAIP but not Survivin, an IAP that prevents mitotic catastrophe and in which antiapoptotic activity is exerted through direct XIAP interaction and stabilization. We found that overexpressed XAF1 down-regulates the protein expression of Survivin. Under these conditions, Survivin expression was restored in the presence of the proteasome inhibitor MG132 or a XIAP RING mutant that is defective in ubiquitin-protein isopeptide ligase (E3) activity, suggesting that XAF1 interaction activates E3 activity of XIAP and targets Survivin by direct ubiquitination. In addition, RNA interference targeting endogenous XIAP protected Survivin degradation by XAF1. Furthermore, interferon-␤-mediated XAF1 induction promoted formation of an endogenous XIAP-XAF1-Survivin complex. This complex facilitated Survivin degradation, which was prevented in XAF1 ؊/؊ stable clones. Altogether, our study demonstrates that XAF1 mediates Survivin down-regulation through a complex containing XIAP, supporting dual roles for XAF1 in apoptosis and mitotic catastrophe. The inhibitor of apoptosis (IAP)5 family members XIAP and Survivin have emerged as promising cancer therapeutic targets (1, 2). Survivin is a structurally and functionally unique IAP in that it contains a single baculoviral IAP repeat and no RING domain and has evolved dual roles in mitosis and apoptosis. XIAP is a potent endogenous inhibitor of apoptosis that functions by binding to and inhibiting caspases. In contrast, the physical interaction between Survivin and caspases appears to be mostly physiologically irrelevant. Instead, the mechanism of Survivin in promoting cytoprotection may lie in its ability to associate with XIAP and enhance its stability by antagonizing function of the RING E3 ligase domain (3).XIAP-associated factor 1 (XAF1) was identified as a nuclear protein that binds to XIAP and suppresses its anti-caspase activity (4, 5), thereby reversing the protective effect of XIAP overexpression in cancer cell lines. The expression of XAF1 is significantly reduced in cancer cell lines (4, 6) and in several primary malignancies (7, 8) as a result of promoter hypermethylation (9, 10). Moreover, reactivation of xaf1 by DNA methylation inhibitors sensitizes cancer cells to apoptosis-inducing agents (11, 12). Conditionally replicative adenovirus encoding XAF1 was found to have potent anti-tumor efficacy in an animal model of colorectal carcinoma (13).Interferons (IFN) are the most broadly active cytokines that...

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role in the cellular response to DNA damage and in radiation resistance. Although much effort has focused on the relationship between ATM and other nuclear signal transducers, little is known about interactions between ATM and mitogenic signaling pathways. In this study, we show a novel relationship between ATM kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), a key mitogenic stimulator. Activation of ATM by radiation down-regulates phospho-ERK1/2 and its downstream signaling via increased expression of mitogenactivated protein kinase phosphatase MKP-1 in both cell culture and tumor models. This dephosphorylation of ERK1/2 is independent of epidermal growth factor receptor (EGFR) activity and is associated with radioresistance. These findings show a new function for ATM in the control of mitogenic pathways affecting cell signaling and emphasize the key role of ATM in coordinating the cellular response to DNA damage.

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<div>Abstract<p>Ataxia telangiectasia mutated (ATM) kinase plays a crucial role in the cellular response to DNA damage and in radiation resistance. Although much effort has focused on the relationship between ATM and other nuclear signal transducers, little is known about interactions between ATM and mitogenic signaling pathways. In this study, we show a novel relationship between ATM kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), a key mitogenic stimulator. Activation of ATM by radiation down-regulates phospho-ERK1/2 and its downstream signaling via increased expression of mitogen-activated protein kinase phosphatase MKP-1 in both cell culture and tumor models. This dephosphorylation of ERK1/2 is independent of epidermal growth factor receptor (EGFR) activity and is associated with radioresistance. These findings show a new function for ATM in the control of mitogenic pathways affecting cell signaling and emphasize the key role of ATM in coordinating the cellular response to DNA damage. (Cancer Res 2006; 66(24): 11554-9)</p></div>

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Supplementary Figures 1-3 from Ataxia Telangiectasia Mutated Down-regulates Phospho-Extracellular Signal-Regulated Kinase 1/2 via Activation of MKP-1 in Response to Radiation

<div>Abstract<p>Ataxia telangiectasia mutated (ATM) kinase plays a crucial role in the cellular response to DNA damage and in radiation resistance. Although much effort has focused on the relationship between ATM and other nuclear signal transducers, little is known about interactions between ATM and mitogenic signaling pathways. In this study, we show a novel relationship between ATM kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), a key mitogenic stimulator. Activation of ATM by radiation down-regulates phospho-ERK1/2 and its downstream signaling via increased expression of mitogen-activated protein kinase phosphatase MKP-1 in both cell culture and tumor models. This dephosphorylation of ERK1/2 is independent of epidermal growth factor receptor (EGFR) activity and is associated with radioresistance. These findings show a new function for ATM in the control of mitogenic pathways affecting cell signaling and emphasize the key role of ATM in coordinating the cellular response to DNA damage. (Cancer Res 2006; 66(24): 11554-9)</p></div>