Members of the tumor necrosis factor (TNF) receptor family and their corresponding ligands are critical regulators of apoptosis and various other cellular processes. Some of the receptors (Fas, TNF-R1, TRAIL-R1, TRAIL-R2, TRAMP/ DR3, DR6, and EDA-R) contain a cytoplasmic region, called the death domain (DD), which is essential for cell death signaling (18,22). Signals emanating from Fas and TNF-R1 have been intensively studied (14). Upon receptor activation, the DD of Fas undergoes direct homotypic interaction with a DD in the adapter protein FADD, while FADD recruitment is indirect (via TRADD) in the case of TNF-R1 (4). The death effector domain (DED) at the amino terminus of FADD then recruits pro-caspase 8 via homotypic interaction with its two DEDs. The high local concentration of caspase 8 zymogens facilitates self-processing and assembly of the mature enzyme. Activated caspase 8 initiates apoptosis by subsequent cleavage of downstream caspases (caspase-3, -6, and -7).Death induced by death receptors is tightly regulated by genes that are activated by the transcription factor NF-B (25). Modulation of the response in favor of NF-B protects cells from apoptosis; failure to do so results in increased cell death. At least six NF-B-responsive genes are involved in this survival amplification loop (26), i.e., those that encode IAP-1 and IAP-2, which block caspase activity (7); that which encodes the Bcl-2 family member A1 (25); and those that encode TRAF-1, TRAF-2 (1), and A20 (21), which are themselves implicated in the NF-B signaling pathway. However, overexpression of all of these genes affords, at best, partial protection, in particular from death triggered by TNF (25). The only known potent inhibitor of death receptor signals is c-FLIP. Two c-FLIPs have been characterized (23, 24). The full-length 55-kDa-long form of FLIP (FLIP L ) exhibits overall structural homology to caspase 8, containing two DEDs that interact with FADD and an inactive caspase-like domain. An alternatively spliced short form of FLIP (FLIP S ) contains only the two DEDs and displays reduced antiapoptotic capacity.We undertook a series of experiments to investigate whether c-FLIP is implicated in the antiapoptotic NF-B response. Here we provide evidence that FLIP expression is upregulated upon the stimulation of several signaling pathways that are known to trigger activation of the transcription factor NF-B. Moreover, we found that cells that were rendered highly sensitive to death ligand-induced apoptosis by blocking NF-B activation could be rescued by expressing FLIP. FLIP may therefore play a key role in the NF-B-mediated control of death signals. MATERIALS AND METHODSAntibodies and materials. Rabbit anti-TRAF2 polyclonal antibody C20, rabbit anti-cIAP1 polyclonal antibody H-85, and mouse anti-TRAF1 monoclonal antibody H3 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Rat anti-cFLIP monoclonal antibody Dave II was from Alexis, Lausen Switzerland, and anti-Phospho-IBa antibody was from Biolab. Anti-tubulin and antiFl...

Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres is unknown. Using FRET-based biosensors to measure localized phosphorylation dynamics in living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules.Accurate chromosome segregation during cell division is essential to maintain genome integrity. Prior to segregation, kinetochores of sister chromatids attach to microtubules from opposite spindle poles (bi-orientation). This configuration is achieved through a trial-and-error process in which correct attachments exert tension across the centromere, which stabilizes kinetochore-microtubule interactions. Incorrect attachments, for example if both sister chromatids attach to a single spindle pole, exert less tension and are destabilized, providing a new opportunity to bi-orient (1,2). How tension is coupled to kinetochore-microtubule stability is not known.The mitotic kinase Aurora B (Ipl1 in budding yeast) localizes to the inner centromere, between sister kinetochores, and destabilizes microtubule attachments by phosphorylating kinetochore substrates, including Dam1 and the Ndc80 complex (3-10). An appealing model is that Aurora B substrates are selectively phosphorylated at incorrect attachments. To test this model we first examined phosphorylation of CENP-A Ser-7, a known kinetochore substrate (11). We used an assay in which Aurora B inhibition leads to a high frequency of syntelic attachment errors, with sister chromatids connected to a single spindle pole (6) (Fig. S1A). We compared phospho-CENP-A staining at correct and incorrect attachments 10 min after removing the reversible Aurora B kinase inhibitor ZM447439 (12), which re-activates Aurora B. Phospho-** Publisher's Disclaimer: This manuscript has been accepted for publication in Science. This version has not undergone final editing.Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the

