The Sno Oncogene Antagonizes Wingless Signaling during Wing Development in Drosophila

The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-β signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

Identification of G protein-coupled receptor genes from the human genome sequence

We have identified novel G protein-coupled receptors (GPCRs) with no introns in the coding region from the human genome sequence: 322 olfactory receptors; 22 taste receptors; 128 registered GPCRs for endogenous ligands; 50 novel GPCR candidates homologous to registered GPCRs for endogenous ligands; and 59 novel GPCR candidates not homologous to registered GPCRs. The total number of GPCRs with and without introns in the human genome was estimated to be approximately 950, of which 500 are odorant or taste receptors and 450 are receptors for endogenous ligands.     

DNA-binding domain mutations in SMAD genes yield dominant-negative proteins or a neomorphic protein that can activate WG target genes in Drosophila

Mutations in SMAD tumor suppressor genes are involved in approximately 140,000 new cancers in the USA each year. At this time, how the absence of a functional SMAD protein leads to a tumor is unknown. However, clinical and biochemical studies suggest that all SMAD mutations are loss-of-function mutations. One prediction of this hypothesis is that all SMAD mutations cause tumors via a single mechanism. To test this hypothesis, we expressed five tumor-derived alleles of human SMAD genes and five mutant alleles of Drosophila SMAD genes in flies. We found that all of the DNA-binding domain mutations conferred gain-of-function activity, thereby falsifying the hypothesis. Furthermore, two types of gain-of-function mutation were identified - dominant negative and neomorphic. In numerous assays, the neomorphic allele SMAD4(100T) appears to be capable of activating the expression of WG target genes. These results imply that SMAD4(100T) may induce tumor formation by a fundamentally different mechanism from other SMAD mutations, perhaps via the ectopic expression of WNT target genes - an oncogenic mechanism associated with mutations in Adenomatous Polyposis Coli. Our results are likely to have clinical implications, because gain-of-function mutations may cause tumors when heterozygous, and the life expectancy of individuals with SMAD4(100T) is likely to be different from those with other SMAD mutations. From a larger perspective, our study shows that the genetic characterization of missense mutations, particularly in modular proteins, requires experimental verification.     

Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway

Interleukin-1 (IL-1) is a proinflammatory cytokine that recognizes a surface receptor complex and generates multiple cellular responses. IL-1 stimulation activates the mitogen-activated protein kinase kinase kinase TAK1, which in turn mediates activation of c-Jun N-terminal kinase and NF-kappaB. TAB2 has previously been shown to interact with both TAK1 and TRAF6 and promote their association, thereby triggering subsequent IL-1 signaling events. The serine/threonine kinase IL-1 receptor-associated kinase (IRAK) also plays a role in IL-1 signaling, being recruited to the IL-1 receptor complex early in the signal cascade. In this report, we investigate the role of IRAK in the activation of TAK1. Genetic analysis reveals that IRAK is required for IL-1-induced activation of TAK1. We show that IL-1 stimulation induces the rapid but transient association of IRAK, TRAF6, TAB2, and TAK1. TAB2 is recruited to this complex following translocation from the membrane to the cytosol upon IL-1 stimulation. In IRAK-deficient cells, TAB2 translocation and its association with TRAF6 are abolished. These results suggest that IRAK regulates the redistribution of TAB2 upon IL-1 stimulation and facilitates the formation of a TRAF6-TAB2-TAK1 complex. Formation of this complex is an essential step in the activation of TAK1 in the IL-1 signaling pathway.     

The photosynthetic performance of a cultivated Japanese green alga Caulerpa lentillifera in response to three different stressors,temperature, irradiance,and desiccation

The effects of temperature, irradiance, and desiccation on the photosynthesis of a cultivated Japanese green alga Caulerpa lentillifera (Caulerpaceae) were determined by a pulse amplitude modulation (PAM)-chlorophyll fluorometer and dissolved oxygen sensors. The photochemical efficiency in the photosystem II (F_v/F_m and ΔF/F_m') during the 72-h temperature exposures (8, 12, 16, 20, 24, 28, 32, 36, and 40°C) was generally stable at 16–32°C but quickly dropped at lower and higher temperatures. The photosynthesis–temperature curve at 200 μmol photons m^?2 s^?1 also revealed that the maximum gross photosynthesis (GP_max) occurred at 30.7°C (30.5–30.9, 95% highest density credible intervals). Photosynthesis–irradiance curves at 16, 24, and 32°C quickly saturated, then expressed photoinhibition, and revealed that the maximum net photosynthetic rates (NP_max) and saturation irradiance (E_k) were highest at 32°C and lowest at 16°C. Continuous 6-h exposure to irradiances of 200 (low) and 400 (high) μmol photons m^?2 s^?1 at 16, 24, and 32°C expressed greater declines in their ΔF/F_m' at 16°C, revealing chronic chilling-light stress. The response to continuous desiccation (~480 min) under 50% humidity at 24°C showed that ΔF/F_m' dropped to zero at 480-min aerial exposure, and the treatments of more than 60-min desiccation did not return to the initial level even after 24-h subsequent rehydration in seawater. Likewise, ΔF/F_m' fell when the absolute water content (AWC) of the frond dropped below AWC of 90% and mostly did not return to the initial level even after 24-h subsequent rehydration in seawater, signifying a low tolerance to desiccation.

     

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.