SUMMARY We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome. This resource provides a powerful tool for biotechnological and eukaryotic cell biology research. Comprehensive gene dispensability comparisons with budding yeast, the first time such studies have been possible between two eukaryotes, revealed that 83% of single copy orthologues in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than non-essential genes to be single copy, broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

The transcription factor nuclear factor-B (NF-B) is activated by a diverse number of stimuli including tumor necrosis factor-␣, interleukin-1, UV irradiation, viruses, as well as receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). NF-B activation by the tumor necrosis factor receptor (TNFR) involves the formation of a multiprotein complex termed a signalosome. Although previous studies have shown that the activated EGFR can induce NF-B, the mechanism of this activation remains unknown. In this study, we identify components of the signalosome formed by the activated EGFR required to activate NF-B and show that, although the activated EGFR uses mechanisms similar to the TNFR, it recruits a distinct signalosome. We show the EGFR forms a complex with a TNFR-interacting protein (RIP), which plays a key role in TNFR-induced NF-B activation, but not with TRADD, an adaptor protein which serves to recruit RIP to the TNFR. Furthermore, we show that the EGFR associates with NF-B-inducing kinase (NIK) and provide evidence suggesting multiprotein complex formation between the EGFR, RIP, and NIK. Using a dominant negative NIK mutant, we show that NIK activation is required for EGFR-mediated NF-B induction. We also show that a S32/36 IB␣ mutant blocks EGFR-induced NF-B activation. Our studies also suggest that a high level of EGFR expression, a frequent occurrence in human tumors, is optimal for epidermal growth factorinduced NF-B activation. Finally, although protein kinase B/Akt has been implicated in tumor necrosis factor and PDGF-induced NF-B activation, our studies do not support a role for this protein in EGFR-induced NF-B activation.The engagement of the epidermal growth factor receptor by its cognate ligand results in the generation of a number of intracellular signals (1). The initial changes induced by ligand binding are receptor dimerization, activation of the kinase activity of the receptor, and autophosphorylation of the receptor on tyrosine residues (2, 3). Autophosphorylation of the receptor results in the creation of docking sites for a number of secondary signaling proteins bearing specific protein interaction domains such as the Src homology 2 domain, which interact specifically with phosphorylated tyrosine residues (4). As a consequence of this interaction, these secondary signaling proteins may themselves become activated and trigger a number of downstream signals. These signaling cascades result in the activation of a number of transcription factors such as AP-1 and STATS (5).The NF-B 1 family of transcription factors plays an important role in inflammatory responses (6). A diverse number of stimuli including cytokines such as TNF␣ and IL-1, UV irradiation, and lipopolysaccharide are known to activate NF-B. In unstimulated cells NF-B is sequestered in the cytoplasm by the IB family of proteins (7). Binding of IB to NF-B masks nuclear localization signals on NF-B and prevents its translocation to the nucleus (8). Stimulation of cells with a diverse array of stimuli results in phos...

