The NF-B 1 family of transcription factors consists of binary complexes of subunits with related promoter-binding and transactivation properties. The p65/RelA, RelB, and c-Rel subunits stimulate transcription, whereas the p50 and p52 subunits serve primarily to bind to DNA (1). The prototypical NF-B complex is the p65-p50 heterodimer (2). NF-B is sequestered in a latent form in the cytoplasm through its interaction with the inhibitory IB proteins. In response to signals, IB kinase is activated, and IB is phosphorylated and degraded, releasing NF-B, which enters the nucleus and binds to DNA (2-5). However, the phosphorylation and degradation of IB and the consequent liberation of NF-B are not sufficient to activate NF-B-dependent transcription, which also relies on a second pathway, which leads to the stimulus-induced phosphorylation of the p65/RelA, RelB, and c-Rel subunits of NF-B (6 -15).Our laboratory (13) and others (7,14) have shown that the pro-inflammatory cytokines IL-1 and TNF induce the phosphorylation and activation of the p65 subunit of NF-B, a pathway distinct from the one leading to IB degradation and NF-B nuclear translocation. Additionally, phosphatidylinositol 3Ј-kinase (PI3K) and the serine/threonine kinase AKT play critical roles in this pathway (13,16,17). Recently, an additional function for IL-1-stimulated PI3K/AKT activation has been reported: phosphorylation of the NF-B p50 subunit in response to these kinases increases the DNA-binding capacity of the NF-B complex (18).Targeted gene disruptions have demonstrated that IKK␤ (but not IKK␣) is largely responsible for cytokine-induced IB degradation and NF-B nuclear translocation (19 -24). However, IKK␣-null mouse embryo fibroblasts (MEFs) are deficient in inducing several NF-B-dependent mRNAs in response to IL-1 and TNF (21). Activated AKT interacts with IKK␣ upon cytokine stimulation and induces the phosphorylation of threonine 23 (25). These findings raise the interesting possibility that, although IKK␣ is dispensable for IB␣ degradation and NF-B nuclear translocation, it may be required in the PI3K/ AKT pathway that leads to the phosphorylation and activation of NF-B. Therefore, we have investigated the roles of the IKK ␣ and ␤ subunits in the IL-1-and TNF-mediated phosphorylation and activation of the p65 subunit of NF-B. EXPERIMENTAL PROCEDURESBiological Reagents and Cell Culture-Recombinant human IL-1␤ was from NCI, National Institutes of Health. Recombinant human TNF was from Preprotech (Rocky Hill, NJ). LY 294002 was from Sigma. Polyclonal anti-IKK␣, anti-IKK␤, anti-IKK␥, anti-p65/RelA, and anti-IB␣ antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, anti-phosphop90 RSK , anti-p90 RSK , anti-phospho-JNK1, and anti-JNK antibodies were from Cell Signaling Technologies (Beverly, MA). Protein A-Sepharose and glutathione-agarose beads were from Amersham Biosciences, Inc. (Buckinghamshire, United Kingdom). Wild-type and...

Our laboratory has delineated that the phosphatidylinositol 3 0 kinase (PI3K)/AKT/IjB kinase (IKK) pathway positively regulates NFjB and b-catenin, both important transcriptional regulators in colorectal cancer (CRC). Therefore, we investigated the effect of inhibiting the PI3K/AKT/IKKa pathway in regulating the inappropriate constitutive activation of NFjB and b-catenin in CRC cell lines. SW480 and RKO CRC cell lines demonstrate constitutive activation of AKT as well as both NFjB-and b-catenin-dependent transcription. The constitutive activation of NFjB-and b-catenin-dependent transcription is inhibited by transiently transfecting either kinase dead (KD) IKKa, which blocks IKKa kinase activity, KD AKT, which blocks AKT activity, or wildtype (WT) PTEN, which inhibits PI3K and AKT activity. The ability of KD IKKa, KD AKT or WT PTEN to decrease b-catenindependent transcription is independent of their effects on NFjB. Inducible expression of either KD IKKa or WT PTEN strongly inhibits both the constitutive NFjB-and b-catenin-dependent promoter and endogenous gene activation. Targeted array-based gene expression analysis of this inducible system reveals that many of the genes downregulated upon inhibition of this pathway are involved in tumor angiogenesis and metastasis. The activation of this pathway and the expression of the three most repressed genes was further analysed in samples of CRC. These results indicate a role of this pathway in controlling gene expression important in tumor progression and metastasis.

I B kinase (IKK), discovered as the major activator of NF-B, plays additional roles in signaling. By using mouse embryo fibroblasts (MEFs) lacking both the ␣ and ␤ subunits of IKK, we find that these proteins are required for induction of a major subset of IFN␥-stimulated genes and that this requirement is independent of NF-B activation. Furthermore, there is no defect in IFN␥-stimulated signal transducer and activator of transcription 1 (Stat1) activation or function in the IKK␣͞␤-null MEFs. Therefore, although activated Stat1 dimers are necessary for the activation of these genes in response to IFN␥, they are not sufficient. These results reveal an important additional pathway for IFN␥-stimulated gene expression in which an NF-B-independent function of IKK is required.

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 A resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.

