EMBO J, 2000 Apr 17, 19(8), 1887 - 99 Association of GCN1-GCN20 regulatory complex with the N-terminus of eIF2alpha kinase GCN2 is required for GCN2 activation; Garcia-Barrio M et al.; Stimulation of GCN4 mRNA translation due to phosphorylation of the alpha-subunit of initiation factor 2 (eIF2) by its specific kinase, GCN2, requires binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like domain in GCN2 . GCN2 function in vivo also requires GCN1 and GCN20, but it was unknown whether these latter proteins act directly to promote the stimulation of GCN2 by uncharged tRNA . We found that the GCN1-GCN20 complex physically interacts with GCN2, binding to the N-terminus of the protein . Overexpression of N-terminal GCN2 segments had a dominant-negative phenotype that correlated with their ability to interact with GCN1-GCN20 and impede association between GCN1 and native GCN2 . Consistently, this Gcn(-) phenotype was suppressed by overexpressing GCN2, GCN1-GCN20 or tRNA(His) . The requirement for GCN1 was also reduced by overexpressing tRNA(His) in a gcn1Delta strain . We conclude that binding of GCN1-GCN20 to GCN2 is required for its activation by uncharged tRNA . The homologous N-terminus of Drosophila GCN2 interacted with yeast GCN1-GCN20 and had a dominant Gcn(-) phenotype, suggesting evolutionary conservation of this interaction. EMBO J, 2000 Apr 17, 19(8), 1873 - 86 A dual role for BBP/ScSF1 in nuclear pre-mRNA retention and splicing; Rutz B et al.; The MSL5 gene, which codes for the splicing factor BBP/ScSF1, is essential in Saccharomyces cerevisiae, yet previous analyses failed to reveal a defect in assembly of (pre)-spliceosomes or in vitro splicing associated with its depletion . We generated 11 temperature-sensitive (ts) mutants and one cold-sensitive (cs) mutant in the corresponding gene and analyzed their phenotypes . While all mutants were blocked in the formation of commitment complex 2 (CC2) at non-permissive and permissive temperature, the ts mutants showed no defect in spliceosome formation and splicing in vitro . The cs mutant was defective in (pre)-spliceosome formation, but residual splicing activity could be detected . In vivo splicing of reporters carrying introns weakened by mutations in the 5' splice site and/or in the branchpoint region was affected in all mutants . Pre-mRNA leakage to the cytoplasm was strongly increased (up to 40-fold) in the mutants . A combination of ts mutants with a disruption of upf1, a gene involved in nonsense-mediated decay, resulted in a specific synthetic growth phenotype, suggesting that the essential function of SF1 in yeast could be related to the retention of pre-mRNA in the nucleus. Cell Growth Differ, 2000 Apr, 11(4), 211 - 9 Binding of 14-3-3beta to the carboxyl terminus of Wee1 increases Wee1 stability, kinase activity, and G2-M cell population; Wang Y et al.; Wee1 protein kinase plays an important regulatory role in cell cycle progression . It inhibits Cdc-2 activity by phosphorylating Tyr15 and arrests cells at G2-M phase . In an attempt to understand Wee1 regulation during cell cycle, yeast two-hybrid screening was used to identify Wee1-binding protein(s) . Five of the eight positive clones identified encode 14-3-3beta . In vivo binding assay in 293 cells showed that both full-length and NH2-terminal truncated Wee1 bind with 14-3-3beta . The 14-3-3beta binding site was mapped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646) . Binding with 14-3-3beta increases the protein level of full-length Wee1 but not of the truncated Wee1 . Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3beta . Increased Wee1 protein level/enzymatic activity is accountable, at least in part, to an increased Wee1 protein half-life when coexpressed with 14-3-3beta . The protein half-life of the NH2-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3beta cotransfection . Biologically, 14-3-3beta/Wee1 coexpression increases the cell population at G2-M phase . Thus, Wee1 binding with 14-3-3beta increases its biochemical activity as well as its biological function . The finding reveals a novel mechanism by which 14-3-3 regulates G2-M arrest and suggests that the NH2-terminal domain of Wee1 contains a negative regulatory sequence that determines Wee1 stability. Cell Mol Biol (Noisy-le-grand), 2000 Mar, 46(2), 407 - 17 Antioxidant role of metallothioneins: a comparative overview; Viarengo A et al.; Metallothioneins (MTs) are sulfhydryl-rich proteins binding essential and non-essential heavy metals . MTs display in vitro oxyradical scavenging capacity, suggesting that they may specifically neutralize hydroxyl radicals . Yet, this is probably an oversimplified view, as MTs represent a superfamily of widely differentiated metalloproteins . MT antioxidant properties mainly derive from sulfhydryl nucleophilicity, but also from metal complexation . Binding of transition metals displaying Fenton reactivity (Fe,Cu) can reduce oxidative stress, whereas their release exacerbates it . In vertebrates, MT gene promoters contain metal (MRE) and glucocorticoid response elements (GRE), Sp and AP sequences, but also antioxidant response elements (ARE) . MT neosynthesis is induced by heavy metals, cytokines, hormones, but also by different oxidants and prooxidants . Accordingly, MT overexpression increases the resistance of tissues and cells to oxidative stress . As for invertebrates, data from the mussel show that MT can actually protect against oxidative stress, but is poorly inducible by oxidants . In yeast, there is a Cu(I)-MT that in contrast to mammalCu-MT exhibits antioxidant activity, possibly due to differences in metal binding domains . Finally, as the relevance of redox processes in cell signaling is becoming more and more evident, a search for MT effects on redox signaling could represent a turning point in the understanding of the functional role of these protein. Cell Mol Biol (Noisy-le-grand), 2000 Mar, 46(2), 367 - 81 Metals and cellular signaling in mammalian cells; DeMoor JM et al.; A number of heavy metals are known to be essential for life, but most of these can also be toxic to cells under certain circumstances, or at elevated levels . Metals can directly induce gene expression through the actions of metal-responsive transcription factors . However, metals can also influence the response to non-metal extracellular signals . Cells respond to extracellular signals through a variety of different, but often interacting, signal transduction pathways . Metals can alter cell behaviour by interacting with transcription factors and transduction molecules, many of which are dependent on metals (primarily zinc) for their action . In addition, metals can affect cells in more nonspecific ways, for example, by inducing a generalized stress response or by cross-linking cell surface thiol groups . The prominent role of zinc in signal transduction combined with low intracellular free zinc levels has lead to the speculation that cellular signaling and gene expression may be regulated, in part, by zinc bioavailability . Experimental modification of the levels of the intracellular metal-binding protein, metallothionein (MT), results in altered responsiveness to extracellular signals . This observation suggests that MT is capable of influencing gene expression, perhaps by regulating the level of intracellular free zinc. Cell Mol Biol (Noisy-le-grand), 2000 Mar, 46(2), 357 - 65 Nuclear trafficking of metallothionein: possible mechanisms and current knowledge; Ogra Y et al.; Although metallothionein (MT) was first characterized as a cytoplasmic protein, it is now known to be localized in the nucleus depending on various cellular events, such as cell proliferation . The suggested roles of karyophilic MT are: to 1) regulate the biological pool of the essential metals zinc (Zn) and copper (Cu), and especially to supply Zn to Zn-requiring enzymes/transcription factors through activated cell proliferation, and 2) to protect DNA from oxidative stress including those caused by antitumor agents . Translocation of MT to the nucleus might be mediated, depending on cellular events, by a structural change in MT itself or through the appearance of nuclear binding proteins . Supporting the former possibility, MT is known to have some structural features, namely, highly conserved lysyl residues, which are anticipated to act as nuclear localization signal (NLS) . In addition, concomitant appearance of non-acetylated MT, without post-translational acetylation, and nuclear localization of MT, have been reported . Supporting the latter possibility, MT-partner proteins might participate in the nuclear trafficking of MT (i.e., an MT-nuclear translocator or a nuclear chaperone of MT) . We now provide an overview of the current knowledge on both mechanisms. Cytogenet Cell Genet, 2000, 88(1-2), 119 - 23 Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1; Quan X et al.; The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development . To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1) . To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically . The radiation map generated in region 2q1 extends its centromeric end of about 150 cR . J Pharmacol Exp Ther, 2000 May, 293(2), 444 - 52 Pancreatic beta-cell K(ATP) channel activity and membrane-binding studies with nateglinide: A comparison with sulfonylureas and repaglinide; Hu S et al.; Nateglinide (A-4166) is an amino acid derivative with insulinotrophic action in clinical development for treatment of type 2 diabetes . The aim of this study was to determine whether nateglinide's interaction at the K(ATP) channel/sulfonylurea receptor underlies its more rapid onset and shorter duration of action in animal models . Binding studies were carried out with membranes prepared from RIN-m5F cells and HEK-293 cells expressing recombinant human sulfonylurea receptor 1 (SUR1) . The relative order for displacement of {(3)H}glibenclamide in competitive binding experiments with RIN-m5F cell membranes was glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-nateglinide > tolbutamide . The results with HEK-293/recombinant human SUR1 cells were similar with the exception that glipizide was more potent than repaglinide . Neither nateglinide nor repaglinide had any effect on the dissociation kinetics for {(3)H}glibenclamide, consistent with both compounds competitively binding to the glibenclamide-binding site on SUR1 . Finally, the inability to measure {(3)H}nateglinide binding suggests that nateglinide dissociates rapidly from SUR1 . Direct interaction of nateglinide with K(ATP) channels in rat pancreatic beta-cells was investigated with the patch-clamp method . The relative potency for inhibition of the K(ATP) channel was repaglinide > glibenclamide > nateglinide . Kinetics of the inhibitory effect on K(ATP) current showed that the onset of inhibition by nateglinide was comparable to glibenclamide but more rapid than that of repaglinide . The time for reversal of channel inhibition by nateglinide was also faster than with glibenclamide and repaglinide . These results suggest that the unique characteristics of nateglinide are largely the result of its interaction at the K(ATP) channel. J Mol Biol, 2000 May 5, 298(3), 521 - 37 Homology-based method for identification of protein repeats using statistical significance estimates; Andrade MA et al.; Short protein repeats, frequently with a length between 20 and 40 residues, represent a significant fraction of known proteins . Many repeats appear to possess high amino acid substitution rates and thus recognition of repeat homologues is highly problematic . Even if the presence of a certain repeat family is known, the exact locations and the number of repetitive units often cannot be determined using current methods . We have devised an iterative algorithm based on optimal and sub-optimal score distributions from profile analysis that estimates the significance