Acta Biochim Biophys Sin (Shanghai), 2004 Jan, 36(1), 42 - 6 Interaction of plasminogen activator inhibitor-2 and proteasome subunit, beta type 1; Fan J et al.; The apoptosis protection by plasminogen activator inhibitor -2(PAI-2) is dependent on a 33 amino acids fragment between helix C and D of PAI-2 which is probably may be due to the interaction of PAI-2 with unknown intracellular proteins . In this study we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cells cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone that encodes 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMbeta1) which plays important roles in NF-kappaB activation . GST-pulldown experiments confirmed the interaction between PAI-2 and PSMB1 in vitro . These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interaction with PSMbeta1. Acta Biochim Biophys Sin (Shanghai), 2004 Jan, 36(1), 11 - 5 Protein product encoded by a human novel gene E9730 enhances AP-1 activity through interacting with Jab1; Wang ZQ et al.; A novel human gene, named E9730 (a clone number of fetal liver cDNA library), has been identified from more than 14,000 expressed sequence tags (ESTs) based on our large scale sequencing of human fetal liver cDNA libraries . Although sequencing of this novel human gene indicates that it is a leucine zipper protein, the function of E9730 and its homongous genes among species is unknown yet . To find out physiological functional clue of E9730, the yeast two-hybrid system was used to screen the E9730-interacting protein(s), and one clone containing a cDNA insert with almost the entire coding sequence (amino acids 39 C335) of human Jab1 (Jun-activating domain binding protein 1) that interacted specifically with E9730 was identified . A specific association between Jab1 and E9730 was shown by co-immunoprecipitation and co-localization experiments . Furthermore, the data indicated that E9730 appeared to enhance Jab1-induced AP-1 activity in a concentration-dependent manner and Jab1 may be involved in the intracellular signaling transduction from E9730 to AP-1. J Biol Chem, 2004 Apr 2, 279(14), 14201 - 6 Epub 2004 Jan 19. Type IV collagen is transcriptionally regulated by Smad1 under advanced glycation end product (AGE) stimulation; Abe H et al.; Prolonged exposure to hyperglycemia is now recognized as the most significant causal factor of diabetic complications . Excessive advanced glycation end products (AGEs) as a result of hyperglycemia in tissues or in the circulation may critically affect the progression of diabetic nephropathy . In diabetic nephropathy, glomerulosclerosis is a typical pathologic feature characterized by the increase of the extracellular matrix (ECM) . We have reported previously that alpha1 type IV collagen (Col4) is one of the major components of ECM, which is up-regulated by AGEs, and that the overexpression of Col4 is transcriptionally regulated by an unknown transcription factor binding to the promoter . Here we identified this protein as Smad1 by yeast one-hybrid screening . Using chromatin immunoprecipitation and reporter assay, we observed that Smad1 directly regulated transcription for Col4 through the binding of Smad1 to the promoter of Col4 . Smad1 was significantly induced along with Col4 in AGE-treated mesangial cells . Moreover, suppression of Smad1 by antisense morpholino resulted in a decrease of AGE-induced Col4 overproduction . To elucidate the interaction between transforming growth factor-beta and Smad1, we investigated whether activin receptor-liked kinase1 (ALK1) was involved in this regulation . AGE stimulation significantly increased the expression of the ALK1 mRNA in mesangial cells . We also demonstrated that Smad1 and ALK1 were highly expressed in human diabetic nephropathy . These results suggest that the modulation of Smad1 expression is responsible for the initiation and progression of diabetic nephropathy and that blocking Smad1 signaling may be beneficial in preventing diabetic nephropathy and other various diabetic complications. J Biol Chem, 2004 Apr 9, 279(15), 15323 - 9 Epub 2004 Jan 19. Self-regulated cleavage of the mitochondrial intramembrane-cleaving protease PARL yields Pbeta, a nuclear-targeted peptide; Sik A et al.; Regulated intramembrane proteolysis (RIP) is an emerging paradigm in signal transduction . RIP is mediated by intramembrane-cleaving proteases (I-CliPs), which liberate biologically active nuclear or secreted domains from their membrane-tethered precursor proteins . The yeast Pcp1p/Rbd1p protein is a Rhomboid-like I-CliP that regulates mitochondrial membrane remodeling and fusion through cleavage of Mgm1p, a regulator of these essential activities . Although this ancient function is conserved in PARL (Presenilins-associated Rhomboid-like protein), the mammalian ortholog of Pcp1p/Rbd1p, the two proteins show a strong divergence at their N termini . However, the N terminus of PARL is significantly conserved among vertebrates, particularly among mammals, suggesting that this domain evolved a distinct but still unknown function . Here, we show that the cytosolic N-terminal domain of PARL is cleaved at positions 52-53 (alpha-site) and 77-78 (beta-site) . Whereas alpha-cleavage is constitutive and removes the mitochondrial targeting sequence, beta-cleavage appears to be developmentally controlled and dependent on PARL I-CliP activity supplied in trans . The beta-cleavage of PARL liberates Pbeta, a nuclear targeted peptide whose sequence is conserved only in mammals . Thus, in addition to its evolutionarily conserved function in regulating mitochondrial dynamics, PARL might mediate a mammalian-specific, developmentally regulated mitochondria-to-nuclei signaling through regulated proteolysis of its N terminus and release of the Pbeta peptide. Phytochemistry, 2004 Jan, 65(2), 159 - 68 cis-3-Hexenal production in tobacco is stimulated by 16-carbon monounsaturated fatty acids; Hong M et al.; Transgenic tobacco plants O9 and T16 expressing the yeast acyl-CoA Delta9 desaturase and an insect acyl-CoA Delta11 desaturase, respectively, displayed altered profiles of fatty acids compared to wild-type tobacco plants and marked increases in cis-3-hexenal, a major leaf volatile derived from alpha-linolenic acid (18:3) . As expected, O9 and T16 plants had increased levels of the major unsaturated fatty acid products formed by the transgenic desaturases they expressed, viz., palmitoleic acid (16:1(Delta9)) and palmitvaccenic acid (16:1(Delta11)), respectively . In addition, levels of 18:3 lipid declined slightly and the pool of free 18:3, which accounts for about 30% of free fatty acids in wild-type plants, disappeared completely in both transgenics . Both O9 and T16 plants were found to have a two-fold increase in 13-lipoxygenase (13-LOX) activity, which catalyzes the first of two steps leading to hexenal production from 18:3 . In O9 and T16 plants, the activity of 9-lipoxygenase and hydroperoxide lyase, the latter catalyzing the formation of cis-3-hexenal from alpha-linolenic acid hydroperoxide, was significantly different from that of the wild-type plants . Although 16:1(Delta9) and 16:1(Delta11) had no direct effects on 13-LOX activity in vitro, cis-3-hexenal production increased in tobacco leaves treated with these fatty acids, suggesting that they may act in vivo by stimulating 13-LOX gene expression. Fungal Genet Biol, 2004 Feb, 41(2), 199 - 212 Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans; Sims AH et al.; The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast . However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms . Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library . An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A . nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source . The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A . nidulans arrays discussed. Trends Cell Biol, 1995 Aug, 5(8), 302 - 5 Anterograde transport through the Golgi complex: do Golgi tubules hold the key? Weidman PJ. Biochemical studies have suggested that anterograde protein transport through the Golgi complex is mediated by coatomer-coated vesicles that bud from one compartment and then transfer to, and fuse with, the next . However, recent genetic studies have shown that coatomer mutations block retrograde, but not anterograde, transport in yeast, calling into question the role of coatomer vesicles in anterograde transport . Peggy Weidman proposes that these findings might be explained if anterograde transport occurs by transient fusion of Golgi tubules and if coatomers have related, but separable, functions in tubule and vesicle dynamics. Trends Cell Biol, 1995 Oct, 5(10), 377 - 80 Inhibition of sphingolipid synthesis: effects on glycosphingolipid-GPI-anchored protein microdomains; Futerman AH; The idea that the transport and sorting of glycosylphosphatidylinositol (GPI)-anchored proteins depends on their interaction with glycosphingolipids was first proposed five or six years ago . Until recently, only circumstantial evidence was available to support this suggestion . During the past year, compelling support for this hypothesis has been provided by observations that inhibition of sphingolipid synthesis reduces the rate of transport of GPI-anchored proteins in yeast, and abolishes the polarized sorting of a GPI-anchored protein in epithelia. Trends Cell Biol, 1992 Dec, 2(12), 363 - 8 An essential role for a protein and lipid kinase complex in secretory protein sorting; Herman PK et al.; Yeast genetics has identified more than 40 genes involved in the biogenesis and maintenance of the yeast lysosome-like vacuole . Recent data on two of these genes, VPS15 and VPS34, are beginning to provide some fundamental insights into the mechanisms governing protein sorting within the eukaryotic secretory pathway . VPS15 and VPS34 encode a novel protein kinase and a phosphatidylinositol 3-kinase, respectively, that function together as components of a membrane-associated signal transduction complex . These studies of the VPS15-VPS34 complex indicate that intracellular protein trafficking decisions may be regulated by protein phosphorylation and phosphatidylinositol signalling events. Trends Cell Biol, 1992 Dec, 2(12), 358 - 60 Phosphatidyl-inositol 3-kinase: a key enzyme in diverse signalling processes; Panayotou G et al.; Phosphatidylinositol (PI) 3-kinase associates in signal-transducing complexes with activated growth factor receptors and other protein tyrosine kinases . The enzyme may also act downstream of receptors that are not tyrosine kinases in terminally differentiated cells . The recent cloning of the catalytic subunit of PI 3-kinase has revealed a structural similarity to a yeast protein important in vacuolar protein sorting . This finding provides some interesting clues to the function of PI3-kinase in diverse cellular responses. Trends Cell Biol, 1992 Mar, 2(3), 77 - 81 A proliferation of cyclins; Lew