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IntroductionNatural killer (NK) cells play key roles in innate and adaptive immune responses during early host defense against infectious pathogens and tumors via 2 major mechanisms: contact-dependent cytotoxicity and cytokine production for immune modulation. [1][2][3][4] Target-cell death is primarily mediated via the granule-exocytosis pathway. NK cells are armed by functional cytotoxic granules containing perforin (Prf1) and granzymes, essential effector molecules for NK-cell cytotoxicity as shown in knockout mice, 4,5 and are triggered to mediate effector activity by receptor ligation. Prf1 facilitates the delivery of granzymes into the cytosol of the target cell, and GzmB, the best-characterized granzyme, cleaves several procaspases, BID, inhibitor of caspase-activated DNase, and other intracellular substrates to initiate the classic apoptotic pathways. [6][7][8][9] Many of the studies of Prf1 and GzmB expression in NK cells have suggested the possible involvement of posttranscriptional regulation. Recently, studies using murine NK cells have shown that acquisition of murine NK-cell cytotoxicity requires the translation of a pre-existing pool of Prf1 and GzmB mRNAs. 4 Despite high basal levels of Prf1 and GzmB mRNA, little protein expression is observed under resting conditions in many types of NK cells, whereas expression of both proteins is up-regulated during activation. 4,10,11 These observations are consistent with a posttranscriptional mechanism operating to allow NK cells to be poised for but to prevent translation before activation, such as silencing by microRNAs. 12,13 microRNAs are an abundant class of endogenous small noncoding RNAs (19-22 nt) generated by sequential processing of primary miRNA transcripts by the ribonuclease Drosha in the nucleus and Dicer1 in the cytoplasm, both of which are essential enzymes in the miRNA biogenesis pathway. In mammals, mature miRNAs are integrated into an RNA-inducing silencing complex, including Argonaute 2 (Ago2), a required endonuclease in the RNA interference pathway, and they associate with 3Ј untranslated regions (UTRs) of specific target mRNAs to down-regulate gene expression by targeting mRNAs for translational suppression or mRNA degradation. [13][14][15][16][17] The involvement of miRNA in immune responses and the development of immune cells from hematopoietic stem cells have been widely investigated by manipulation of specific miRNA levels 13,18 or by disruption of molecules involved in biogenesis and activity of all miRNAs, such as Arg, 19 Drosha, 20 and Dicer. [21][22][23][24] Recently, characterization of NK cells from mice with conditional deletion of Dicer and DiGeorge syndrome critical region 8 were reported, with evidence of impairments in NK-cell activation, survival, and function during viral infection. 24 These genetic studies have suggested miRNAs play essential roles in immune cell development and function. 13,14,25 Despite evidence for a broad impact in regulation of immune function, the molecular mechanism, importance, and biologic si...

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Development of oligodendrocytes and the generation of myelin internodes within the spinal cord depends on regional signals derived from the notochord and axonally derived signals. Neuregulin 1 (NRG)-1, localized in the floor plate as well as in motor and sensory neurons, is necessary for normal oligodendrocyte development. Oligodendrocytes respond to NRGs by activating members of the erbB receptor tyrosine kinase family. Here, we show that erbB2 is not necessary for the early stages of oligodendrocyte precursor development, but is essential for proligodendroblasts to differentiate into galactosylcerebroside-positive (GalC+) oligodendrocytes. In the presence of erbB2, oligodendrocyte development is normal. In the absence of erbB2 (erbB2−/−), however, oligodendrocyte development is halted at the proligodendroblast stage with a >10-fold reduction in the number of GalC+ oligodendrocytes. ErbB2 appears to function in the transition of proligodendroblast to oligodendrocyte by transducing a terminal differentiation signal, since there is no evidence of increased oligodendrocyte death in the absence of erbB2. Furthermore, known survival signals for oligodendrocytes increase oligodendrocyte numbers in the presence of erbB2, but fail to do so in the absence of erbB2. Of the erbB2−/− oligodendrocytes that do differentiate, all fail to ensheath neurites. These data suggest that erbB2 is required for the terminal differentiation of oligodendrocytes and for development of myelin.

RhoB, a tumor suppressor, has emerged as an interesting cancer target, and extensive studies aimed at understanding its role in apoptosis have been performed. In our study, we investigated the involvement of RhoB-interacting molecules in apoptosis. To identify RhoB-interacting proteins, we performed yeast-two hybrid screening assays using RhoB as a bait and isolated TNFAIP1, a TNFa-induced protein containing the BTB/POZ domain. The interaction between RhoB and TNFAIP1 was demonstrated in vivo through coimmunoprecipitation studies and in vitro binding assays. RFP-TNFAIP1 was found to be partially colocalized with EGFP-RhoB. The partial colocalization of RhoB and TNFAIP1 in endosomes suggests that RhoB-TNFAIP1 interactions may have a functional role in apoptosis. TNFAIP1 elicited proapoptotic activity, while simultaneous expression of RhoB and TNFAIP1 resulted in a dramatic increase in apoptosis in HeLa cells. Furthermore, knockdown of RhoB using siRNA clearly rescued cells from apoptosis induced by TNFAIP1. This finding suggests that interactions between RhoB and TNFAIP1 are crucial for induction of apoptosis in HeLa cells. The observation of increased SAPK/JNK phosphorylation in apoptotic cells and the finding that a JNK inhibitor suppressed apoptosis indicates that SAPK/JNK signaling may be involved in apoptosis induced by RhoB-TNFAIP1 interactions. In conclusion, we found that RhoB interacts with TNFAIP1 to regulate apoptosis via a SAPK/ JNK-mediated signal transduction mechanism. ' 2009 UICC

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