of all repeats that are detected in a single sequence . This procedure allows the identification of homologues at alignment scores lower than the highest optimal alignment score for non-homologous sequences . The method has been used to investigate the occurrence of eleven families of repeats in Saccharomyces cerevisiae, Caenorhabditis elegans and Homo sapiens accounting for 1055, 2205 and 2320 repeats, respectively . For these examples, the method is both more sensitive and more selective than conventional homology search procedures . The method allowed the detection in the SwissProt database of more than 2000 previously unrecognised repeats belonging to the 11 families . In addition, the method was used to merge several repeat families that previously were supposed to be distinct, indicating common phylogenetic origins for these families . Arch Biochem Biophys, 2000 Apr 15, 376(2), 439 - 48 N-(cyclohexanecarboxyl)-O-phospho-l-serine, a minimal substrate for the dual-specificity protein phosphatase IphP; Savle PS et al.; Three dual-specific phosphatases {DSPs}, IphP, VHR, and Cdc14, and three protein-tyrosine phosphatases {PTPs}, PTP-1B, PTP-H1, and Tc-PTPa, were challenged with a set of low molecular weight phosphoesters to probe the factors underlying the distinct substrate specificities displayed by these two mechanistically homologous families of protein phosphatases . It was observed that beta-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs . While DSPs tended to hydrolyze alpha-naphthyl phosphate at rates comparable to that of the beta-isomer, the PTPs PTP-1B and Tc-PTPa did not . PTP-H1, however, displayed high alpha-naphthyl phosphatase activity . Intriguingly, PTP-H1 also displayed much higher protein-serine phosphatase activity in vitro, 0.2-0.3% that toward equivalent tyrosine phosphorylated proteins, than did PTP-1B or Tc-PTPa . The latter two PTPs discriminated between the serine- and tyrosine-phosphorylated forms of two test proteins by factors of >/=10(4)-10(6) . While free phosphoserine represented an extremely poor substrate for all of the DSPs examined, the addition of a hydrophobic "handle" to form N-(cyclohexanecarboxyl)-O-phospho-l-serine produced a compound that was hydrolyzed by IphP with high efficiency, i.e., at a rate comparable to that of free phosphotyrosine or p-nitrophenyl phosphate . VHR also hydrolyzed N-(cyclohexanecarboxyl)-O-phospho-l-serine (1 mM) at a rate approximately one-tenth that of beta-naphthyl phosphate . None of the PTPs tested exhibited significant activity against this compound . However, N-(cyclohexanecarboxyl)-O-phospho-l-serine did not prove to be a universal substrate for DSPs as Cdc14 displayed little propensity to hydrolyze it . Nucleic Acids Res . 2000 May 15;28(10):E48. In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes; Brugere JF et al.; A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and {alpha-(32)P}dCTP incorporation mediated by the Klenow fragment . Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resulting from a single restriction of the whole genome and (ii) the DDIC (double digestion of isolated chromosome)-PFGE which is the eukaryotic counterpart of complete/complete 2D-PFGE in bacterial genomics. Cancer Gene Ther, 2000 Feb, 7(2), 339 - 46 Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts; Marangoni E et al.; Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation . The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line . Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA . The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics . After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population . The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control . Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours . This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells . This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy. Mol Endocrinol, 2000 Apr, 14(4), 545 - 53 Activation of estrogen receptor-alpha by the heavy metal cadmium; Stoica A et al.; Previous studies from this laboratory have shown that the heavy metal cadmium (Cd) mimics the effects of estradiol in estrogen-responsive breast cancer cell lines . To understand the mechanism by which cadmium activates estrogen receptor-alpha (ER-alpha), the ability of cadmium to bind to and activate wild-type and various mutants of ER-alpha was examined . When tested in transient cotransfection assays in COS-1 cells, cadmium concentrations as low as 10(-11) M activated ER-alpha . Scatchard analysis employing either purified human recombinant ER-alpha or extracts from ER-containing MCF-7 cells demonstrated that l09Cd binds to the ER with an equilibrium dissociation constant of approximately 4 to 5 x 10(-10) M . Cadmium also blocks the binding of estradiol to ER-alpha in a noncompetitive manner (K(i) = 2.96 x 10(-10) M), suggesting that the heavy metal interacts with the hormone-binding domain of the receptor . To study the role of the hormone-binding domain in cadmium activation, COS-1 cells were transiently cotransfected with GAL-ER, a chimeric receptor containing the DNA-binding domain of the transcription factor GAL4 and the hormone-binding domain of ER-alpha, and a GAL4-responsive reporter gene . Treatment of the transfected cells with either 10(-6) M cadmium or 10(-9) M estradiol resulted in a 4-fold increase in reporter gene activity . The effect of cadmium on the chimeric receptor was blocked by the antiestrogen, ICI-164,384, suggesting that cadmium activates ER-alpha through an interaction with the hormone-binding domain of the receptor . Transfection and binding assays with ER-alpha mutants identified C381, C447, E523, H524, and D538 as possible interaction sites of cadmium with the hormone-binding domain of ER-alpha. J Cell Sci, 2000 May, 113 ( Pt 10), 1747 - 58 Dictyostelium DdCP224 is a microtubule-associated protein and a permanent centrosomal resident involved in centrosome duplication; Graf R et al.; A cDNA encoding a 224-kDa Dictyostelium discoideum centrosomal protein (DdCP224) was isolated by immunoscreening . DdCP224 was detected at the centrosome and, more weakly, along microtubules throughout the entire cell cycle . Centrosomal localization does not require microtubules, suggesting that DdCP224 is a genuine centrosomal component . DdCP224 exhibits sequence identity to a weakly conserved class of microtubule-associated proteins including human TOGp and yeast Stu2p . Stu2p has a size of only approximately 100 kDa and corresponds to the N-terminal half of DdCP224 . The functions of the N- and C-terminal halves of DdCP224 were investigated in the corresponding GFP-fusion mutants . Surprisingly, the N-terminal construct showed only cytosolic localization, whereas the C-terminal construct localized exclusively to the centrosome . This is unexpected because Stu2p is localized at the spindle pole body . Full-length DdCP224-GFP was present both at centrosomes and along microtubules . Furthermore, it bound to microtubules in vitro, unlike the two truncated mutants . Thus centrosome binding is determined by the C-terminal half and microtubule binding may require the interaction of the N- and C-terminal halves . Interestingly, cells expressing full-length DdCP224-GFP exhibit supernumerary centrosomes and show a cytokinesis defect, suggesting that DdCP224 plays an important role in centrosome duplication . These features are unique among the known centrosomal proteins. J Cell Sci, 2000 May, 113 ( Pt 10), 1687 - 94 Association of human ubiquitin-conjugating enzyme CDC34 with the mitotic spindle in anaphase; Reymond F et al.; Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function . Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression . We find that CDC34 protein is constitutively expressed during all stages of the cell cycle . Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm . The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments . Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34's cell cycle function in Saccharomyces cerevisiae . Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with (beta)-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis . Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells. Biochem J, 2000 May 1, 347 Pt 3, 749 - 55 Involvement of NRAMP1 from Arabidopsis thaliana in iron transport; Curie C et al.; Nramp genes code for a widely distributed class of proteins involved in a variety of processes, ranging from the control of susceptibility to bacterial infection in mammalian cells and taste behaviour in Drosophila to manganese uptake in yeast . Some of the NRAMP proteins in mammals and in yeast are capable of transporting metal ions, including iron . In plants, iron transport was shown to require a reduction/Fe(II) transport system . In Arabidopsis thaliana this process involves the IRT1 and Fro2 genes . Here we report the sequence of five NRAMP proteins from A . thaliana . Sequence comparison suggests that there are two classes of NRAMP proteins in plants: A . thaliana (At) NRAMP1 and Oriza sativa (Os) NRAMP1 and 3 (two rice isologues) represent one class, and AtNRAMP2-5 and OsNRAMP2 the other . AtNramp1 and OsNramp1 are able to complement the fet3fet4 yeast mutant defective both in low- and high-affinity iron transports, whereas AtNramp2 and OsNramp2 fail to do so . In addition, AtNramp1 transcript, but not AtNramp2 transcript, accumulates in response to iron deficiency in roots but not in leaves . Finally, overexpression of AtNramp1 in transgenic A . thaliana plants leads to an increase in plant resistance to toxic iron concentration . Taken together, these results demonstrate that AtNramp1 participates in the control of iron homoeostasis in plants. Biochem J, 2000 May 1, 347 Pt 3, 703 - 9 Conserved sequences in the beta subunit of archaeal and eukaryal translation initiation factor 2 (eIF2), absent from eIF5, mediate interaction with eIF2gamma; Thompson GM et al.; The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode . This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome . Here we provide a comparative sequence analysis of the beta subunit of eIF2 and its archaeal counterpart (aIF2beta) . aIF2beta differs from eIF2beta in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B . The remaining sequences are highly conserved, and are shared with eIF5 . Previously isolated mutations in the yeast eIF2beta, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2beta, but not in eIF5 . We show that the sequence of eIF2beta homologous to aIF2beta is sufficient for binding eIF2gamma, the only subunit with which it interacts, and comprises, at the most, 78 residues . eIF5 does not interact with eIF2gamma, despite its similarity with eIF2beta, probably because of a gap in homology in this region . These observations have implications for the evolution of the mechanism of translation initiation. J Cell Biol, 2000 Apr 17, 149(2), 423 - 30 Suppression of Pyk2 kinase and cellular activities by FIP200; Ueda H et al.; Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways . We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase-interacting protein of 200 kD) by using a yeast two-hybrid screen . In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK . However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm . FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays . FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases . Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2 . Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200-Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells . Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain. J Cell Biol, 2000 Apr 17, 149(2), 249 - 54 The transcriptional coactivator CBP interacts with beta-catenin to activate gene expression; Takemaru KI et al.; Beta-catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors . Although it has been suggested that the COOH-terminal region of beta-catenin functions as an activation domain, the mechanisms of activation remain unclear . To screen for potential transcriptional coactivators that bind to the COOH-terminal region of beta-catenin, we used a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane . Using this system, we isolated the CREB-binding protein (CBP) . Armadillo (Arm) repeat 10 to the COOH terminus of beta-catenin is involved in binding to CBP, whereas beta-catenin interacts directly with the CREB-binding domain of CBP . Beta-catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo . Conversely, beta-catenin-dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP . The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A . These findings suggest that CBP provides a link between beta-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of beta-catenin. FEBS Lett, 2000 Apr 14, 471(2-3), 141 - 6 FHA domain boundaries of the dun1p and rad53p cell cycle checkpoint kinases; Hammet A et al.; Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains . Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues) . In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks . This indicates that the non-catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase. FEBS Lett, 2000 Apr 14, 471(2-3), 133 - 6 Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana; Kohler C et al.; The recently identified cyclic nucleotide-gated ion channels (AtCNGCs) from Arabidopsis thaliana have the ability to bind calmodulin . Using two different methods, we mapped the binding site of AtCNGC1 to the last predicted alpha helix of the cyclic nucleotide binding domain . This is in contrast to CNGCs from animals, where the calmodulin binding site is located in the N-terminus, implying that different mechanisms for CNGC modulation have evolved in animals and plants . Furthermore, we demonstrate that AtCNGC1 and AtCNGC2 have different calmodulin binding affinities and we provide evidence for target specificities among calmodulin isoforms. Hum Mol Genet, 2000 May 1, 9(8), 1245 - 9 A mutation in the human heme A:farnesyltransferase gene (COX10 ) causes cytochrome c oxidase deficiency; Valnot I et al.; Cytochrome c oxidase (COX) defects are found in a clinically and genetically heterogeneous group of mitochondrial disorders . To date, mutations in only two nuclear genes causing COX deficiency have been described . We report here a genetic linkage study of a consanguineous family with an isolated COX defect and subsequent identification of a mutation in a third nuclear gene causing a deficiency of the enzyme . A genome-wide search for homozygosity allowed us to map the disease gene to chromosome 17p13.1-q11.1 (Z (max)= 2.46; theta = 0.00 at the locus D17S799) . This region encompasses two genes, SCO1 and COX10, encoding proteins involved in COX assembly . Mutation analysis followed by a complementation study in yeast permitted us to ascribe the COX deficiency to a homozygous missense mutation in the COX10 gene . This gene encodes heme A:farnesyltransferase, which catalyzes the first step in the conversion of protoheme to the heme A prosthetic groups of the enzyme . All three nuclear genes now linked to isolated COX deficiency are involved in the maturation and assembly of COX, emphasizing the major role of such genes in COX pathology. Hum Mol Genet, 2000 May 1, 9(8), 1195 - 200 Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis of Refsum's disease; Jansen GA et al.; Refsum's disease (RD) is an inherited neurological syndrome biochemically characterized by the accumulation of phytanic acid in plasma and tissues . Patients with RD are unable to degrade phytanic acid due to a deficient activity of phytanoyl-CoA hydroxyl-ase (PhyH), a peroxisomal enzyme catalysing the first step of phytanic acid alpha-oxidation . To enable mutation analysis of RD at the genome level, we have elucidated the genomic organization of the PHYH gene . The gene is approximately 21 kb and contains nine exons and eight introns . Mutation analysis of PHYH cDNA from 22 patients with RD revealed 14 different missense mutations, a 3 bp insertion, and a 1 bp deletion, which were all confirmed at the genome level . A 111 bp deletion identified in the PHYH cDNA of several patients with RD was due to either one of two different mutations in the same splice acceptor site, which result in skipping of exon 3 . Six mutations, including a large in-frame deletion and five missense mutations, were expressed in the yeast Saccharomyces cerevisiae to study their effect on PhyH activity . The results showed that all these mutations lead to an enzymatically inactive PhyH protein. Nature, 2000 Apr 6, 404(6778), 622 - 5 XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis; Maiorano D et al.; In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins . Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref . 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases . The functional assembly of MCM proteins into chromatin corresponds to replication licensing . Removal of these proteins from chromatin in S phase is crucial in origins firing regulation . We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus . This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates . Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication . XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis . Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin . These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase. Glycobiology, 2000 Apr, 10(4), 403 - 12 Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme; Tessier DC et al.; The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransferase (UGGT) has the unique property of recognizing incompletely folded glycoproteins and, if they carry an N -linked Man(9)GlcNAc(2)oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose . Using peptide sequence information, we have isolated the complete cDNA of rat liver UGGT and expressed it in insect cells . The cDNA specifies an open reading frame which codes for a protein of 1527 residues including an 18 amino acid signal peptide . The protein has a C-terminal tetrapeptide (HEEL) characteristic of endoplasmic reticulum luminal proteins . The purified recombinant enzyme shows the same preference for unfolded polypeptides with N -linked Man(9)GlcNAc(2)glycans as the enzyme purified from rat liver . A genetically engineered Saccharomyces cerevisiae strain capable of producing glyco-proteins with Man(9)GlcNAc(2)core oligosaccharides was constructed and secreted acid phosphatase (G0-AcP) was purified . G0-AcP was used as an acceptor glycoprotein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin . Recombinant rat UGGT has a K (m) of 44 microM for UDP-glucose . A proteolytic fragment of UGGT was found to retain enzymatic activity thus localizing the catalytic site of the enzyme to the C-terminal 37 kDa of the protein . Using site-directed mutagenesis and photoaffinity labeling, we have identified residues D1334, D1336, Q1429, and N1433 to be necessary for the catalytic activity of the enzyme. J Biol Chem, 2000 Jun 23, 275(25), 18767 - 76 Stabilization of MyoD by direct binding to p57(Kip2); Reynaud EG et al.; Recent data have demonstrated the role of Cdk1- and Cdk2-dependent phosphorylation of MyoD(Ser200) in the regulation of MyoD activity and protein turnover . In the present study, we show that in presence of p57(Kip2), MyoD(Ala200), a MyoD mutant that cannot be phosphorylated by cyclin-Cdk complexes, displayed activity 2-5-fold higher than of MyoD(Ala200) alone in transactivation of muscle-specific genes myosin heavy chain, creatine kinase, and myosin light chain 1 . Furthermore, p57(Kip2) increases the levels of MyoD(Ala200) in cotransfected cells . This result implies that p57(Kip2) may regulate MyoD through a process distinct from its function as a cyclin-dependent kinase inhibitors . We report that overexpression of p57(Kip2) increased the half-life of MyoD(Ala200) . This increased half-life of MyoD involves a physical interaction between MyoD and p57(Kip2) but not with p16(Ink4a), as shown by cross-immunoprecipitation not only on overexpressed proteins from transfected cells, but also on endogenous MyoD and p57(Kip2) from C2C12 myogenic cells . Mutational and functional analyses of the two proteins show that the NH(2) domain of p57(Kip2) associates with basic region in the basic helix-loop-helix domain of MyoD . Competition/association assays and site-directed mutagenesis of the NH(2) terminus of p57(Kip2) identified the intermediate alpha-helix domain, located between the Cdk and the cyclin binding sites, as essential for MyoD interaction . These data show that the alpha-helix domain of p57(Kip2), which is conserved in the Cip/Kip proteins, is implicated in protein-protein interaction and confers a specific regulatory mechanism, outside of their Cdk-inhibitory activity, by which the p57(Kip2) family members positively act on myogenic differentiation. J Biol Chem, 2000 Jun 23, 275(25), 19288 - 96 Structure-function analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p; Girrbach V et al.; Protein O-mannosylation is an essential protein modification . It is initiated at the endoplasmic reticulum by a family of dolichyl phosphate-mannose:protein O-mannosyltransferases (Pmts), which is evolutionarily conserved from yeast to humans . Saccharomyces cerevisiae Pmt1p is an integral membrane protein of the endoplasmic reticulum . ScPmt1p forms a complex with ScPmt2p that is required for maximum transferase activity . Recently, we proposed a seven-transmembrane structural model for ScPmt1p . A large, hydrophilic, endoplasmic reticulum-oriented segment is flanked by five amino-terminal and two carboxyl-terminal membrane-spanning domains . Based on this model, a structure-function analysis of ScPmt1p was performed . Deletion mutagenesis identified the N-terminal third of the transferase as being essential for the formation of a functional ScPmt1p-ScPmt2p complex . Deletion of the central hydrophilic loop eliminates mannosyltransferase activity, but not ScPmt1p-ScPmt2p interactions . Alignment of all fully characterized PMT family members revealed that this central loop region contains three highly conserved peptide motifs, which can be considered as signatures of the PMT family . In addition, a number of invariant amino acid residues were identified throughout the entire protein sequence . In order to evaluate the functional significance of these conserved residues site-directed mutagenesis was performed . We show that several amino acid substitutions in the conserved motifs significantly reduce ScPmt1p activity . Further, the invariant residues Arg-64, Glu-78, Arg-138, and Leu-408 are essential for ScPmt1p function . In particular, Arg-138 is crucial for ScPmt1p-ScPmt2p complex formation. J Biol Chem, 2000 Jun 16, 275(24), 18070 - 8 Glc7p protein phosphatase inhibits expression of glutamine-fructose-6-phosphate transaminase from GFA1; Zheng J et al.; Inhibitor-1 (I-1) is a specific inhibitor of protein phosphatase-1 (PP1) . We assayed the ability of I-1 to inhibit Saccharomyces cerevisiae PP1, Glc7p, in vivo . Glc7p like other PP1 catalytic subunits associates with a variety of noncatalytic subunits, and Glc7p holoenzymes perform distinct physiological roles . Our results show that I-1 inhibits Glc7p holoenzymes that regulate transcription and mitosis, but holoenzymes responsible for meiosis and glycogen metabolism were unaffected . Additionally, we exploited a genetic screen for mutants that were dependent on I-1 to grow . This scheme can identify processes that are negatively regulated by Glc7p-catalyzed dephosphorylation . In this paper I-1-dependent gfa1 mutations were analyzed in detail . GFA1 encodes glutamine-fructose-6-phosphate transaminase . One or more phosphorylated proteins activate GFA1 transcription because the pheromone