DJ et al.; Cyclins are regulatory subunits of the serine/threonine protein kinases that play key roles in cell cycle control . The roster of known cyclins has expanded significantly in the past year, revealing a large and very diverse family of proteins . Although cyclins were originally characterized by their periodic accumulation during interphase and destruction in mitosis (these were the 'mitotic' cyclins that control entry into mitosis), the newly identified cyclins do not conform to this pattern . Here we review what is known about the functions of the nonmitotic cyclins in yeast and in mammalian cells. Trends Cell Biol, 1993 Oct, 3(10), 330 - 2 Three clathrin-dependent budding steps and cell polarity; Riezman H; Clathrin plays an important role in the vesicular transport of proteins in all eukaryotes, but the precise steps in which it is involved may not be identical in all of them . Here, I put forward the hypothesis that three distinct clathrin-dependent budding events are common to all eukaryotes and have the following distinctive features: the first requires actin, the second is used for targeting soluble hydrolases from the Golgi to the hydrolytic compartment, and the third uses a tyrosine localization signal to concentrate membrane proteins . I suggest that the latter budding step is found on endosomes in yeast and is used for retrieval of membrane proteins back to the Golgi . Several testable predictions arise from this hypothesis as well as a possible evolutionary scenario concerning the origin of basolateral and apical plasma membranes in multicellular organisms. Trends Cell Biol, 1991 Dec, 1(6), 165 - 71 Vesicle budding: insights from cell-free assays; Melancon P et al.; Transfer of proteins and lipids between the various membrane-bound subcellular compartments of the eukaryotic cell is mediated by transport vesicles . The development of cell-free assays has allowed rapid progress towards a molecular description of the formation, or budding, of these vesicles . This article reviews and integrates data obtained from various yeast and mammalian systems on molecules involved in the budding reaction. Trends Cell Biol, 1993 Aug, 3(8), 268 - 72 Mx proteins: GTPases with antiviral activity; Staeheli P et al.; Mx proteins are synthesized in interferon-treated vertebrate cells . They have attracted much attention because some of them can block the multiplication of influenza A virus and certain other negative-stranded RNA viruses . Recently, Mx proteins have been shown to be GTPases with significant homology to dynamins and yeast VPS1, enzymes involved in intracellular protein trafficking . Several biochemical properties of dynamin and VPS1 are similar to those of Mx, promoting new speculation about how Mx proteins might interfere with virus multiplication. Trends Cell Biol, 1993 Nov, 3(11), 381 - 5 Profilin as a potential mediator of membrane-cytoskeleton communication; Machesky LM et al.; Profilin, the prototype actin-monomer-sequestering protein, has recently emerged as a multifunctional protein with several different activities . Genetic evidence in yeast and flies confirms that profilin is required for a normal actin cytoskeleton, while biochemical evidence suggests a role in regulating phosphoinositide signalling . New studies suggest that profilin may interact with other ligands, and even its role in regulating actin polymerization is now being re-evaluated. Trends Cell Biol, 1993 May, 3(5), 156 - 61 Flagellar regeneration in Chlamydomonas: a model system for studying organelle assembly; Johnson KA et al.; How do the many different components of an organelle assemble into a functional structure at an appropriate place and time? Flagellar regeneration by the biflagellate green alga Chlamydomonas is one experimental system in which genetics, biochemistry and ultrastructural analysis are being combined to investigate the assembly of a microtubule-containing organelle . Recent advances in the molecular biology of this 'green yeast' have made possible several new approaches to the problem of flagellar assembly; insights from these new approaches are the focus of this review. Trends Cell Biol, 1994 Mar, 4(3), 96 - 101 Cold fission: splitting the pombe cell at room temperature; Fankhauser C et al.; The mechanisms responsible for cytokinesis and its coordination with other events of the cell cycle are poorly understood . Genetic studies of cytokinesis in fission yeast are one useful approach to this problem . A number of conditional mutants of fission yeast that show defects in the formation of the septum of cytokinesis have been identified . Cloning of the genes affected in these mutants has begun to shed light upon the elements required to direct the construction of the division septum and also upon how the initiation of septum formation may be coordinated with mitosis. Trends Cell Biol, 1991 Nov, 1(5), 117 - 21 Cyclin-dependent kinases: a new cell cycle motif? Pines J, Hunter T. Increasing evidence suggests that the eukaryotic cell cycle is controlled at several checkpoints by different members of a novel class of protein kinase, the cyclin-dependent kinases . To phosphorylate their substrates, these enzymes bind to proteins of the cyclin family--proteins that are synthesized and degraded at specific points in each cell cycle . The most well known of these kinases is the 34 kDa product of the cdc2 gene in fission yeast, p34cdc2; however, several putative cyclin-dependent kinases have now been cloned or identified . Some of these closely resemble p34cdc2 . Here we review these new proteins, their potential roles in the cell cycle and the cyclins with which they may interact. Trends Cell Biol, 1995 May, 5(5), 197 - 201 In search of a function for centrins; Schiebel E et al.; Spindle pole bodies, basal bodies and centrosomes are morphologically quite different structures that nevertheless perform similar microtubule-organizing functions in diverse cell types . The recent discoveries that both centrins and gamma-tubulin are common components of these structures suggest a molecular basis for their common functions . The role of centrins is just beginning to be investigated . These filament-associated proteins bind Ca2+ . The filaments contract at least in certain circumstances by an ATP-independent mechanism . However, yeast centrin is clearly involved in the duplication of the spindle pole body . A common molecular mechanism may underlie these two apparently different functions. Trends Cell Biol, 1995 May, 5(5), 189 - 91 The importance of importin; Adam SA; The accumulation of karyophilic proteins in the nucleus requires cytoplasmic factors . Cell-free systems that reconstitute nuclear protein import have been used to identify several of these factors and to define the biochemical requirements for the import process . Recently, one factor has been purified and cloned from Xenopus and identified as a homologue of the 'suppressor of RNA polymerase l' (SRP1) gene originally described in yeast . This factor belongs to a closely related group of proteins that may share similar functions in nuclear protein transport. Plant J, 2004 Feb, 37(3), 426 - 38 Definition and interactions of a positive regulatory element of the Arabidopsis INNER NO OUTER promoter; Meister RJ et al.; INNER NO OUTER (INO) expression is limited to the abaxial cell layer of the incipient and developing outer integument in Arabidopsis ovules . Using deletion analysis of the previously defined INO promoter (P-INO), at least three distinct regions that contribute to the endogenous INO expression pattern were identified . One such positive element, designated POS9, which comprises at least three distinct subelements, was found to include sufficient information to duplicate the INO expression pattern when four or more copies were used in conjunction with a heterologous minimal promoter . While known regulators of INO, including INO, SUPERMAN, BELL1, and AINTEGUMENTA, did not detectably interact with POS9 in yeast one-hybrid assays, two groups of proteins that interact specifically with POS9 were identified in one-hybrid library screens . Members of one group include C2H2 zinc finger motifs . Members of the second group contain a novel, conserved DNA-binding region and were designated the BASIC PENTACYSTEINE (BPC) proteins on the basis of conserved features of this region . The BPC proteins are nuclear localized and specifically bind in vitro to GA dinucleotide repeats located within POS9 . The widespread expression patterns of the BPCs and the large number of GA repeat potential target sequences in the Arabidopsis genome indicate that BPC proteins may affect expression of genes involved in a variety of plant processes. Biochem J, 2004 May 1, 379(Pt 3), 687 - 95 DES2 protein is responsible for phytoceramide biosynthesis in the mouse small intestine; Omae F et al.; The C-4 hydroxylation of sphinganine and dihydroceramide is a rate-limiting reaction in the biosynthesis of phytosphingolipids . Mouse DES1 (MDES1) cDNA homologous to the Drosophila melanogaster degenerative spermatocyte gene-1 (des-1) cDNA leads to sphingosine Delta4-desaturase activity, and another mouse homologue, MDES2, has bifunctional activity, producing C-4 hydroxysphinganine and Delta4-sphingenine in yeast {Ternes, Franke, Zahringer, Sperling and Heinz (2002) J . Biol . Chem . 277, 25512-25518} . Here, we report the characterization of mouse DES2 (MDES2) using an in vitro assay with a homogenate of COS-7 cells transfected with MDES2 cDNA and N -octanoyl-sphinganine and sphinganine as substrates . MDES2 protein prefers dihydroceramide as a substrate to sphinganine, and exhibits dihydroceramide Delta4-desaturase and C-4 hydroxylase activities . MDES2 mRNA content was high in the small intestine and abundant in the kidney . In situ hybridization detected signals of MDES2 mRNA in the crypt cells . Immunohistochemistry using an anti-MDES2 peptide antibody stained the crypt cells and the adjacent epithelial cells . These results suggest that MDES2 is the dihydroceramide C-4 hydroxylase responsible for the biosynthesis of enriched phytosphingoglycolipids in the microvillous membranes of intestinal epithelial cells. Proteomics, 2004 Jan, 4(1), 226 - 34 In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics; Ippel JH et al.; Isotope labelling of proteins is important for progress in the field of structural proteomics . It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins . The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media . This method has several drawbacks . Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes . The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins . A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv . Elkana) . Two plants were grown at low costs using potassium-{(15)N}-nitrate as the sole nitrogen source . At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled . Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers . NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure . The presented method of isotope labelling is easily modified to label other plants . Its envisioned impact in the field of structural proteomics of plants is discussed. Mol Cell Biol, 2004 Feb, 24(3), 1174 - 87 Human RNPS1 and its associated factors: a versatile alternative pre-mRNA splicing regulator in vivo; Sakashita E et al.; Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently . To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library . Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2 beta (hTra2 beta; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles) . The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2 beta, respectively . Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo . Overexpression of RNPS1 in HeLa cells induced exon skipping in a model beta-globin pre-mRNA and a human tra-2 beta pre-mRNA . Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase gamma-subunit pre-mRNA . The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54 . RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs. Mol Cell Biol, 2004 Feb, 24(3), 1081 - 95 Extracellular signal-regulated kinase 2 interacts with and is negatively regulated by the LIM-only protein FHL2 in cardiomyocytes; Purcell NH et al.; The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis . The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized . Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes . This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells . In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form . ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein . Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2 . The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2 . However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter . Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation . Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling. Mol Cell Biol, 2004 Feb, 24(3), 1058 - 69 Identification of MoKA, a novel F-box protein that modulates Krüppel-like transcription factor 7 activity; Smaldone S et al.; KLF7, a member of the Kruppel-like transcription factor family, is believed to regulate neurogenesis and cell cycle progression . Here, a yeast two-hybrid screen for KLF7 cofactors in the developing nervous system identified a novel 140-kDa protein named MoKA, for modulator of KLF7 activity . Interaction between MoKA and KLF7 was confirmed by the in vitro glutathione S-transferase pull-down assay and by coimmunoprecipitation of the proteins overexpressed in mammalian cells . Functional assays documented that MoKA is a KLF7 coactivator, and in situ hybridizations identified the developing nervous system and the adult testes as two sites of MoKA and Klf7 coexpression . Chromatin immunoprecipitation experiments demonstrated KLF7 binding to the p21(WAF1/Cip1) gene while transient transfection assays documented KLF7 stimulation of the p21(WAF1/Cip1) proximal promoter . Additional tests revealed that distinct structural motifs of MoKA direct interaction with KLF7 and shuttling between the nucleus and cytoplasm of asynchronously cycling cells . Altogether, our results strongly suggest that MoKA and KLF7 interact functionally to regulate gene expression during cell differentiation and identify the cell cycle regulator p21(WAF1/Cip1) as one of the targeted genes. Trends Plant Sci, 2004 Jan, 9(1), 33 - 41 Getting the message across: how do plant cells exchange macromolecular complexes? Oparka KJ. A major pathway for macromolecular exchange in plants involves plasmodesmata (PD), the small pores that connect adjoining cells . This article considers the nature of macromolecular complexes (MCs) that pass through PD and the pathways and mechanisms that guide them to the PD pore . Recent cell-biological studies have identified proteins involved in the directional trafficking of MCs to PD, and yeast two-hybrid studies have isolated novel host proteins that interact with viral movement proteins . Collectively, these studies are yielding important clues in the search for components that compose the plant intercellular MC trafficking pathway . Here, they are placed in the context of a functional model that links the cytoskeleton, chaperones and secretory pathway in the intercellular trafficking of MCs. Exp Cell Res, 2004 Feb 1, 293(1), 43 - 57 The human small glutamine-rich TPR-containing protein is required for progress through cell division; Winnefeld M et al.; Eukaryotic organisms from yeast to human harbor genes encoding the small glutamine-rich tetratricopeptide repeat-containing (SGT) protein . Work presented here demonstrated the presence of human SGT (hSGT) protein in a panel of human cell lines and throughout the cell cycle . To identify cellular processes in which hSGT is involved, knock down populations were analyzed which were generated through transfection of hsgt-specific small interfering RNA . Most strikingly, depletion of hSGT led to reduced proliferation of the affected cell populations while the mitotic index was increased . Time-lapse video microscopy revealed that cells from hSGT-depleted populations were unable to complete cell division due to mitotic arrest which was frequently followed by cell death . Further evidence for a role in cell division was given by the accumulation of hSGT in the midzone and the midbody, and by a mitosis-specific migration pattern of hSGT as detected by Western blotting after SDS-PAGE or two-dimensional gel electrophoresis . In conclusion, results obtained in this study demonstrate that hSGT protein is a constitutive component of all human cell lines tested and that this protein is essential for successful completion of cell division. Eur J Biochem, 2004 Feb, 271(3), 628 - 36 Interaction between the 2'-5' oligoadenylate synthetase-like protein p59 OASL and the transcriptional repressor methyl CpG-binding protein 1; Andersen JB et al.; The human 2'-5' oligoadenylate synthetases (OAS) form a conserved family of interferon-induced proteins consisting of four genes: OAS1, OAS2, OAS3 and the 2'-5' oligoadenylate synthetase-like gene (OASL) . When activated by double-stranded RNA, OAS1-3 polymerize ATP into 2'-5'-linked oligoadenylates; 2'-5'-linked oligoadenylates, in turn, activate a latent endoribonuclease that degrades viral and cellular RNAs . In contrast, while the p59 OASL protein is highly homologous to the OAS family (45% identity), its 350 amino acid N-terminal domain lacks 2'-5' oligoadenylate synthetase activity . A C-terminal 164 amino acid domain, which is 30% homologous to a tandem repeat of ubiquitin, further distinguishes the p59 OASL protein and suggests that it serves a biological role which is distinct from other OAS family members . To dissect the function of p59 OASL, we utilized the yeast two-hybrid system to identify interacting proteins . Methyl CpG-binding protein 1 (MBD1), which functions as a transcriptional repressor, was identified as a strong p59 OASL interactor . Interestingly, like p59 OASL, transcription of the MBD1 gene was induced by interferon, indicating that these genes are co-ordinately regulated . The interaction was confirmed in vitro and in vivo and was mapped to the ubiquitin-like domain of p59 OASL . The p59 OASL-MBD1 interaction was specific, because p59 OASL did not interact with any of the other MBD family members and MBD1 did not interact with OAS1 . These findings link the p59 OASL with MBD1 transcriptional control in the context of an interferon-stimulated cell, and provide the basis for future studies to examine the functional role of this interaction. Comp Med, 2003 Dec, 53(6), 642 - 8 Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9; Cho KI et al.; Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner . Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells . A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice . The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9 . Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively . Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281 . On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region . The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes . The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus . Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans. Nippon Yakurigaku Zasshi, 2003 Nov, 122 Suppl, 30P - 32P {Functional analysis of SIR2}; Horio Y et al.; The yeast silent information regulator 2 (Sir2) is an NAD-dependent histone deacetylase and silences transcription at the mating type loci, telomeres and the ribosomal DNA . Over-expression of Sir2 extends the life span of yeast and C . elegans, while Sir2 knockout shortened yeast life span to about 50% . Mammalian cells also express several Sir2 homologues . However, most of physiological functions of Sir2 homologues seem to be unknown . We found that Sir2 alpha (SIRT1) was highly expressed during embryogenesis . In the adult mouse brain, ependymal cells and some cells of subventricular zone expressed Sir2 alpha . To identify proliferating neural cells in the adult brain, BrdU was administered in the drinking water for 2 weeks . BrdU-positive and nestin-positive cells expressed Sir2 alpha . Furthermore, neurosphere cultured from E14 mouse striatum expressed Sir2 alpha . The expression of Sir2 alpha in neurosphere disappeared after differentiation . Nicotinamide, splitomicin and sirtinol, potent inhibitors of Sir2 alpha, inhibited the growth of neurosphere . FACS analysis showed that sirtinol did not increase sub-G1 population of cells . Differentiation of neurosphere into neuron and oligodendrocyte was inhibited by the addition of nicotinamide, splitomicin or sirtinol in the differentiation medium . These results suggest that Sir2 alpha has a pivotal role in the proliferation and differentiation of neural stem cells. EMBO J, 2004 Jan 28, 23(2), 439 - 49 Epub 2004 Jan 15. The Arabidopsis homologue of Xrcc3 plays an essential role in meiosis; Bleuyard JY et al.; The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair . Rad51-like proteins have been identified from yeast (Rad55, Rad57 and Dmc1) to vertebrates (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3 and Dmc1) . These Rad51-like proteins are all members of the genetic recombination and DNA damage repair pathways . The sequenced genome of Arabidopsis thaliana encodes putative homologues of all six vertebrate Rad51-like proteins . We have identified and characterized an Arabidopsis mutant defective for one of these, AtXRCC3, the homologue of XRCC3 . atxrcc3 plants are sterile, while they have normal vegetative development . Cytological observation shows that the atxrcc3 mutation does not affect homologous chromosome synapsis, but leads to chromosome fragmentation after pachytene, thus disrupting both male and female gametogenesis . This study shows an essential role for AtXrcc3 in