Large numbers of inhibitors for polo-like kinases and aurora kinases are currently being evaluated as anticancer drugs. Interest in these drugs is fuelled by the idea that these kinases have unique functions in mitosis. Within the polo-like kinase family, the emphasis for targeted therapies has been on polo-like kinase 1 (PLK1), and in the aurora kinase family drugs have been developed to specifically target aurora kinase A (AURKA; also known as STK6) and/or aurora kinase B (AURKB; also known as STK12). Information on the selectivity of these compounds in vivo is limited, but it is likely that off-target effects within the same kinase families will affect efficacy and toxicity profiles. In addition, it is becoming clear that interplay between polo-like kinases and aurora kinases is much more extensive than initially anticipated, and that both kinase families are important factors in the response to classical chemotherapeutics that damage the genome or the mitotic spindle. In this Review we discuss the implications of these novel insights on the clinical applicability of polo-like kinase and aurora kinase inhibitors.

The cytokine IL-2 plays a very important role in the proliferation and survival of activated T cells. These effects of IL-2 are dependent on signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. We and others have shown that PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). In this study we have investigated the role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival. We show that IL-2 regulates phosphorylation of FoxO3 in a PI3K-dependent fashion. Phosphorylation and inactivation of FoxO3 appears to play an important role in IL-2-mediated T cell survival, because mere activation of FoxO3 is sufficient to trigger apoptosis in T cells. Indeed, active FoxO3 can induce expression of IL-2-regulated genes, such as the cdk inhibitor p27Kip1 and the proapoptotic Bcl-2 family member Bim. Furthermore, we show that IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, we propose that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival.

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Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

SummaryPrecise control of the attachment strength between kinetochores and spindle microtubules is essential to preserve genomic stability. Aurora B kinase has been implicated in regulating the stability of kinetochore-microtubule attachments but its relevant kinetochore targets in cells remain unclear. Here, we identify multiple serine residues within the N-terminus of the kinetochore protein Hec1 that are phosphorylated in an Aurora-B-kinase-dependent manner during mitosis. On all identified target sites, Hec1 phosphorylation at kinetochores is high in early mitosis and decreases significantly as chromosomes bi-orient. Furthermore, once dephosphorylated, Hec1 is not highly rephosphorylated in response to loss of kinetochore-microtubule attachment or tension. We find that a subpopulation of Aurora B kinase remains localized at the outer kinetochore even upon Hec1 dephosphorylation, suggesting that Hec1 phosphorylation by Aurora B might not be regulated wholly by spatial positioning of the kinase. Our results define a role for Hec1 phosphorylation in kinetochore-microtubule destabilization and error correction in early mitosis and for Hec1 dephosphorylation in maintaining stable attachments in late mitosis.

Genetic evidence is mounting that survivin plays a crucial role in mitosis, but its exact role in human cell division remains elusive. We show that mammalian cells lacking survivin are unable to align their chromosomes, fail to recruit Aurora B to kinetochores and become polyploid at a very high frequency. Survivin‐depleted cells enter mitosis with normal kinetics, but are delayed in prometaphase in a BubR1/Mad2‐dependent fashion. Nonetheless, these cells exit mitosis prior to completion of chromosome congression and without sister chromatid segregation, indicating that the spindle assembly checkpoint is not fully functional. Indeed, in survivin‐depleted cells, BubR1 and Mad2 are prematurely displaced from kinetochores, yet no tension is generated at kinetochores. Importantly, these cells fail to respond to drugs that prevent tension, but do arrest in mitosis after depolymerization of the mitotic spindle. This demonstrates that survivin is not required for initial checkpoint activation, or for sustained checkpoint activation by loss of microtubules. However, stable association of BubR1 to kinetochores and sustained checkpoint signalling in response to lack of tension crucially depend on survivin.