response and Pkc1p/mitogen-activated protein kinase pathways positively regulate GFA1 transcription . Our findings show that an I-1-sensitive Glc7p holoenzyme reduces GFA1 transcription . Therefore, GFA1 is a member of a growing list of genes that are negatively regulated by Glc7p dephosphorylation. J Biol Chem, 2000 Jun 9, 275(23), 17229 - 32 Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis; Chung N et al.; Sphingolipids are essential eukaryotic membrane lipids that are structurally and metabolically conserved through evolution . Sphingolipids have also been proposed to regulate eukaryotic stress responses as novel second messengers . Here we show that, in Saccharomyces cerevisiae, phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways . Inactivation of serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress . Addition of phytosphingosine bypasses the requirement for serine palmitoyltransferase and restores proteolysis . Phytosphingosine-induced proteolysis requires multiubiquitin chain formation through the stress-responsive lysine 63 residue of ubiquitin . We propose that heat stress increases phytosphingosine and activates ubiquitin-dependent proteolysis. J Mol Biol, 2000 Apr 14, 297(5), 1171 - 82 Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase; Modis Y et al.; Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA . This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules . High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction . In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket . A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding . The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction . The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction . The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom . Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface . This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks . Nature, 2000 Mar 30, 404(6777), 510 - 4 DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation; Difilippantonio MJ et al.; Cancer susceptibility genes have been classified into two groups: gatekeepers and caretakers . Gatekeepers are genes that control cell proliferation and death, whereas caretakers are DNA repair genes whose inactivation leads to genetic instability . Abrogation of both caretaker and gatekeeper function markedly increases cancer susceptibility . Although the importance of Ku80 in DNA double-strand break repair is well established, neither Ku80 nor other components of the non-homologous end-joining pathway are known to have a caretaker role in maintaining genomic stability . Here we show that mouse cells deficient for Ku80 display a marked increase in chromosomal aberrations, including breakage, translocations and aneuploidy . Despite the observed chromosome instabilities, Ku80-/- mice have only a slightly earlier onset of cancer . Loss of p53 synergizes with Ku80 to promote tumorigenesis such that all Ku80-/- p53-/- mice succumb to disseminated pro-B-cell lymphoma before three months of age . Tumours result from a specific set of chromosomal translocations and gene amplifications involving IgH and c-Myc, reminiscent of Burkitt's lymphoma . We conclude that Ku80 is a caretaker gene that maintains the integrity of the genome by a mechanism involving the suppression of chromosomal rearrangements. J Cell Biochem, 2000 Apr, 77(3), 345 - 60 High levels of intracellular polyamines promote histone acetyltransferase activity resulting in chromatin hyperacetylation; Hobbs CA et al.; Polyamines stimulate expression of a variety of genes, including many implicated in cell proliferation . Indeed, aberrant expression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, plays a causal role in tumorigenesis . Gene activity is influenced by dynamic changes in acetylation of nucleosomal histones . Although polyamines influence the histone acetyltransferase and deacetylase activities in cell-free systems, their ability to modulate these enzymes in live cells has never been established . To examine the effects of elevated intracellular levels of ODC and polyamines on gene transcription and histone acetylation, cells were infected with a retrovirus containing a cDNA for ODC . ODC overexpression potentiated the stimulatory effects of histone deacetylase inhibitors on reporter gene expression beyond that promoted by ODC or inhibitor treatment alone . Indeed, elevated intracellular levels of ODC promoted hyperacetylation of histones in several epidermal and fibroblast cell types . The ODC-mediated increase in acetylated histones was abrogated when cells were treated with alpha-difluoromethylornithine, a specific inhibitor of ODC activity, implying a distinct role for polyamines . Specifically, polyamines were found to enhance the action of histone acetyltransferases either directly or indirectly . Our studies document effects of elevated intracellular polyamine levels on histone acetylation in proliferating cells, suggesting a mechanism by which altered polyamine biosynthesis contributes to aberrant expression of genes, facilitating tumor growth . In addition, these studies may have implications for the development of drugs designed to regulate enzymes that modify the acetylation status of histones . Int J Cancer, 2000 May 1, 86(3), 318 - 24 Mutation analysis of replicative genes encoding the large subunits of DNA polymerase alpha and replication factors A and C in human sporadic colorectal cancers; Popanda O et al.; We examined cDNAs of the catalytic subunit of DNA polymerase alpha (185 kDa), the 70 kDa subunit of replication protein A (single-stranded DNA-binding protein) and the 140 kDa subunit of replication factor C for mutations . Surgical specimens from 12 patients with sporadic colon cancer and normal mucosae from the same patients were investigated . In addition, we analyzed 3 human colon cancer cell lines that exhibited defects in mismatch repair (DLD-1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29, SW480 and SW620) . For detection of mutations, we used reverse transcription of mRNA, amplification of cDNAs by PCR, analysis of single-strand conformation polymorphism and DNA sequencing . Eleven colon cancers and 6 colon cancer cell lines were analyzed for DNA polymerase alpha . Only 2 silent point mutations were detected, in 1 colon carcinoma and in cell line HCT116 . Two sequence alterations of the 70 kDa subunit of replication factor A were identified in 15 specimens (9 colon carcinomas and 6 cell lines) . Colon carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in codon 351, which caused a Thr-->Ala exchange . In carcinomas CC5MA and CC8MA, a TCC-->TCT (Ser-->Ser) transition in codon 352 was observed . The deviations in codons 351 and 352 occurred in both cancer tissues and normal mucosae, suggesting a genetic polymorphism . No mutation was found in the 140 kDa subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines) . Point mutations were identified in the p53 tumor-suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens . We did not find tumor-associated DNA sequence alterations that resulted in amino acid changes in the DNA replication genes analyzed . We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication proteins . Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 350 - 4 Alternative splicing of murine SorCS leads to two forms of the receptor that differ completely in their cytoplasmic tails; Hermey G et al.; We report the identification of a splice variant of SorCS, a member of the family of VPS10 domain containing receptors . These type I transmembrane proteins share the presence of internalization signals in their cytoplasmic tail as one common characteristic . We show that an alternatively spliced transcript of SorCS is generated by differential processing of a composite internal/terminal exon . This splice variant encodes a protein with an N-terminal VPS10 domain followed by a leucine-rich module and a transmembrane domain identical with the already described SorCS protein, but a divergent cytoplasmic tail . In contrast to the known intracellular regions of the related receptors, this splice variant contains no internalization or sorting signals. Biochim Biophys Acta, 2000 Apr 12, 1484(2-3), 287 - 97 Genetic evidence for a multi-subunit complex in the O-methyltransferase steps of coenzyme Q biosynthesis; Hsu AY et al.; Coq3 O-methyltransferase carries out both O-methylation steps in coenzyme Q (ubiquinone) biosynthesis . The degree to which Coq3 O-methyltransferase activity and expression are dependent on the other seven COQ gene products has been investigated . A panel of yeast mutant strains harboring null mutations in each of the genes required for coenzyme Q biosynthesis (COQ1-COQ8) have been prepared . Mitochondria have been isolated from each member of the yeast coq mutant collection, from the wild-type parental strains and from respiratory deficient mutants harboring deletions in ATP2 or COR1 genes . These latter strains constitute Q-replete, respiratory deficient controls . Each of these mitochondrial preparations has been analyzed for COQ3-encoded O-methyltransferase activity and steady state levels of Coq3 polypeptide . The findings indicate that the presence of the other COQ gene products is required to observe normal levels of O-methyltransferase activity and the Coq3 polypeptide . However, COQ3 steady state RNA levels are not decreased in any of the coq mutants, relative to either wild-type or respiratory deficient control strains, suggesting either a decreased rate of translation or a decreased stability of the Coq3 polypeptide . These data are consistent with the involvement of the Coq polypeptides (or the Q-intermediates formed by the Coq polypeptides) in a multi-subunit complex . It is our hypothesis that a deficiency in any one of the COQ gene products results in a defective complex in which the Coq3 polypeptide is rendered unstable. Biochim Biophys Acta, 2000 Apr 12, 1484(2-3), 93 - 106 Recent advances in the study of prenylated proteins; Sinensky M; Post-translational modification of proteins with isoprenoids was first recognized as a general phenomenon in 1984 . In recent years, our understanding, including mechanistic studies, of the enzymatic reactions associated with these modifications and their physiological functions has increased dramatically . Of particular functional interest is the role of prenylation in facilitating protein-protein interactions and membrane-associated protein trafficking . The loss of proper localization of Ras proteins when their farnesylation is inhibited has also permitted a new target for anti-malignancy pharmaceuticals . Recent advances in the enzymology and function of protein prenylation are reviewed in this article. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3919 - 24 Glucocorticoids repress NF-kappaB-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell; De Bosscher K et al.; Glucocorticoids (GCs) are used to combat inflammatory diseases . Their beneficial effect relies mainly on the inhibition of NF-kappaB- and/or AP-1-driven proinflammatory gene expression . Previously, we have shown that GCs repress tumor necrosis factor-induced IL-6 gene expression by an NF-kappaB-dependent nuclear mechanism without changing the DNA-binding capacity of NF-kappaB or the expression levels of the cytoplasmic inhibitor of NF-kappaB (IkappaB-alpha) . In the present work, we investigate the effect of GC repression on different natural and/or recombinant NF-kappaB-driven reporter gene constructs in the presence of increasing amounts of various coactivator molecules, such as CREB-binding protein (CBP), p300, and SRC-1 . We found that GCs maintain their repressive capacities, irrespective of the amount of cofactor present in the cell . Similar results were obtained for the reciprocal transrepression of a GC receptor (GR) element-driven reporter gene by p65 . We demonstrate that neither the expression levels of p65 and CBP nor their physical association are affected by activated GR . Using Gal4 chimeras, we show that repression by GCs is specific for p65-mediated transactivation, ruling out competition for limiting nuclear factors as the major underlying mechanism of gene repression . In addition, the transactivation potential of a point-mutated Gal4-p65 variant with a decreased CBP interaction capability is still repressed by GR . Finally, we present evidence that the specificity of GC repression on p65-driven gene expression is codetermined by the TATA box context. Mol Microbiol, 2000 Mar, 35(6), 1295 - 311 Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance; Bialek-Wyrzykowska U et al.; The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast . The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane . C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins . The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes . What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown . Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature . A shift to the restrictive temperature does not alter exponential growth during the first 3 h . The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C . In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point . Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity . Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization. Mol Microbiol, 2000 Mar, 35(6), 1277 - 85 The mitochondrial protein targeting suppressor (mts1) mutation maps to the mRNA-binding domain of Npl3p and affects translation on cytoplasmic polysomes; Gratzer S et al.; In all eukaryotic organisms, messenger RNA (mRNA) is synthesized in the nucleus and then exported to the cytoplasm for translation . The export reaction requires the concerted action of a large number of protein components, including a set of shuttle proteins that can exit and re-enter the nucleus through the nuclear pore complex . Here, we show that, in Saccharomyces cerevisiae, the shuttle protein Npl3p leaves the nuclear pore complex entirely and continues to function in the cytoplasm . A mutation at position 219 in its RNA-binding domain leaves Npl3p lingering in the cytoplasm associated with polysomes . Yeast cells expressing the mutant Npl3(L-219S) protein show alterations in mRNA stability that can affect protein synthesis . As a result, defects in nascent polypeptide targeting to subcellular compartments such as the mitochondria are also suppressed. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4046 - 51 SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage; Mao Y et al.; Ubiquitin/26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage . In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1/Smt3p, a ubiquitin-like protein . This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1/Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells . (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1/Smt3p . (iii) TOP1 physically interacts with UBC9 . (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9/SUMO-1 may be involved in the repair of TOP1-mediated DNA damage. Plant Physiol, 2000 Apr, 122(4), 1447 - 55 The sucrose transporter of celery . Identification and expression during salt stress; Noiraud N et al.; In celery (Apium graveolens L.), long-distance transport of reduced carbon occurs both in the form of sucrose (Suc) and mannitol . The presence of mannitol has been related to the resistance of celery to salt stress . To investigate the transport events occurring during salt stress, we have cloned the H(+)/Suc transporter of celery AgSUT1 (A . graveolens Suc uptake transport 1) from a mature leaf cDNA library . The function of the encoded protein was confirmed by expression in yeast . AgSUT1 is a H(+)/Suc transporter with a high affinity for Suc (K(m) of 139 microM) . Another closely related cDNA (AgSUT2) was also identified . AgSUT1 is mainly expressed in mature leaves and phloem of petioles, but also in sink organs such as roots . When celery plants were subjected to salt stress conditions (30 d watering with 300 mM NaCl) favoring mannitol accumulation (J.D . Everard, R . Gucci, S.C . Kann, J.A . Flore, W.H . Loescher {1994} Plant Physiol 106: 281-292), AgSUT1 expression was decreased in all organs, but markedly in roots . The results are discussed in relation to the physiology of celery. Plant J, 2000 Mar, 21(5), 455 - 67 Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation; Weschke W et al.; In order to understand sucrose transport in developing seeds of cereals at the molecular level, we cloned from a caryopses library two cDNAs encoding sucrose transporters, designated HvSUT1 and HvSUT2 . Sucrose uptake activity was confirmed by heterologous expression in yeast . Both transporter genes are expressed in maternal as well as filial tissues . In a series of in situ hybridizations we analysed the cell type-specific expression in developing seeds . HvSUT1 is preferentially expressed in caryopses in the cells of the nucellar projection and the endospermal transfer layer, which represent the sites of sucrose exchange between the maternal and the filial generation and are characterized by transfer cell formation . HvSUT2 is expressed in all sink and source tissues analysed and may have a general housekeeping role . The rapid induction of HvSUT1 gene expression in caryopses at approximately 5-6 days after fertilization coincides with increasing levels of sucrose as well as sucrose synthase mRNA and activity, and occurs immediately before the onset of rapid starch accumulation within the endosperm . Starch biosynthesis requires sucrose to be imported into the endosperm, as direct precursor for starch synthesis and to promote storage-associated processes . We discuss the possible role of HvSUT1 as a control element for the endospermal sucrose concentration. Plant J, 2000 Mar, 21(5), 401 - 8 Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product; Reidt W et al.; The Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-specific seed proteins . To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis-motif, conserved in many seed-specific promoters, a promoter analysis as well as band-shift experiments were performed . The analysis of promoter mutants revealed the structural requirements for the function of the RY cis-element . It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif . Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis-motif is a target for both transcription factors . For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established . The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development. Plant J, 2000 Feb, 21(4), 379 - 85 The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin-dependent kinase activity in vitro; Lui H et al.; Cyclin-dependent kinases (CDKs) are important regulators of the eukaryotic cell division cycle . To study protein-protein interactions involving plant CDKs, the Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two-hybrid system . Here we report on the isolation of ICK2, and show that it interacts with Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt . ICK2 contains a carboxy-terminal domain related to that of ICK1, a previously described CDK inhibitor from Arabidopsis, and to the CDK-binding domain of the mammalian inhibitor p27Kip1 . Outside of this domain, ICK2 is distinct from ICK1, p27Kip1, and other proteins . At nanogram levels (8 nM), purified recombinant ICK2 inhibits p13Suc1-associated histone H1 kinase activity from Arabidopsis tissue extracts, demonstrating that it is a potent inhibitor of plant CDK activity in vitro . ICK2 mRNA was present in all tissues analysed by Northern hybridization, and its distribution was distinct from that of ICK1 . These results demonstrate that plants possess a family of differentially regulated CDK inhibitors that contain a conserved carboxy terminal but with distinct amino terminal regions. Proc Natl Acad Sci U S A, 2000 Apr 25, 97(9), 4609 - 14 The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane; Gillece P et al.; Peptides and misfolded secretory proteins are transported efficiently from the endoplasmic reticulum (ER) lumen to the cytosol, where the proteins are degraded by proteasomes . Protein export depends on Sec61p, the ribosome-binding core component of the protein translocation channel in the ER membrane . We found that prebinding of ribosomes abolished export of a glycopeptide from yeast microsomes . Deletion of SSH1, which encodes a ribosome-binding Sec61p homologue in the ER, had no effect on glycopeptide export . A collection of cold-sensitive sec61 mutants displayed a variety of phenotypes: two mutants strongly defective in misfolded protein export from the ER, sec61-32 and sec61-41, displayed only minor peptide export defects . Glycopeptide export was severely impaired, however, in several sec61 mutants that were only marginally defective in misfolded protein export . In addition, a mutation in SEC63 strongly reduced peptide export from the ER . ER-luminal ATP was required for both misfolded protein and glycopeptide export . We conclude that the protein translocation channel in the ER membrane mediates glycopeptide transport across the ER membrane. Mol Cell Biol, 2000 May, 20(9), 3086 - 96 Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase; Zou L et al.; In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase . S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase . The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin . Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G(1)-S transition in an S-CDK-dependent manner . During S phase, Cdc45p associates with different replication origins at specific times . The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins . These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing . The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of ARS1, a potential origin DNA-unwinding element . Cdc45p is required for recruiting DNA polymerase alpha onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase epsilon only during S phase . These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin. Mol Cell Biol, 2000 May, 20(9), 2996 - 3003 Ku recruits the XRCC4-ligase IV complex to DNA ends; Nick McElhinny SA et al.; Genetic experiments have determined that Ku, XRCC4, and ligase IV are required for repair of double-strand breaks by the end-joining pathway . The last two factors form a tight complex in cells . However, ligase IV is only one of three known mammalian ligases and is intrinsically the least active in intermolecular ligation; thus, the biochemical basis for requiring this ligase has been unclear . We demonstrate here a direct physical interaction between the XRCC4-ligase IV complex and Ku . This interaction is stimulated once Ku binds to DNA ends . Since XRCC4-ligase IV alone has very low DNA binding activity, Ku is required for effective recruitment of this ligase to DNA ends . We further show that this recruitment is critical for efficient end-joining activity in vitro . Preformation of a complex containing Ku and XRCC4-ligase IV increases the initial ligation rate 20-fold, indicating that recruitment of the ligase is an important limiting step in intermolecular ligation . Recruitment by Ku also allows XRCC4-ligase IV to use Ku's high affinity for DNA ends to rapidly locate and ligate ends in an excess of unbroken DNA, a necessity for end joining in cells . These properties are conferred only on ligase IV, because Ku does not similarly interact with the other mammalian ligases . We have therefore defined cell-free conditions that reflect the genetic requirement for ligase IV in cellular end joining and consequently can explain in molecular terms why this factor is required. Mol Cell Biol, 2000 May, 20(9), 2970 - 83 Domains in the SPT5 protein that modulate its transcriptional regulatory properties; Ivanov D et al.; SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II . SPT4 and SPT5 are involved in both 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein . Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5 . In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein . We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein . Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation . These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties. Genetics, 2000 Mar, 154(3), 1203 - 11 The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis; Carney GE et al.; Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone . Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins . We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations . EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain . In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis . EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity . Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages . Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis. Genetics, 2000 Mar, 154(3), 1125 - 39 An mre11 mutant of Coprinus cinereus has defects in meiotic chromosome pairing, condensation and synapsis; Gerecke EE et al.; The rad11 gene of the basidiomycete Coprinus cinereus is required for the completion of meiosis and for survival after gamma irradiation . We have cloned the rad11 gene and shown that it is a homolog of MRE11, a gene required for meiosis and DNA repair in numerous organisms . The expression of C . cinereus mre11 is induced during prophase I of meiosis and following gamma irradiation . The gene encodes a predicted polypeptide of 731 amino acids, and the mre11-1 (rad11-1) mutation is a single base pair change that results in a stop codon after amino acid 315 . The mre11-1 mutant shows enhanced sensitivity to ionizing radiation, but no enhanced sensitivity to UV radiation . It shows a delay in fruitbody formation and a reduction in the number of mushrooms formed per dikaryon . The mre11-1 mutant also has several meiotic defects . Pachytene chromatin condensation is disrupted, and although some meiotic cells appear to achieve metaphase I condensation, no further meiotic progression is observed . The mre11-1 mutant also fails to undergo proper chromosome synapsis; neither axial elements nor mature synaptonemal complexes are complete . Finally, meiotic homolog pairing is reduced in the mre11-1 mutant . Thus, in C . cinereus, Mre11 is required for meiotic DNA metabolism. Genomics, 2000 Mar 15, 64(3), 298 - 306 The structure and expression of the murine wildtype p53-induced phosphatase 1 (Wip1) gene; Choi J et al.; The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner . We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA . The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA . The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa . Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level . The 5'-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene . The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species . Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues . Wip1 has been mapped near the p53 gene on mouse chromosome 11 . Nucleic Acids Res, 2000 May 1, 28(9), 2012 - 7 Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIalpha; Kim YC et al.; To investigate the function of a DNA topoisomerase III enzyme in Caenorhabditis elegans, the full-length cDNA of C.elegans DNA topoisomerase IIIalpha was cloned . The deduced amino acid sequence exhibited identities of 48 and 39% with those of human DNA topoisomerase IIIalpha and Saccharomyces cerevisiae DNA topoisomerase III, respectively . The overexpressed polypeptide showed an optimal activity for removing negative DNA supercoils at a relatively high temperature of 52-57 degrees C, which is similar to the optimum temperatures of other eukaryotic DNA topoisomerase III enzymes . When topoisomerase IIIalpha expression was interfered with by a cognate double-stranded RNA injection, pleiotropic phenotypes with abnormalities in germ cell proliferation, oogenesis and embryo-genesis appeared . These phenotypes were well correlated with mRNA expression localized in the meiotic cells of gonad and early embryonic cells. Nucleic Acids Res, 2000 May 1, 28(9), 1986 - 93 Identification of a transcription factor IIIA-interacting protein; Moreland RJ et al.; Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes . The protein from vertebrates has nine contiguous Cys(2)His(2)zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation . In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners . A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA . This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined from expressed sequence tags . In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno-precipitation of the complex using anti-TFIIIA-intP antibody . Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co-chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins . In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene. Nucleic Acids Res, 2000 May 1, 28(9), 1955 - 62 Human Krüppel-like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription; van Vliet J et al.; CACCC-boxes are recognised by transcription factors of the Sp/Kruppel-like Factor (Sp1/KLF) family . Here we describe one member of this family, KLF8/ZNF741/BKLF3 (KLF8) . KLF8 contains a characteristic C-terminal DNA-binding domain comprised of three Kruppel-like zinc fingers, but also has limited homology to another family member, KLF3/Basic Kruppel-like Factor (KLF3/BKLF), in its N-terminus . Most significantly, it shares with KLF3/BKLF a Pro-Val-Asp-Leu-Ser/Thr motif . In KLF3/BKLF this motif mediates contact with the co-repressor protein C-terminal Binding Protein (CtBP) . We demonstrate that the KLF8 Pro-Val-Asp-Leu-Ser motif also contacts CtBP . We show that the N-terminus of KLF8 functions as a repression domain and that its activity relies on the integrity of the CtBP recognition motif . We demonstrate that the zinc fingers of KLF8 recognize CACCC elements in DNA and that full-length KLF8 can repress a CACCC-dependent promoter . Finally we determine that KLF8 is broadly expressed in human tissues . These results establish KLF8 as a CACCC-box binding protein that associates with CtBP and represses transcription. Glia, 2000 May, 30(3), 209 - 18 Transplantation of human olfactory ensheathing cells elicits remyelination of demyelinated rat spinal cord; Kato T et al.; Human olfactory ensheathing cells (OECs) were prepared from adult human olfactory nerves, which were removed during surgery for frontal base tumors, and were transplanted into the demyelinated spinal cord of immunosuppressed adult rats . Extensive remyelination was observed in the lesion site: In situ hybridization using a human DNA probe (COT-1) indicated a similar number of COT-1-positive cells and OEC nuclei within the repaired lesion . The myelination was of a peripheral type with large nuclei and cytoplasmic regions surrounding the axons, characteristic of Schwann cell and OEC remyelination . These results provide evidence that adult human OECs are able to produce Schwann cell-like myelin sheaths around demyelinated axons in the adult mammalian CNS in vivo. Crit Rev Biochem Mol Biol, 2000, 35(1), 1 - 33 Ku autoantigen: a multifunctional DNA-binding protein; Tuteja R et al.; Ku is a heterodimeric protein composed of approximately 70- and approximately 80-kDa subunits (Ku70 and Ku80) originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases . Ku has high binding affinity for DNA ends and that is why originally it was known as a DNA end binding protein, but now it is known to also bind the DNA structure at nicks, gaps, hairpins, as well as the ends of telomeres . It has been reported also to bind with sequence specificity to DNA and with weak affinity to RNA . Ku is an abundant nuclear protein and is present in vertebrates, insects, yeast, and worms . Ku contains ssDNA-dependent ATPase and ATP-dependent DNA helicase activities . It is the regulatory subunit of the DNA-dependent protein kinase that phosphorylates many proteins, including SV-40 large T antigen, p53, RNA-polymerase II, RP-A, topoisomerases, hsp90, and many transcription factors such as c-Jun, c-Fos, oct-1, sp-1, c-Myc, TFIID, and many more . It seems to be a multifunctional protein that has been implicated to be involved directly or indirectly in many important cellular metabolic processes such as DNA double-strand break repair, V(D)J recombination of immunoglobulins and T-cell receptor genes, immunoglobulin isotype switching, DNA replication, transcription regulation, regulation of heat shock-induced responses, regulation of the precise structure of telomeric termini, and it also plays a novel role in G2 and M phases of the cell cycle . The mechanism underlying the regulation of all the diverse functions of Ku is still obscure. Cardiovasc Drugs Ther, 2000 Feb, 14(1), 31 - 9 Coordinate regulation of metabolic enzyme encoding genes during cardiac development and following carvedilol therapy in spontaneously hypertensive rats; Sack MN et al.; Fuel substrate utilization is highly regulated during cardiac development and with the onset of cardiac hypertrophy . Glucose and lactate are the predominant fuel substrates utilized during cardiac development . Postnatally, a switch occurs so that fatty acids become the chief energy substrate in the nonfed adult mammalian heart . A reversion back towards fetal energy metabolism occurs with the development of cardiac hypertrophy . To evaluate the role of this substrate preference switch in the development of cardiac hypertrophy, the molecular regulation directing these switches is being explored . Thus, we have begun by defining the temporal expression patterns of genes encoding key rate-controlling enzymes directing major fuel substrate metabolism during cardiac development, with pressure-overload-induced cardiac hypertrophy, and following antihypertensive therapy in spontaneously hypertensive rats . The genes encoding the fatty acid and adult enriched rate-controlling glycolytic enzymes are expressed at low levels in the fetal and neonatal rat heart . The genes encoding these enzymes are significantly and coordinately upregulated (> or = 70%) in adult rat hearts compared to the fetal expression patterns . A reciprocal and coordinate downregulation (> or = 40% reduction) of the fatty acid and adult enriched glycolytic enzyme encoding genes are observed with the induction of pressure-overload-induced hypertrophy in spontaneously hypertensive rats compared to Wistar-Furth normotensive control rats . Antihypertensive therapy with carvedilol, a vasodilating alpha- and beta-adrenoreceptor antagonist, attenuates this reversion of the metabolic gene expression pattern towards fetal levels compared to placebo-treated littermate controls . This coordinate developmental and hypertrophy-induced regulation of genes that encode enzymes controlling both fatty acid and glycolytic catabolic pathways in the heart implicates potential mutual/overlapping regulatory signaling proteins within their gene regulatory programs . These gene regulatory pathways need to be identified and modulated in order to characterize the functional role of fuel substrate metabolism in cardiac development and with the induction of cardiac hypertrophy. Trends Biochem Sci, 2000 Apr, 25(4), 200 - 4 Recombination in telomere-length maintenance; Kass-Eisler A et al.; Although telomerase is the major mechanism for telomere elongation in most cells, telomerase-independent mechanisms of telomere maintenance can allow cell survival . Yeast cells that lack telomerase maintain telomere length through a form of recombination known as gene conversion . Understanding the role that telomeric recombination might play in mammalian cells has important implications for