meiosis in plants and possibly in other higher eukaryotes . Furthermore, atxrcc3 cells and plants are hypersensitive to DNA-damaging treatments, supporting the involvement of this Arabidopsis Rad51-like protein in recombinational repair. Cancer Biol Ther, 2004 Mar, 3(3), 289 - 92 Epub 2004 Mar 10. Telomerase is frequently activated in tumors with microsatellite instability; Ibanez de Caceres I et al.; Telomeres are specialized structures at the ends of eukaryotic chromosomes that are required for the complete replication and stability of naturally occurring chromosome ends . Telomere stabilization is critical for the unlimited cellular proliferation that is necessary for tumor formation . While most tumors achieve telomere stabilization through activation of telomerase, a subset of tumors utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain chromosome termini . Tumors utilizing ALT for telomere preservation will likely be refractory to treatment with telomerase inhibitors . Furthermore, tumors carrying mutations that predispose a cell to utilize ALT may activate this pathway when challenged by telomerase inhibition . Mutation of the mismatch repair (MMR) pathway enhances telomerase independent survival in yeast, with the survivors using recombination-based pathways for telomere maintenance . One possibility is that mutation of the MMR pathways alleviates suppression of recombination, thereby abrogating the need for telomerase activation . If true, one might predict an increased frequency of tumors harboring MMR mutation to use ALT for telomere maintenance . Here we characterized tumors with and without MMR mutation for the presence of telomerase activity versus ALT . We found similarly frequent activation of telomerase in tumors with and without MMR mutation, suggesting that human tumors with MMR mutation may respond favorably to treatment with telomerase inhibitors. Cell Cycle, 2004 Mar, 3(3), 300 - 4 Epub 2004 Mar 01. Genetic dissection of mammalian Cdc7 kinase: cell cycle and developmental roles; Kim JM et al.; Cdc7, originally discovered by Hartwell as a budding yeast mutant that arrests immediately before the onset of S phase, is conserved through evolution and plays essential roles in initiation of mitotic DNA replication . Inducible inactivation of Cdc7 in mouse embryonic stem cells leads to rapid cessation of DNA synthesis and the subsequent activation of checkpoint responses, resulting in p53 activation and eventually p53-mediated apoptosis . This indicates a requirement of Cdc7 kinase for ongoing replication of mammalian genomes, and loss of Cdc7 kinase presumably generates arrested replication fork signals . Cdc7-/- mice or embryonic fibroblast cells (MEFs) expressing a low level of transgene-encoded Cdc7 protein are viable but exhibit reduced body size with impaired germ cell development and decreased cell proliferation . Interestingly, these phenotypes are largely corrected by the presence of an additional copy of the transgene, resulting in increased level of Cdc7 expression . This indicates the requirement of a critical level of Cdc7 for normal cell proliferation and development of specific organs . These results from mammals will be discussed in conjunction with the pleiotropic effects of Cdc7 mutation observed in yeasts. J Biol Chem, 2004 Apr 2, 279(14), 13540 - 6 Epub 2004 Jan 15. Organization and function of the small Tim complexes acting along the import pathway of metabolite carriers into mammalian mitochondria; Muhlenbein N et al.; Tim9, Tim10a, and Tim10b are members of the family of small Tim proteins located in the intermembrane space of mammalian mitochondria . In yeast, members of this family act along the TIM22 import pathway during import of metabolite carriers and other integral inner membrane proteins . Here, we show that the human small proteins form two distinct hetero-oligomeric complexes . A 70-kDa complex that contains Tim9 and Tim10a and a Tim9-10a-10b that is part of a higher molecular weight assembly of 450 kDa . This distribution among two complexes suggests Tim10b to be the functional homologue of yeast Tim12 . Both human complexes are tightly associated with the inner membrane and, compared with yeast, soluble 70-kDa complexes appear to be completely absent in the intermembrane space . Thus, the function of soluble 70-kDa complexes as trans-site receptors for incoming carrier proteins is not conserved from lower to higher eukaryotes . During import, the small Tim complexes directly interact with human adenine nucleotide translocator (ANT) in transit in a metal-dependent manner . For insertion of carrier preproteins into the inner membrane, the human small Tim proteins directly interact with human Tim22, the putative insertion pore of the TIM22 translocase . However, in contrast to yeast, only a small fraction of Tim9-Tim10a-Tim10b complex is in a stable association with Tim22 . We conclude that different mechanisms and specific requirements for import and insertion of mammalian carrier preproteins have evolved in higher eukaryotes. Exp Hematol, 2004 Jan, 32(1), 113 - 21 Coexistence of phosphotyrosine-dependent and -independent interactions between Cbl and Bcr-Abl; Gaston I et al.; Cbl is one of the major tyrosine-phosphorylated proteins in Bcr-Abl-expressing cells . A direct association between the SH2 domain of Bcr-Abl and tyrosine-phosphorylated Cbl has been demonstrated . The purpose of this study was to determine if and how unphosphorylated Cbl and Bcr-Abl may associate.Interactions between Cbl and Bcr-Abl were investigated in yeast two- and three-hybrid systems, gel overlay assays, and immunoprecipitates from mammalian cells expressing wild-type and the Y177F mutant of Bcr-Abl.No direct interaction between Bcr-Abl and unphosphorylated Cbl was observed . Bcr-Abl did, however, associate with Grb2, an adaptor protein that binds tyrosine 177 of Bcr-Abl . Additionally, Grb2 interacted with Cbl . In a yeast three-hybrid assay, Grb2 mediated an interaction between Cbl and Bcr-Abl that was dependent on a functional Grb2 binding site . This interaction was confirmed in vitro using purified proteins . In cells expressing Bcr-Abl with a mutation in the Grb2 binding site, binding of Cbl to Bcr-Abl was significantly reduced, but Cbl tyrosine phosphorylation was maintained . Imatinib treatment of these cells further reduced but did not abrogate Cbl binding, reflecting residual kinase activity.Multiple phosphotyrosine-dependent and -independent interactions stabilize the interaction between Cbl and Abl . Grb2 or another, yet unidentified, protein may mediate an initial interaction between Cbl and Bcr-Abl that is independent of Cbl tyrosine phosphorylation . Following this initial interaction, Cbl can then become tyrosine phosphorylated and interact with the SH2 domain of Bcr-Abl, further stabilizing the complex. J Pharmacol Exp Ther, 2004 Apr, 309(1), 340 - 7 Epub 2004 Jan 14. Cyclohexyl-octahydro-pyrrolo{1,2-a}pyrazine-based inhibitors of human N-myristoyltransferase-1; French KJ et al.; N-myristoyltransferase (NMT) is an emerging therapeutic target that catalyzes the attachment of myristate to the N terminus of an acceptor protein . We have developed a medium-throughput assay for screening potential small molecule inhibitors of human NMT-1 consisting of recombinant enzyme, biotinylated peptide substrate, and {3H}myristoyl-CoA . Approximately 16,000 diverse compounds have been evaluated, and significant inhibition of NMT was found with 0.8% of the compounds . From these hits, we have identified the cyclohexyl-octahydropyrrolo{1,2-a}pyrazine (COPP) chemotype as inhibitory toward human NMT-1 . Thirty-two compounds containing this substructure inhibited NMT-1, with IC(50) values ranging from 6 microM to millimolar concentrations, and a quantitative structure-activity relationship equation (r(2) = 0.72) was derived for the series . The most potent inhibitor (24, containing 9-ethyl-9H-carbazole) demonstrated competitive inhibition for the peptide-binding site of NMT-1 and noncompetitive inhibition for the myristoyl-CoA site . Computational docking studies using the crystal structure of the highly homologous yeast NMT confirmed that 24 binds with excellent complementarity to the peptide-binding site of the enzyme . To evaluate the ability of 24 to inhibit NMT activity in intact cells, monkey CV-1 cells expressing an N-myristoylated green fluorescent protein (GFP) fusion protein were treated with a known NMT inhibitor or with 24 . Each compound caused the redistribution of GFP from the plasma membrane to the cytosol . Furthermore, 24 inhibits cancer cell proliferation at doses similar to those that inhibit protein myristoylation . Overall, these studies establish an efficient assay for screening for inhibitors of human NMT and identify a novel family of inhibitors that compete at the peptide-binding site and have activity in intact cells. Water Res, 2004 Feb, 38(3), 733 - 9 Effects of chlorine on the decrease of estrogenic chemicals; Lee BC et al.; The effects of chlorination on the elimination of three estrogenic chemicals such as 17beta-estradiol, nonylphenol and bis-phenol A were investigated using yeast two-hybrid assay (YTA), estrogen receptor (ER) competition assay (ER-CA), and high-performance liquid chromatography/mass spectrometry (LC/MS) . The results of YTA, ER-CA and the analysis of LC/MS indicated that the estrogenic activity of the above-mentioned three endocrine disruptors were significantly reduced as a result of chlorination . The decrease in estrogenic activity paralleled a decrease in estrogenic chemicals under the influence of free chlorine . One common characteristic of estrogenic chemicals is the presence of a phenolic ring . Considering that a phenolic ring is likely to undergo some sort of transformation in an aqueous chlorination solution, the above-mentioned results may be applied to the rest of the estrogenic chemicals in natural waters. Dev Cell, 2004 Jan, 6(1), 69 - 78 Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis; Nakamura A et al.; Translational control is a critical process in the spatio-temporal restriction of protein production . In Drosophila oogenesis, translational repression of oskar (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation . This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive . Here, we report that an ovarian protein, Cup, is required to repress precocious osk translation . Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins . A mutant Cup protein lacking this sequence fails to repress osk translation in vivo . Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner . These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex. Dev Cell, 2004 Jan, 6(1), 1 - 3 Molecular rearrangements within the nuclear pore complexes: a new way to regulate nucleocytoplasmic transport; Doye V; Until now, regulation of nucleocytoplasmic transport of macromolecules has been thought to occur mainly through modifications of the cargo molecules . However, in the December 26 issue of Cell, Makhnevych et al . describe a novel mechanism controlling cell cycle progression in yeast that involves subtle molecular rearrangements within the nuclear pore complexes. Genes Cells, 2004 Jan, 9(1), 1 - 14 Mammalian septin Sept2 modulates the activity of GLAST, a glutamate transporter in astrocytes; Kinoshita N et al.; Sept2 is a member of the septin family of GTPases . Septins form filaments in a GTP-form dependent manner, and are involved in cytokinesis from yeast to mammals; however, some mammalian septins, including Sept2, are expressed in the brain, a tissue in which almost all the cells are postmitotic . Recently, some functions of mammalian septin other than cytokinesis such as vesicle transport have been reported . However, mammalian septin's physiological functions are still unclear . The present study revealed that Sept2 co-localizes with the astrocyte glutamate transporter GLAST in the Bergmann glial processes facing axons and synapses . Biochemical analyses demonstrated that Sept2 bound directly to the carboxy-terminal region of GLAST in a GDP-form dependent manner . Expression of constitutive GDP-form Sept2 mutant reduced the glutamate uptake activity of GLAST via internalization of GLAST from cell surface . Thus Sept2 may regulate GLAST-mediated glutamate uptake by astrocytes, which is important for appropriate transmitter signalling in the cerebellum. Pflugers Arch, 2004 Mar, 447(6), 840 - 4 Epub 2004 Jan 14. The mitochondrial outer membrane is not a major diffusion barrier for ADP in mouse heart skinned fibre bundles; Kongas O et al.; The response of mitochondrial oxygen consumption to ADP in saponin-skinned cardiac fibre bundles has an apparent Km an order of magnitude higher than that in isolated mitochondria . Here we report that incubating skinned cardiac fibre bundles from wild-type mice or double-knockout mice lacking both cytosolic and mitochondrial creatine kinase (CK) with CK and creatine or with yeast hexokinase and glucose as extramitochondrial ADP-producing systems decreases the apparent Km of the bundles for ADP severalfold . We conclude that the affinity of mitochondria for ADP in mouse heart is of the same order of magnitude as that of isolated mitochondria, while the high apparent Km of the bundles is caused by diffusion gradients outside the mitochondria. Nucleic Acids Res, 2004 Jan 13, 32(1), 307 - 15 Print 2004. Secondary structure models of the nuclear internal transcribed spacer regions and 5.8S rRNA in Calciodinelloideae (Peridiniaceae) and other dinoflagellates; Gottschling M et al.; Secondary structure models of the 5.8S rRNA and both internal transcribed spacers (ITS1 and ITS2) are proposed for Calciodinelloideae (Peridiniaceae) and are also plausible for other dinoflagellates . The secondary structure of the 5.8S rRNA corresponds to previously developed models, with two internal paired regions and at least one 5.8S rRNA-28S rRNA interaction . A general secondary structure model of ITS1 for Calciodinelloideae (and other dinoflagellates), consisting of an open multibranch loop with three major helices, is proposed . The homology of these paired regions with those found in other taxa, published in previous studies (e.g . yeast, green algae and Platyhelmithes) remains to be determined . Finally, a general secondary structure model of ITS2 for Calciodinelloideae (and other dinoflagellates) is reconstructed . Based on the 5.8S rRNA-28S rRNA interaction, it consists of a closed multibranch loop, with four major helices . At least helix III and IV have homology with paired regions found in other eukaryotic taxa (e.g . yeast, green algae and vertebrates) . Since the secondary structures of both ITS regions are more conserved than the nucleotide sequences, their analysis helps in understanding molecular evolution and increases the number of structural characters . Thus, the structure models developed in this study may be generally useful for future phylogenetic analyses. Hum Mol Genet, 2004 Mar 1, 13(5), 495 - 507 Epub 2004 Jan 13. Muscleblind protein, MBNL1/EXP, binds specifically to CHHG repeats; Kino Y et al.; Myotonic dystrophy (DM) type 1 is caused by an expansion of a CTG repeat in the DMPK gene and type 2 by a CCTG repeat in the ZNF9 gene . Previous reports have suggested that transcripts containing expanded CUG/CCUG repeats might have toxic gain-of-function effects, probably affecting the function of RNA-binding proteins in the pathogenesis of DM . Here, it was attempted to compare the RNA-binding properties of three proteins, CUG-BP, MBNL1/EXP and PKR, which have previously been suggested to interact with CUG repeats . MBNL1, but not CUG-BP or PKR, interacted with both CUG and CCUG repeats in a yeast three-hybrid system . By using various synthetic RNAs, it was found that MBNL1 specifically interacts with repetitive sequences summarized as CHHG and CHG repeats, where H is A, U or C . Interestingly, MBNL1 did not interact with a genuine double-stranded RNA comprising CAG/CUG repeats, suggesting that MBNL1 prefers bulge-containing double-stranded RNAs . Deletion analysis indicates a difference in RNA-binding abilities among splice variants of MBNL1 . It was also found that MBNL1 can bind to repetitive motifs in ZNF9, which contain a minimal length of CCUG repeats with non-CCUG insertions. J Biol Chem, 2004 Mar 26, 279(13), 12685 - 94 Epub 2004 Jan 13. Characterization of the Drosophila sphingosine kinases and requirement for Sk2 in normal reproductive function; Herr DR et al.; Sphingosine kinase is a highly conserved enzyme that catalyzes the synthesis of sphingosine 1-phosphate and reduces cellular levels of sphingosine and ceramide . Although ceramide is pro-apoptotic and sphingosine is generally growth-inhibitory, sphingosine 1-phosphate signaling promotes cell proliferation, survival, and migration . Sphingosine kinase is thus in a strategic position to regulate important cell fate decisions which may contribute to normal animal development . To facilitate studies examining the potential role of sphingosine kinase and long chain base metabolism in Drosophila development, we characterized two putative Drosophila sphingosine kinase genes, Sk1 and Sk2 . Both genes functionally and biochemically complement a yeast sphingosine kinase mutant, express predominantly cytosolic activities, and are capable of phosphorylating a range of endogenous and non-endogenous sphingoid base substrates . The two genes demonstrate overlapping but distinct temporal and spatial expression patterns in the Drosophila embryo, and timing of expression is consistent with observed changes in long chain base levels throughout development . A null Sk2 transposon insertion mutant demonstrated elevated long chain base levels, impaired flight performance, and diminished ovulation . This is the first reported mutation of a sphingosine kinase in an animal model; the associated phenotypes indicate that Sk1 and Sk2 are not redundant in biological function and that sphingosine kinase is essential for diverse physiological functions in this organism. J Biol Chem, 2004 Apr 9, 279(15), 15091 - 5 Epub 2004 Jan 13. Alpha1-syntrophin modulates turnover of ABCA1; Munehira Y et al.; ABCA1 (ATP-binding cassette transporter A1) mediates the release of cellular cholesterol and phospholipid to form high density lipoprotein . Functions of ABCA1 are highly regulated at the transcriptional and post-transcriptional levels, and the synthesized ABCA1 protein turns over rapidly with a half-life of 1-2 h . To examine whether the functions of ABCA1 are modulated by associated proteins, a yeast two-hybrid library was screened with the C-terminal 120 amino acids of ABCA1 . Two PDZ (PSD95-Discs large-ZO1) proteins, alpha1-syntrophin and Lin7, were found to interact with ABCA1 . Immunoprecipitation revealed that alpha1-syntrophin interacted with ABCA1 strongly and that the interaction was via the C-terminal three amino acids SYV of ABCA1 . Co-expression of alpha1-syntrophin in human embryonic kidney 293 cells retarded degradation of ABCA1 and made the half-life of ABCA1 five times longer than in the cells not expressing alpha1-syntrophin . This effect is not common among PDZ-containing proteins interacting with ABCA1, because Lin7, which was also found to interact with the C terminus region of ABCA1, did not have a significant effect on the half-life of ABCA1 . Co-expression of alpha1-syntrophin significantly increased the apoA-I-mediated release of cholesterol . ABCA1 was co-immunoprecipitated with alpha1-syntrophin from mouse brain . These results suggest that alpha1-syntrophin is involved in intracellular signaling, which determines the stability of ABCA1 and modulates cellular cholesterol release. J Biol Chem, 2004 Mar 26, 279(13), 13027 - 34 Epub 2004 Jan 13. RanBPM is a phosphoprotein that associates with the plasma membrane and interacts with the integrin LFA-1; Denti S et al.; Integrin adhesion receptors can act as signaling receptors that transmit information from the extracellular environment to the interior of the cell, affecting many fundamental cellular processes, such as cell motility, proliferation, differentiation, and survival . Integrin signaling depends on the formation of organized sub-membrane complexes that comprise cytoskeletal, adapter, and signaling molecules . The identification of molecules that interact with the cytoplasmic domain of integrins has been the focus of research aimed to elucidating the mechanistic basis of integrin signal transduction . We have identified RanBPM as a novel interactor of the beta(2) integrin LFA-1 in a yeast-two-hybrid screen . In the same assay, RanBPM also interacted with the beta(1) integrin cytoplasmic domain . We demonstrate that RanBPM is a peripheral membrane protein and that integrins and RanBPM interact in vitro and in vivo and co-localize at the cell membrane . We find that RanBPM is phosphorylated on serine residues; phosphorylation of RanBPM is increased by stress stimuli and decreased by treatment with the p38 kinase inhibitor SB203580 . Transfection of RanBPM synergizes with LFA-1-mediated adhesion in the transcriptional activation of an AP-1-dependent promoter, indicating that the two proteins interact functionally as well . We suggest that RanBPM may constitute a molecular scaffold that contributes to coupling LFA-1 and other integrins with intracellular signaling pathways. J Biol Chem, 2004 Apr 16, 279(16), 16670 - 6 Epub 2004 Jan 13. Structure and DNA-binding sites of the SWI1 AT-rich interaction domain (ARID) suggest determinants for sequence-specific DNA recognition; Kim S et al.; ARID (AT-rich interaction domain) is a homologous family of DNA-binding domains that occur in DNA-binding proteins from a wide variety of species, ranging from yeast to nematodes, insects, mammals, and plants . SWI1, a member of the SWI/SNF