A novel fraction of c-phycocyanin from the thermophilic cyanobacterium Thermosynechcoccus vulcanus, with an absorption maxima blue-shifted to 612 nm (PC 612 ), has been purified from allophycocyanin and crystallized. The crystals belong to the P6 3 space group with cell dimensions of 153 Å ؋ 153 Å ؋ 59 Å with a single (␣␤) monomer in the asymmetric unit, resulting in a solvent content of 65%, and diffract to 2.7 Å. The PC 612 crystal structure has been determined by molecular replacement and refined to a crystallographic R-factor of 20.9% (R free ‫؍‬ 27.8%). The crystal packing in this form shows that the PC 612 form of phycocyanin does not associate into hexamers and that its association with adjacent trimers in the unit cell is very different from that found in a previously determined structure of the normal form of T. vulcanus phycocyanin, which absorbs at 620 nm. Analysis of the PC 612 structure shows that the ␣ subunits, which typically form the interface between two trimers within a hexamer, have a high degree of flexibility, as indicated by elevated B-factors in portions of helices B, E, and G. Examination of calculated electron density omit maps shows that unlike all other structures of phycobiliproteins determined so far, the Asn␤ 72 residue is not methylated, explaining the blueshift in its absorption spectra. On the basis of the results presented here, we suggest that this new form of trimeric phycocyanin may constitute a special minor component of the phycobilisome and may form the contact between the phycocyanin rods and the allophycocyanin core.Cyanobacteria and red algae efficiently harvest light used for photo-induced electron transfer by a variety of pigment-protein complexes called phycocbilisomes (PB) 1 (reviewed in Refs. 1-5).The PB is the largest type of all photosynthetic antenna pigment-protein complexes attached to photosynthetic reaction centers. The sizes of such complexes vary between species and growth conditions and can easily reach molecular masses in excess of 2 MDa. The self-association of the protein subunits has been studied and documented for a variety of species and shows different complex forms. All pigment-binding protein species show a canonical first level quaternary structure of the association of two pigment-binding subunits termed ␣ and ␤ into the basic (␣␤) monomer. The monomers further assemble into higher organizational levels of (␣␤) 3 trimers and (␣␤) 6 hexamers. The trimers (and hexamers) are round disks with dimensions of about 110 ϫ 30 Å (or 60 Å for the hexamers). The hexamer encloses a large internal cavity with triangular shaped openings on both sides. The most prevalent PB forms, found in many cyanobacteria, are made up of a core assembly of three hexamers of allophycocyanin (APC) that are arranged with their ring planes perpendicular to the membrane surface directly above Photosystem II complexes (4). On this core are arranged six rod-like structures, made up of phycocyanin (PC) hexamers. In some species, and under certain environmental conditions, addition...

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Previously, we found that the protein kinase C (PKC) inhibitor H7 stimulates p53 to accumulate in a form incapable of inducing transcription from p53-dependent promoters. We concluded that H7 inhibits constitutive C-terminal phosphorylation of p53, which regulates its turnover in unstressed cells. We now show that p53 and its inhibitor MDM2 (HDM2 in human cells) are together in the nuclei of H7-treated cells and can be co-immunoprecipitated. Despite this association of p53 with the ubiquitin ligase MDM2, ubiquitinated p53 was not detected in H7-treated cells. Furthermore, co-treatment with H7 and the proteosome inhibitor LLnL prevented the accumulation of ubiquitinated p53 that was observed in cells treated solely with LLnL. In addition, treatment of cells with the PKC activator phorbol ester stimulated the ubiquitination of p53 and reduced its ability to accumulate after stress. H7 did not induce the phosphorylation of human p53 on Ser-15 (Ser-18 in mouse protein), a modification that occurs in response to DNA damage and leads to the release of MDM2 and to transactivation by p53. We conclude that phosphorylation of the C-terminal domain of p53 by PKC increases its ubiquitination and degradation in unstressed cells.

Anomalies in homocysteine (HCY) and folate metabolism are associated with common birth defects and adult diseases, several of which can be suppressed with dietary folate supplementation. Although supplementation reduces the occurrence and severity of neural tube defects (NTDs), many cases are resistant to these beneficial effects. The basis for variable response and biomarkers that predict responsiveness are unknown. Crooked-tail (Cd) mutant mice are an important model of folate-responsive NTDs. To identify features that are diagnostic for responsiveness versus resistance to dietary folate supplementation, we surveyed metabolite and expression levels in liver samples from folate-supplemented, folate-reduced and control diets in Cd mutant and wild-type adult females. Cd homozygotes had normal total homocysteine (tHcy) levels suggesting that folate suppresses NTDs through a mechanism that does not involve modulating serum tHcy levels. Instead, parallel changes in metabolite and expression profiles in folate-supplemented Cd/Cd homozygotes and folate-reduced+/+and Cd/+mice suggest that Crooked-tail homozygotes have a defect in the utilization of intracellular folate. Then, by combining these expression and metabolite profile results with published results for other models and their controls, two clusters were found, one of which included several folate-responsive NTD models and the other previously untested and presumably folate-resistant models. The predictive value of these profiles was verified by demonstrating that NTDs of Ski-/-mutant mice, whose profile suggested resistance to folate supplementation, were not suppressed with dietary folate supplementation. These results raise the possibility of using metabolite and expression profiles to distinguish folate-responsive and resistance adult females who are at risk for bearing fetuses with an NTD.