cancer therapeutics. FEMS Microbiol Lett, 2000 Apr 15, 185(2), 255 - 61 Interaction of the Neurospora crassa heat shock factor with the heat shock element during heat shock and different developmental stages; Meyer U et al.; The interaction of the heat shock factor (HSF) with the heat shock element (HSE) was determined by a non-radioactive electrophoretic mobility shift assay, in order to analyze HSF regulation in Neurospora crassa . HSF binds to HSE under normal, non-stress conditions and is thus constitutively trimerized . Upon heat shock, the HSF-HSE complex shows a retarded mobility . This was also observed in Saccharomyces cerevisiae, where this mobility shift was shown to be due to HSF phosphorylation {Sorger and Pelham (1988) Cell 54, 855-864} . In N . crassa, HSE-dependent electrophoretic mobility shift is temperature- and time-dependent . Under normal growth conditions, the HSF is located in the cytoplasm as well as in the nucleus . In germinating conidia the HSF shows a retarded mobility typical for heat shock even at normal growth temperatures . No HSF-dependent mobility shift was detectable in aerial hyphae. Curr Opin Genet Dev, 2000 Apr, 10(2), 187 - 92 Promoter targeting and chromatin remodeling by the SWI/SNF complex; Peterson CL et al.; The SWI/SNF complex is a 2 MDa multi-subunit DNA-dependent ATPase that contributes to the regulation of gene transcription by altering chromatin structure . Recent studies have revealed that the SWI/SNF complex is targeted to promoters via direct interactions with transcription activators and have provided insights into mechanisms by which the complex alters nucleosome structure and contributes to the remodeling of chromatin. Biotechnol Prog, 2000 Mar-Apr, 16(2), 228 - 37 Optimal design of protein production plants with time and size factor process models; Montagna JM et al.; In this work we propose an optimization model for the design of a biotechnological multiproduct batch plant . A first level of detail posynomial model is constructed for each unit, as well as decisions regarding the structural optimization of the plant . A particular feature of this model is that it contains composite units in which semicontinuous items operate on the material contained by batch items . This occurs in the purification steps, in particular with the microfilters operating between retentate and permeate vessels, and with the homogenizer and ultrafilters operating on the material contained in a batch holding vessel . Also, the unit models rely on batch operating time expressions that depend on both the batch size and the size of semicontinuous items . The model takes into account all of the available options to increase the efficiency of the batch plant design: unit duplication in-phase and out-of-phase and intermediate storage tanks . The resulting mathematical model for the minimization of the plant capital cost is a mixed integer non-linear program (MINLP), which is solved to global optimality with an implementation of the outer approximation/ equality relaxation/ augmented penalty (OA/ER/AP) method . A plant that produces four recombinant proteins in eight processing stages is used to illustrate the proposed approach . An interesting feature of this example is that it represents an attempt to standardize a plant for the production of both therapeutic and nontherapeutic proteins; the model applied is generic and can thus be applied to any such modular plant . Results indicate that the best solution in terms of minimal capital cost contains no units in parallel and with intermediate storage tank allocation. J Biol Chem, 2000 Jun 9, 275(23), 17827 - 37 Interactions of CCCH zinc finger proteins with mRNA . Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA; Lai WS et al.; Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA . TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA . In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish . We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells . Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays . We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays . In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells . Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively. J Cell Sci, 2000 May, 113 ( Pt 9), 1611 - 21 Determination of cell polarity in germinated spores and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP homolog; Wendland J et al.; In the filamentous ascomycete Ashbya gossypii, like in other filamentous fungi onset of growth in dormant spores occurs as an isotropic growth phase generating spherical germ cells . Thereafter, a switch to polarized growth results in the formation of the first hyphal tip . The initial steps of hyphal tip formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi . We investigated whether phenotypic similarities between these distinct events extended to the molecular level . To this end we isolated and characterized the A . gossypii homolog of the Saccharomyces cerevisiae BEM2 gene which is part of a network of rho-GTPases and their regulators required for bud emergence and bud growth in yeast . Here we show that the AgBem2 protein contains a GAP- (GTPase activating protein) domain for rho-like GTPases at its carboxy terminus, and that this part of AgBem2p is required for complementation of an Agbem2 null strain . Germination of spores resulted in enlarged Agbem2 germ cells that were unable to generate the bipolar branching pattern found in wild-type germ cells . In addition, mutant hyphae were swollen due to defects in polarized cell growth indicated by the delocalized distribution of chitin and cortical actin patches . Surprisingly, the complete loss of cell polarity which lead to spherical hyphal tips was overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips . In conclusion these results and other findings demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require members of &rgr;-GTPase modules. J Mol Endocrinol, 2000 Apr, 24(2), 165 - 82 Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors; Sartorius CA et al.; Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD(PR)) containing two highly conserved zinc fingers . DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors . We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD(PR )fusion protein . Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADP-ribose) polymerase . The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK(CS) . DNA-PK that was 'pulled-down' by DBD(PR) on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK(CS), (2) transphosphorylate DBD(PR), and (3) phosphorylate a DNA-PK-specific p53 peptide substrate . DNA-PK was also able to associate with the DBD of the yeast activator GAL4 . However, neither a PR DBD mutant lacking a structured first zinc finger (DBD(CYS)) nor the core DBD of the estrogen receptor (DBD(ER)) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs . Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/DNA-PK complex can assemble in vivo . These data show that DNA-PK and DBD(PR) interact, that DBD(PR) is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription. Hum Mol Genet, 2000 Mar 22, 9(5), 821 - 8 The mouse neurological mutant flailer expresses a novel hybrid gene derived by exon shuffling between Gnb5 and Myo5a; Jones JM et al.; Exon shuffling is thought to be an important mechanism for evolution of new genes . Here we show that the mouse neurological mutation flailer (flr) expresses a novel gene that combines the promoter and first two exons of guanine nucleotide binding protein beta 5 (Gnb5) with the C-terminal exons of the closely linked Myosin 5A (MyoVA) gene (Myo5a) . The flailer protein, which is expressed predominantly in brain, contains the N-terminal 83 amino acids of Gnb5 fused in-frame with the C-terminal 711 amino acids of MyoVA, including the globular tail domain that binds organelles for intracellular transport . Biochemical and genetic studies indicate that the flailer protein competes with wild-type MyoVA in vivo, preventing the localization of smooth endoplasmic reticulum vesicles in the dendritic spines of cerebellar Purkinje cells . The flailer protein thus has a dominant-negative mechanism of action with a recessive mode of inheritance due to the dependence of competitive binding on the ratio between mutant and wild-type proteins . The chromosomal arrangement of Myo5a upstream of Gnb5 is consistent with non-homologous recombination as the mutational mechanism . To our knowledge, flailer is the first example of a mammalian mutation caused by germ line exon shuffling between unrelated genes. Hum Mol Genet, 2000 Mar 22, 9(5), 735 - 44 Batten disease: evaluation of CLN3 mutations on protein localization and function; Haskell RE et al.; Juvenile neuronal ceroid lipofuscinosis (JNCL), Batten disease, is an autosomal recessive lysosomal storage disease associated with mutations in CLN3 . CLN3 has no known homology to other proteins and a function has not yet been described . The predominant mutation in CLN3 is a 1.02 kb genomic deletion that accounts for nearly 85% of the disease alleles . In this mutation, truncation of the protein by a premature stop codon results in the classical phenotype . Additional missense and nonsense mutations have been described . Some missense substitutions result in a protracted phenotype, with delays in the onset of classical clinical features, whereas others lead to classical JNCL . In this study, we examined the effect of naturally occurring point mutations on the intracellular localization of CLN3 and their ability to complement the CLN3-deficient yeast, btn1-Delta . We also examined a putative farnesylation motif thought to be involved in CLN3 trafficking . All of the point mutations, like wild-type CLN3, were highly associated with lysosome-associated membrane protein II in non-neuronal cells and with synaptophysin in neuronal cell lines . In the yeast functional assay, point mutations correlating with a mild phenotype also demonstrated CLN3 activity, whereas the mutations associated with severe disease failed to restore CLN3 function completely . CLN3 with a mutation in the farnesylation motif trafficked normally but was functionally impaired . These data suggest that these clinically relevant point mutations, causative of Batten disease, do not affect protein trafficking but rather exert their effects by impairing protein function. Mol Biol Cell, 2000 Apr, 11(4), 1401 - 19 EMB-30: an APC4 homologue required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans; Furuta T et al.; Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans . Germline-specific emb-30 mutant alleles block the meiotic divisions . Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I . As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur . Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis . Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting . By further reducing maternal emb-30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-to-anaphase transition in many, if not all, cells . Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M-phase progression . A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery . This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development . Positional cloning established that emb-30 encodes the likely C . elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition . Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism. Mol Biol Cell, 2000 Apr, 11(4), 1305 - 13 Chromosome condensation factor Brn1p is required for chromatid separation in mitosis; Ouspenski II et al.; This work describes BRN1, the budding yeast homologue of Drosophila Barren and Xenopus condensin subunit XCAP-H . The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation . Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase . Chromatid cohesion before anaphase is properly maintained in the mutants . Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle . Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis . Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP-G . Overexpression of SMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex. J Biol Chem, 2000 Jun 2, 275(22), 16443 - 9 Evidence for simultaneous protein interactions between human Rad51 paralogs; Schild D et al.; In yeast, the Rad51-related proteins include Rad55 and Rad57, which form a heterodimer that interacts with Rad51 . Five human Rad51 paralogs have been identified (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2, and Rad51D/Rad51L3), and each interacts with one or more of the others . Previously we reported that HsRad51 interacts with XRCC3, and Rad51C interacts with XRCC3, Rad51B, and HsRad51 . Here we report that in the yeast two-hybrid system, Rad51D interacts with XRCC2 and Rad51C . No other interactions, including self-interactions, were found, indicating that the observed interactions are specific . The yeast Rad51 interacts with human Rad51 and XRCC3, suggesting Rad51 conservation since the human yeast divergence . Data from yeast three-hybrid experiments indicate that a number of the pairs of interactions between human Rad51 paralogs can occur simultaneously . For example, Rad51B expression enhances the binding of Rad51C to XRCC3 and to HsRad51D, and Rad51C expression allows the indirect interaction of Rad51B with Rad51D . Experiments using 6xHis-tagged proteins in the baculovirus system confirm several of our yeast results, including Rad51B interaction with Rad51D only when Rad51C is simultaneously expressed and Rad51C interaction with XRCC2 only when Rad51D is present . These results suggest that these proteins may participate in one complex or multiple smaller ones. J Biol Chem, 2000 May 26, 275(21), 15605 - 8 The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase; Steinberg SJ et al.; Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur . A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization . We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative . When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non-isotopic and radioactive assays . Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate . Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells . Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 190 - 8 The ZIP family of metal transporters; Guerinot ML; Members of the ZIP gene family, a novel metal transporter family first identified in plants, are capable of transporting a variety of cations, including cadmium, iron, manganese and zinc . Information on where in the plant each of the ZIP transporters functions and how each is controlled in response to nutrient availability may allow the manipulation of plant mineral status with an eye to (1) creating food crops with enhanced mineral content, and (2) developing crops that bioaccumulate or exclude toxic metals. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 140 - 51 Sodium transport in plant cells; Blumwald E et al.; Salinity limits plant growth and impairs agricultural productivity . There is a wide spectrum of plant responses to salinity that are defined by a range of adaptations at the cellular and the whole-plant levels, however, the mechanisms of sodium transport appear to be fundamentally similar . At the cellular level, sodium ions gain entry via several plasma membrane channels . As cytoplasmic sodium is toxic above threshold levels, it is extruded by plasma membrane Na(+)/H(+) antiports that are energized by the proton gradient generated by the plasma membrane ATPase . Cytoplasmic Na(+) may also be compartmentalized by vacuolar Na(+)/H(+) antiports . These transporters are energized by the proton gradient generated by the vacuolar H(+)-ATPase and H(+)-PPiase . Here, the mechanisms of sodium entry, extrusion, and compartmentation are reviewed, with a discussion of recent progress on the cloning and characterization, directly in planta and in yeast, of some of the proteins involved in sodium transport. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 52 - 78 Molecular aspects of higher plant P-type Ca(2+)-ATPases; Geisler M et al.; Recent genomic data in the model plant Arabidopsis thaliana reveal the existence of at least 11 Ca(2+)-ATPase genes, and an analysis of expressed sequence tags suggests that the number of calcium pumps in this organism might be even higher . A phylogenetic analysis shows that 11 Ca(2+)-ATPases clearly form distinct groups, type IIA (or ECA for ER-type Ca(2+)-ATPase) and type IIB (ACA for autoinhibited Ca(2+)-ATPase) . While plant IIB calcium pumps characterized so far are localized to internal membranes, their animal homologues are exclusively found in the plasma membrane . However, Arabidopsis type IIB calcium pump isoforms ACA8, ACA9 and ACA10 form a separate outgroup and, based on the high molecular masses of the encoded proteins, are good candidates for plasma membrane bound Ca(2+)-ATPases . All known plant type IIB calcium ATPases seem to employ an N-terminal calmodulin-binding autoinhibitor . Therefore it appears that the activity of type IIB Ca(2+)-ATPases in plants and animals is controlled by N-terminal and C-terminal autoinhibitory domains, respectively . Possible functions of plant calcium pumps are described and - beside second messenger functions directly linked to calcium homeostasis - new data on a putative involvement in secretory and salt stress functions are discussed. J Biol Chem, 2000 May 19, 275(20), 15157 - 65 Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex; Zhang Z et al.; Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX) . The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial . We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system . Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7 . Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides . The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system . The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator . HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast . Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX . In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway . Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX. J Biol Chem, 2000 May 26, 275(21), 16373 - 81 The C/EBP bZIP domain can mediate lipopolysaccharide induction of the proinflammatory cytokines interleukin-6 and monocyte chemoattractant protein-1; Hu HM et al.; C/EBPalpha, beta, and delta are all expressed by bone marrow-derived macrophages . Ectopic expression of any of these transcription factors is sufficient to confer lipopolysaccharide (LPS)-inducible expression of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to a B lymphoblast cell line, which normally lacks C/EBP factors and does not display LPS induction of proinflammatory cytokines . Thus, the activities of C/EBPalpha, beta, and delta are redundant in regard to expression of IL-6 and MCP-1 . Surprisingly, the bZIP region of C/EBPbeta, which lacks any previously described activation domains, can also confer LPS-inducible expression of IL-6 and MCP-1 in stable transfectants . Transient transfections reveal that the bZIP regions of C/EBPbeta, C/EBPdelta, and, to a lesser extent, C/EBPalpha can activate the IL-6 promoter and augment its induction by LPS . Furthermore, the transdominant inhibitor, LIP, can activate expression from the IL-6 promoter . The ability of the C/EBPbeta bZIP region to activate the IL-6 promoter in transient transfections is completely dependent upon an intact NF-kappaB-binding site, supporting a model where the bZIP protein primarily functions to augment the activity of NF-kappaB . Replacement of the leucine zipper of C/EBPbeta with that of GCN4 yields a chimeric protein that can dimerize and specifically bind to a C/EBP consensus sequence, but shows a markedly reduced ability to activate IL-6 and MCP-1 expression . These results implicate the leucine zipper domain in some function other than dimerization with known C/EBP family members, and suggest that C/EBP redundancy in regulating cytokine expression may result from their highly related bZIP regions. J Biol Chem, 2000 Jun 2, 275(22), 16954 - 62 Hydrophobic residues Phe751 and Leu753 are essential for STAT5 transcriptional activity; Callus BA et al.; One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and activators of transcription (STAT) family members . It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes . To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding . Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12-amino acid region essential for STAT5 transactivation . Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein . Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain . Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation . In contrast, the non-acidic residues were crucial for transactivation . Mutating either Phe(751) or Leu(753) to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery. J Biol Chem, 2000 Jul 14, 275(28), 21477 - 85 Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase; Wu Y et al.; PTEN/MMAC is a phosphatase that is mutated in multiple human tumors . PTEN/MMAC dephosphorylates 3-phosphorylated phosphatidylinositol phosphates that activate AKT/protein kinase B (PKB) kinase activity . AKT/PKB is implicated in the inhibition of apoptosis, and cell lines and tumors with mutated PTEN/MMAC show increased AKT/PKB kinase activity and resistance to apoptosis . PTEN/MMAC contains a PDZ domain-binding site, and we show here that the phosphatase binds to a PDZ domain of membrane-associated guanylate kinase with inverted orientation (MAGI) 3, a novel inverted membrane-associated guanylate kinase that localizes to epithelial cell tight junctions . Importantly, MAGI3 and PTEN/MMAC cooperate to modulate the kinase activity of AKT/PKB . These data suggest that MAGI3 allows for the juxtaposition of PTEN/MMAC to phospholipid signaling pathways involved with cell survival. J Biol Chem, 2000 Jul 14, 275(28), 21331 - 9 Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1); Vitale N et al.; Activation of ADP-ribosylation factors (ARFs) is mediated by guanine nucleotide-exchange proteins, which accelerate conversion of inactive ARF-GDP to active ARF-GTP . ARF domain protein (ARD1), a 64-kDa GTPase with a C-terminal ADP-ribosylation factor domain, is localized to lysosomes and the Golgi apparatus . When ARD1 was used as bait to screen a human liver cDNA library using the yeast two-hybrid system, a cDNA for cytohesin-1, a approximately 50-kDa protein with ARF guanine nucleotide-exchange protein activity, was isolated . In this system, ARD1-GDP interacted well with cytohesin-1 but very poorly with cytohesin-2 . In agreement, cytohesin-1, but not cytohesin-2, markedly accelerated {(35)S}guanosine 5'-3-O-(thio)triphosphate binding to ARD1 . The effector region of the ARF domain of ARD1 appeared to be critical for the specific interaction with cytohesin-1 . Replacement of single amino acids in the Sec7 domains of cytohesin-1 and -2 showed that residue 30 is critical for specificity . In transfected COS-7 cells, overexpressed ARD1 and cytohesin-1 were partially colocalized, as determined by confocal fluorescence microscopy . It was concluded that cytohesin-1 is likely to be involved in ARD1 activation, consistent with a role for ARD1 in the regulation of vesicular trafficking. J Biol Chem, 2000 Jun 23, 275(25), 18670 - 5 Cellular mechanisms regulating protein phosphatase-1 . A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit; Connor JH et al.; Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1) . To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed . Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1 . Both mutants were effectively inhibited by I-1 . Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition . Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2 . Si