protein complex that is involved in chromatin remodeling during transcription, contains the ARID motif . The ARID domain of human SWI1 (also known as p270) does not select for a specific DNA sequence from a random sequence pool . The lack of sequence specificity shown by the SWI1 ARID domain stands in contrast to the other characterized ARID domains, which recognize specific AT-rich sequences . We have solved the three-dimensional structure of human SWI1 ARID using solution NMR methods . In addition, we have characterized nonspecific DNA binding by the SWI1 ARID domain . Results from this study indicate that a flexible, long, internal loop in the ARID motif is likely to be important for sequence-specific DNA recognition . The structure of the human SWI1 ARID domain also represents a distinct structural subfamily . Studies of ARID indicate that the boundary of DNA binding structural and functional domains can extend beyond the sequence homologous region in a homologous family of proteins . Structural studies of homologous domains such as the ARID family of DNA-binding domains should provide information to better predict the boundary of structural and functional domains in structural genomic studies. Biol Cell, 2003 Dec, 95(9), 595 - 602 The Drosophila kinesin-I associated protein YETI binds both kinesin subunits; Wisniewski TP et al.; The microtubule-based motor kinesin-I is essential for the intracellular transport of membrane-bound organelles in the Drosophila nervous system and female germ line . A number of studies have demonstrated that kinesin-I binds to its intracellular cargos through protein-protein interactions between the kinesin tail domain and proteins on the cargo surface . To identify proteins that mediate or regulate kinesin-cargo interactions, we have performed yeast two-hybrid screens of a Drosophila embryonic cDNA library, using the tetratricopeptide repeats of the kinesin light chain and amino acids 675-975 of the kinesin heavy chain as baits . One of the proteins we have identified is YETI . Interestingly, YETI has the unique ability to bind specifically to both subunits of the kinesin tail domain . An epitope-tagged YETI fusion protein, when expressed in Drosophila S2 cultured cells, binds to kinesin-I in copurification assays, suggesting that YETI-kinesin-I interactions are context-independent . Immunostaining of cultured cells expressing YETI shows that YETI accumulates in the nucleus and cytosol . YETI is evolutionarily conserved, and its yeast homolog (AOR1) may have a role in regulating cytoskeletal dynamics or intracellular transport . Collectively, these results demonstrate that YETI interacts with both kinesin subunits of the kinesin tail domain, and is potentially involved in kinesin-dependent transport pathways. J Gen Virol, 2004 Jan, 85(Pt 1), 119 - 30 The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7; Kay-Jackson PC et al.; The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function . Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells . In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments . Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction . A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues . Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7 . In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories . These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network . Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals. J Biol Chem, 2004 Mar 26, 279(13), 12734 - 43 Epub 2004 Jan 12. Membrane-type 1 matrix metalloproteinase cytoplasmic tail-binding protein-1 is a new member of the Cupin superfamily . A possible multifunctional protein acting as an invasion suppressor down-regulated in tumors; Uekita T et al.; Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is an enzyme that promotes tumor cell invasion in tissues . Although the proteolytic activity of MT1-MMP is indispensable for invasion, it is also regulated by functions of the cytoplasmic tail . In this study we obtained a new human gene whose product binds to the tail sequence in yeast . The product, MTCBP-1, is a 19-kDa protein that belongs to the newly proposed Cupin superfamily composed of proteins with diverse functions . MTCBP-1 expressed in cells formed a complex with MT1-MMP and co-localized at the membrane . It was also detected in both the cytoplasm and nucleus, where MT1-MMP does not exist . In human tumor cell lines MTCBP-1 expression was significantly low compared with non-transformed fibroblasts, and enforced expression of MTCBP-1 inhibited the activity of MT1-MMP in promoting cell migration and invasion . MTCBP-1 showed significant homology to the bacterial aci-reductone dioxygenase, which is an enzyme in methionine metabolism . The C-terminal part of MTCBP-1 is identical to Sip-L, which is reported to be important for human hepatitis C virus replication . Thus, MTCBP-1 may have multiple functions other than the regulation of MT1-MMP, which presumably depends on the subcellular compartment. J Biol Chem, 2004 Apr 2, 279(14), 13934 - 43 Epub 2004 Jan 12. Marlin-1, a novel RNA-binding protein associates with GABA receptors; Couve A et al.; GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system . Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established . Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons . Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons . Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons . Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1 . Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers . Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission. J Biol Chem, 2004 Apr 2, 279(14), 13953 - 61 Epub 2004 Jan 12. Gene expression changes associated with the endoplasmic reticulum stress response induced by microsomal cytochrome p450 overproduction; Szczesna-Skorupa E et al.; Induction of drug-metabolizing microsomal cytochromes p450 (p450s) results in a striking proliferation of the smooth endoplasmic reticulum (ER) . Overexpression of P450s in yeast and cultured cells produces a similar response . The signals mediating this process are not known but probably involve signal transduction pathways involved in the unfolded protein response (UPR) or the ER overload response (EOR) . We have examined the temporal response of specific genes in these pathways and genes globally to overexpression of p450 in cultured cells . Activity of NFkappaB, an EOR component, was substantially increased by overexpression of full-length p450 2C2 or a chimera with the 28-amino acid signal anchor sequence of p450 2C2 in HepG2 cells, and the activation correlated temporally with the accumulation of p450 in the cells . In the UPR pathway, activation of the transcription factor XBP1 by IRE1 also correlated with the accumulation of p450 in the cells, and in contrast, maximum activation of the BiP/grp78 promoter preceded the accumulation . Differential effects of expression of p450 on apoptosis were observed in nonhepatic COS1 and hepatic HepG2 cells . In COS1 cells, apoptosis was induced, and this correlated with sustained activation of the pro-apoptotic JNK pathway, induction of CHOP, and an absence of the increased NFkappaB activity . In HepG2 cells, JNK was only transiently activated, and CHOP expression was not induced . As assessed by DNA microarray analysis, up-regulation of signaling genes was predominant including those involved in anti-apoptosis and ER stress . These results suggest that both the EOR and UPR pathways are involved in the cellular response to induction of p450 expression and that in hepatic cells genes are also induced to block apoptosis, which may be a physiologically relevant response to prevent cell death during xenobiotic induced expression of p450 in the liver. Environ Sci Technol, 2003 Dec 15, 37(24), 5665 - 70 Products of aqueous chlorination of 17beta-estradiol and their estrogenic activities; Hu J et al.; To assess the estrogenic activity potentially stemming from 17beta-estradiol (E2) in drinking water, ESI-LC-MS was used to identifythe products of its aqueous chlorination under the following conditions: 50 microg/L E2, 1.46 mg/L sodium hypochlorite, pH 7.5, 25 degrees C . Seven products, including 2,4-dichloro-17beta-estradiol, monochloroestrone, 2,4-dichloroestrone, and the four byproducts such as 4-{2-(2,6-dichloro-3-hydroxyphenyl)ethyl}-7alpha-methyloctahydroinden-5-one (product C in the text) were identified in chlorinated E2 solution . The estrogenic activities of the aqueous chlorinated E2 solution at 10, 30, 60, 120, and 180 min contact time were assessed by a yeast two-hybrid system based on the ligand-dependent interaction of two proteins, a human estrogen receptor (ER) and a coactivator . All five solutions elicited transcriptional activation induction . The maximal beta-galactosidase activities induced by the chlorinated solution at 10, 30, and 60 min were similar and slightly lower than those before chlorination, while the activities of the chlorinated solution at 120 and 180 min were about 40% of those before chlorination . Finally, 4-chloro-17beta-estradiol (4-chloro-E2) (we failed to synthesize the 2-chloroestrone (2-chloro-E1)), 2,4-dichloro-17beta-estradiol (2,4-dichloro-E2), and 2,4-dichloroestrone (2,4-dichloro-E1) were synthesized, and product C was fractionated by HPLC . It was found that 4-chloro-E2 elicited strong estrogenic activity, at almost the same level as that of estrone (EC50 = 10(2) nM), while 2,4-dichloro-E2 elicited weaker beta-galactosidase activity compared with that of 4-chloro-E2 . The EC50 was ca . 10(3) nM . The maximal beta-galactosidase activity for 2,4-dichloro-E1 was lower than that of 2,4-dichloro-E2, while its EC50 was similar to that of 2,4-dichloro-E2 . In addition, product C, 4-{2-(2,6-dichloro-3-hydroxyphenyl)ethyl}-7alpha-methyloctahydroinden-5-one, induced high beta-galactosidase activity at the relatively higher concentration of 3.5 x 10(5) nM . On the basis of the dose-response curve of a single byproduct of chlorinated E2, the estrogenic activity at 120 and 180 min appears to be induced mainly by 2,4-dichloro-E2 and 2,4-dichloro-E1. J Clin Microbiol, 2004 Jan, 42(1), 115 - 8 Direct comparison of the BACTEC 9240 and BacT/ALERT 3D automated blood culture systems for candida growth detection; Horvath LL et al.; A direct comparison of two automated blood culture systems was conducted to compare their ability to detect Candida growth . The systems evaluated were the BACTEC 9240 (Bactec) and BacT/ALERT 3D (BacT) . The aerobic, anaerobic, and mycology media for each system were evaluated: Bactec Plus Aerobic/F, Plus Anaerobic/F, and Myco/F Lytic bottles, respectively, and BacT FA, SN, and MB bottles, respectively . Each blood culture bottle was inoculated with fresh blood from healthy donors . Fifty isolates of Candida spp . were used . The six different blood culture bottles were each inoculated with 1000 yeasts per bottle and then incubated in the corresponding automated system . The BacT detected growth of 90% (135 of 150) of Candida pathogens, while Bactec detected 66% (100 of 150) . Growth was detected in all BacT and Bactec mycology bottles, all BacT aerobic bottles, and by terminal subculture of all bottles . Sixty-five of 300 (22%) bottles had no growth detected; 50 from the Bactec (5 aerobic and 45 anaerobic) and 15 from the BacT (all anaerobic) . Terminal subculture of "negative" bottles demonstrated viable yeast growth from all 65 bottles, representing 65 false-negatives . The mean time to growth detection in the BacT system was 25.62 h while the Bactec was 27.30 h (P < 0.01) . Both automated blood culture systems detected all episodes of simulated candidemia when specialized mycology media were used . However, when only standard aerobic and anaerobic media were used, the BacT performed better than the Bactec in overall growth detection, time to growth detection, and number of false-negatives. Biochem Biophys Res Commun, 2004 Jan 30, 314(1), 268 - 76 Rpp20 interacts with SMN and is re-distributed into SMN granules in response to stress; Hua Y et al.; Spinal muscular atrophy (SMA) is a neurodegenerative disorder resulting from homozygous loss of the SMN1 gene . To investigate SMN functions, we undertook the yeast two-hybrid screens and identified Drosophila Rpp20, a subunit of the RNase P and RNase MRP holoenzymes, to interact with the Drosophila SMN protein . Interaction between human SMN and Rpp20 was validated by in vitro binding assays and co-immunoprecipitation . The exons 3-4 of SMN are necessary and sufficient for binding to Rpp20 . Binding efficiency between Rpp20 and SMNs with mutations in the Y-G domain is abrogated or reduced and correlated with severity of SMA disease . Immunofluorescence results indicate that Rpp20 is diffusely distributed throughout the cytoplasm with higher concentration observed in the nucleus . However, in response to stress, SMN forms aggregates and redistributes Rpp20 into punctuated cytoplasmic SMN granules . Our findings suggest a possible functional association of SMN with RNase P and RNase MRP complexes. Biochem Biophys Res Commun, 2004 Jan 30, 314(1), 97 - 103 ATBF1 enhances the suppression of STAT3 signaling by interaction with PIAS3; Nojiri S et al.; ATBF1 was first discovered as a suppressor of AFP expression in hepatocytes . It is present in brain, adult liver, lung, and gastro-intestinal tract . Recently, it has been reported that ATBF1 regulates myoblastic differentiation and interacts with v-Myb in regulation of its transactivation . Using the yeast two-hybrid system, we searched for protein-protein interactions to uncover new functions for ATBF1 . We present here experimental evidence that ATBF1 is a new regulatory factor for STAT3-mediated signal transduction through its interaction with PIAS3 . PIAS3 was thus identified as an ATBF1-binding protein . In co-transfection experiments, the full-length ATBF1 was found to form complexes with PIAS3 in Hep G2 cells . In the luciferase assay, ATBF1 was found to have no influence on STAT3 signaling induced by IL-6 stimulation, but it did synergistically enhance PIAS3 inhibition of activated STAT3 . In conclusion, ATBF1 can suppress the IL-6-mediated cellular response by acting together with PIAS3. Cell Death Differ, 2004 Apr, 11(4), 448 - 57 Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells; Kanzawa T et al.; Autophagy is originally named as a process of protein recycling . It begins with sequestering cytoplasmic organelles in a membrane vacuole called autophagosome . Autophagosomes then fuse with lysosomes, where the materials inside are degraded and recycled . To date, however, little is known about the role of autophagy in cancer therapy . In this study, we present that temozolomide (TMZ), a new alkylating agent, inhibited the viability of malignant glioma cells in a dose-dependent manner and induced G2/M arrest . At a clinically achievable dose (100 microM), TMZ induced autophagy, but not apoptosis in malignant glioma cells . After the treatment with TMZ, microtubule-associated protein light-chain 3 (LC3), a mammalian homologue of Apg8p/Aut7p essential for amino-acid starvation-induced autophagy in yeast, was recruited on autophagosome membranes . When autophagy was prevented at an early stage by 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, not only the characteristic pattern of LC3 localization, but also the antitumor effect of TMZ was suppressed . On the other hand, bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, that prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes, sensitized tumor cells to TMZ by inducing apoptosis through activation of caspase-3 with mitochondrial and lysosomal membrane permeabilization, while LC3 localization pattern stayed the same . These results indicate that TMZ induces autophagy in malignant glioma cells . Application of an autophagy inhibitor that works after the association of LC3 with autophagosome membrane, such as bafilomycin A1, is expected to enhance the cytotoxicity of TMZ for malignant gliomas. EMBO J, 2004 Jan 14, 23(1), 212 - 20 Epub 2004 Jan 08. Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway; Miyauchi H et al.; The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans . This regulatory process involves the activity of DAF-16, a forkhead transcription factor . Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells . Here we show that Akt activity increases along with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells . Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur . FOXO3a influences p53 activity by regulating the level of reactive oxygen species . These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology. J Urol, 2004 Feb, 171(2 Pt 1), 907 - 10 Inorganic selenium retards progression of experimental hormone refractory prostate cancer; Corcoran NM et al.; PURPOSE: The development of hormone refractory prostate cancer marks the onset of the terminal phase of the disease . Despite the use of traditional chemotherapeutic drugs as well as many novel agents life expectancy is not significantly increased beyond palliative care alone . Selenium is a micronutrient that is incorporated into a number of essential enzymes and a minimum intake is necessary for the maintenance of health . In the last few years evidence has accumulated from case-control and limited randomized control data that supranutritional doses of selenium could inhibit the progression of prostate cancer . While much attention has focused on its use as a chemopreventive agent, its use as specific therapy has been limited . We hypothesized that dietary supplementation of selenium would inhibit the progression of hormone refractory prostate cancer in an experimental model . MATERIALS AND METHODS: We established orthotopic PC3 tumors in the prostates of 6-week-old male nude mice and fed them a baseline selenium replete diet (0.07 ppm), supplementing intake with different forms of selenium (sodium selenate, selenomethionine, methylselenocysteine and selenized yeast) at 2 different concentrations (0.3 and 3 ppm) in drinking water . RESULTS: Inorganic selenium (sodium selenate) significantly retarded the growth of primary prostatic tumors and the development of retroperitoneal lymph node metastases, which was associated with a decrease in angiogenesis . CONCLUSIONS: High dose dietary supplementation of inorganic selenium inhibits the progression of hormone refractory prostate cancer, which is due at least in part to a decrease in angiogenesis. Gene Ther, 2004 Jan, 11(2), 142 - 51 5-Fluorocytosine increases the toxicity of Wnt-targeting replicating adenoviruses that express cytosine deaminase as a late gene; Fuerer C et al.; Clinical studies with oncolytic adenoviruses have shown that existing viruses are safe but lack efficacy . To selectively increase the toxicity of oncolytic adenoviruses targeting colon tumours, we have inserted the yeast cytosine deaminase gene (yCD) after the fibre gene in the major late transcript . yCD was expressed using either an internal ribosome entry site (IRES) or by alternative splicing of a new exon analogous to the Ad41 long fibre exon . The IRES-CD virus gave higher yCD expression on Western blots . Both approaches result in yCD expression restricted to the period after viral DNA replication . Viral burst size was reduced by less than approximately 10-fold by 5-fluorocytosine (5-FC), showing that expression of yCD as a late gene is compatible with virus replication . Cytopathic effect assays in colon cancer cell lines showed that both yCD viruses have approximately 10-fold increased toxicity in the presence of the prodrug 5-FC, which is converted to 5-fluorouracil (5-FU) by yCD . Toxicity was higher following addition of 5-FC immediately after infection . The largest gain in toxicity was seen in HT29 colon cancer cells, which are the least permissive colon cancer cells for the parental virus, indicating that the new 5-FC/yCD viruses may have broader applications for colon cancer therapy than their predecessors. Cell Cycle, 2004 Feb, 3(2), 182 - 8 Structure predictions and interaction studies indicate homology of separase N-terminal regulatory domains and Drosophila THR; Jager H et al.; The final resolution of sister chromatid cohesion during mitotic and meiotic divisions is mediated by activation of separase which cleaves a cohesin complex subunit . The structural basis of separase regulation is unknown . Separases from different eukaryotes share almost no sequence similarity, especially within the large N-terminal domain that precedes the protease domain except in Drosophila melanogaster . Moreover, sequence similarity among securin proteins, which associate as regulatory subunits with separase, is restricted to the signals that promote the mitotic degradation required for separase activation . Here, we address the surprising divergence of separase and securin sequences . The absence of an extended N-terminal separase domain in dipteran species is shown to be correlated with the expression of an extra regulatory subunit (THR) . The interactions of THR with separase and securin in Drosophila melanogaster are analogous to those of the human N-terminal separase domain with its C-terminal domain and securin . Even heterologous interactions between Drosophila and human separase complex components occur in yeast two-hybrid experiments . Tertiary structure predictions reveal alpha-alpha superhelix folds in both THR and the N-terminal domains of nondipteran separases . The compatibility of these folds with a wide range of primary sequences has likely allowed the rapid divergence of THR/N-terminal separase sequences and securins, which contact this region. Curr Biol, 2004 Jan 6, 14(1), 69 - 74 A potential tension-sensing mechanism that ensures timely anaphase onset upon metaphase spindle orientation; Rajagopalan S et al.; The spindle orientation checkpoint (SOC) in fission yeast has been proposed to delay metaphase-to-anaphase transition when the spindle poles are misaligned with respect to the long axis of the cell . This checkpoint is activated in the absence of either an actomyosin division ring or astral microtubules . Although the SOC could be overridden in the absence of the transcription factor Atf1p, its mechanistic nature remained unclear . Here, we show that the SOC-triggered metaphase delay depends on a subset of the spindle assembly checkpoint (SAC) components Mph1p and Bub1p . Based on this finding and a detailed imaging of the spindle orientation process, we hypothesized that the spindle pole might contain proteins capable of sensing the achievement of spindle alignment . We identified the kendrin-like spindle pole body resident Pcp1p as a candidate molecule . A targeted mutation in its central domain specifically triggered the SOC in spite of the presence of oriented spindles, causing a metaphase delay that could be relieved in the absence of Mph1p, Bub1p, and Atf1p . Thus, Pcp1p might provide a link between the mechanical process of spindle alignment and the signal transduction that initiates anaphase. J Biomol Screen, 2003 Dec, 8(6), 676 - 84 A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions; Nieuwenhuijsen BW et al.; To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GalphaZ and RGS-Z1 . Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions . They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene . Both the firefly and Renilla luciferase reporter genes demonstrated a 40- to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains . Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed . The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts. J Cell Sci, 2004 Feb 1, 117(Pt 4), 619 - 29 Epub 2004 Jan 06. A role for the spectrin superfamily member Syne-1 and kinesin II in cytokinesis; Fan J et al.; Expression of a dominant negative fragment of the spectrin family member Syne-1 causes an accumulation of binucleate cells, suggesting a role for this protein in cytokinesis . An association of this fragment with the C-terminal tail domain of the kinesin II subunit KIF3B was identified by yeast two-hybrid and co-precipitation assays, suggesting that the role of Syne-1 in cytokinesis involves an interaction with kinesin II . In support of this we found that (1) expression of KIF3B tail domain also gives rise to multinucleate cells, (2) both Syne-1 and KIF3B localize to the central spindle and midbody during cytokinesis in a detergent resistant and ATP sensitive manner and (3) Syne-1 localization is blocked by expression of KIF3B tail . Also, membrane vesicles containing syntaxin associate with the spindle midbody with identical properties . We conclude that Syne-1 and KIF3B function together in cytokinesis by facilitating the accumulation of membrane vesicles at the spindle midbody. J Autoimmun, 2004 Feb, 22(1), 53 - 63 Epitopes recognised by tissue transglutaminase antibodies in coeliac disease; Nakachi K et al.; The interaction between IgA tissue transglutaminase (tTG) antibodies (Abs) and 35S-labelled tTG produced in a transcription/translation (TnT) system with various amino acid (aa) deletions has been studied . These experiments showed that the tTG N-terminal aa 1-89 were important for tTG Ab binding in all 15 coeliac disease sera studied and the central residues (aa 401-491) were important for binding of tTG Abs in all but one sera . The contribution of C-terminal residues to tTG Ab binding varied in different coeliac sera but overall was less than the contributions of the N terminal and central regions.Mouse monoclonal antibodies (MAbs) to tTG were produced and the tTG aa sequences recognised by the MAbs determined using modified 35S-labelled tTG proteins . Analysis of the inhibiting effects of patient sera tTG Ab on binding of tTG MAbs to tTG confirmed the importance of the N-terminal and central regions of tTG in forming serum tTG Ab binding sites.Recombinant human tTG was expressed in yeast and purified to better than 95% homogeneity using MAb affinity chromatography as a final purification step . This material was highly suitable for use in an ELISA for tTGAb. Cell Signal, 2004 Apr, 16(4), 515 - 20 Translocated in liposarcoma (TLS) is a substrate for fibroblast growth factor receptor-1; Klint P et al.; Binding of fibroblast growth factor (FGF) to the high affinity receptor-1 (FGFR-1) leads to activation of its endogenous tyrosine kinase activity . A number of substrates for the FGFR-1 kinase have been identified . Among those, FGF receptor-substrate-2 (FRS-2) was identified by virtue of its interaction with p13suc, a yeast protein involved in cell cycle regulation . We have used immobilized p13suc to identify a new substrate for FGRF-1, which is identical to "translocated in liposarcoma" (TLS) . TLS is a RNA/DNA-binding protein which occurs in fusion products with different transcription factors in a variety of solid tumours . We show that TLS is tyrosine phosphorylated in intact cells by a number of different growth factors, indicating a role in growth regulation. J Hepatol, 2003, 39 Suppl 1, S70 - 6 Hepatitis B vaccines; Shouval D; Yeast-derived hepatitis B vaccines, containing the small HBV envelope protein SHBAg, are immunogenic, safe and cost-effective in prevention of hepatitis B virus infection in neonates, children and adults . Newly developed pre-S/S hepatitis B vaccines may play a role in inducing fast and augmented seroconversion rates in special risk groups. J Biol Chem, 2004 Mar 19, 279(12), 10848 - 54 Epub 2004 Jan 05. Repression of promoter activity by CNOT2, a subunit of the transcription regulatory Ccr4-not complex; Zwartjes CG et al.; The evolutionary conserved Ccr4-Not complex controls mRNA metabolism at multiple levels in eukaryotic cells . Genetic analysis of not mutants in yeast identifies a negative role in transcription, which is dependent on core promoter structure . To obtain direct support for this we targeted individual core subunits of the human Ccr4-Not complex to promoters in transient transfections of human cells . In this experimental setup we found that the CNOT2 and CNOT9(hRcd1/hCaf40) subunits act as repressors of reporter gene activity . Interestingly, recruitment of other Ccr4-Not subunits did not affect the reporter gene . The major repression function of CNOT2 is localized in a specialized protein motif, the Not-Box . This conserved motif is present in all CNOT2 orthologs and surprisingly also in CNOT3 orthologs . Repression by the Not-Box was sensitive to treatment with the histone deacetylase inhibitor trichostatin A . In addition, mutation of a canonical TATA-box enhanced repression . Our experiments show for the first time direct regulation of promoter activity by components of the Ccr4-Not complex. J Biol Chem, 2004 Mar 26, 279(13), 12883 - 9 Epub 2004 Jan 05. Ras GTPase-activating protein binds to Akt and is required for its activation; Yue Y et al.; RasGAP (Ras GTPase-activating protein) is a negative regulator as well as a downstream effector of Ras . To identify partners of RasGAP we used it as the bait in a yeast two-hybrid screen . This resulted in discovering its interaction with Akt . Overexpression of RasGAP or a mutant lacking the GTPase-activating domain (nGAP) enhanced phosphorylation and activity of Akt, which was dependent on the upstream integrin-linked kinase . Also, nGAP protected the cells against staurosporin-induced apoptosis through an Akt-dependent pathway . To determine the role of RasGAP in receptor-mediated activation of Akt, we used short hairpin RNA interference to knock out endogenous RasGAP expression . Although this procedure resulted in enhanced Ras activity, it inhibited Akt phosphorylation . Thus, we propose that Ras-GAP interacts with Akt and is necessary for its activation, possibly via integrin-linked kinase-mediated phosphorylation of Ser-473 . The data suggest that this effect is independent of Ras activity. FEBS Lett, 2004 Jan 2, 556(1-3), 281 - 6 Predominant expression of Sir2alpha, an NAD-dependent histone deacetylase, in the embryonic mouse heart and brain; Sakamoto J et al.; Sir2 is an NAD-dependent histone deacetylase that functions in longevity, gene silencing, heterochromatin formation, DNA repair, and suppression of DNA recombination in yeast . The mammalian homolog Sir2alpha (SIRT1) has been shown to inhibit p53-dependent apoptosis, but its physiological roles are still not known . We found that the level of Sir2alpha expression during embryogenesis was high . The highest Sir2alpha mRNA expression was detected as early as embryonic day (E) 4.5 . Although the level was down-regulated during embryogenesis, a high level of expression was still found in the late embryonic stage (E18.5) . In embryos, Sir2alpha was expressed at high levels in the heart, brain, spinal cord, and dorsal root ganglia . The expression levels in these organs were high on E10.5-E13.5 and low on E16.5 . Quantitative reverse transcription polymerase chain reaction showed a 60% reduction in Sir2alpha mRNA content in the heart between E12.5 and E14.5 . After E14.5, the expression level in the heart remained constant up to 27 months of age . The expression pattern of Sir2alpha protein in embryonic hearts was consistent with that of mRNA . These results suggest new roles of Sir2alpha not only in early embryogenesis but also in cardiogenesis and neurogenesis with a stage-specific manner. FEBS Lett, 2004 Jan 2, 556(1-3), 187 - 92 Gbetagamma subunits stimulate p21-activated kinase 1 (PAK1) through activation of PI3-kinase and Akt but act independently of Rac1/Cdc42; Menard RE et al.; The p21-activated kinase (PAK) family is homologous to the yeast sterile 20 (Ste20) and regulates a wide variety of cellular responses, including cell morphology, proliferation, and survival . In this study we examined the activation of PAK1 by Gbetagamma subunits . Co-transfection of COS7 cells with Gbeta1gamma2 or Gbeta1gamma5 was sufficient to induce agonist-independent activation of PAK1 . Expression of dominant/negative Rac, Cdc42, or Ras did not inhibit this Gbetagamma-dependent activation . Wortmannin, which inhibits phosphoinositide 3-kinase (PI3-kinase) activity, and expression of a dominant/negative form of Akt were sufficient to abrogate the activation of PAK1 that was induced by Gbetagamma . These results reveal that stimulation of PAK1 by Gbetagamma can occur via a PI3-kinase and Akt pathway that does not require Rac1 or Cdc42. Biochem Biophys Res Commun, 2004 Jan 23, 313(4), 969 - 76 Human zinc finger protein 161, a novel transcriptional activator of the dopamine transporter; Lee KH et al.; The dopamine transporter (DAT) terminates dopaminergic neurotransmission via reuptake of released dopamine into presynaptic neurons . We have cloned 2.5 kb of the regulatory region upstream of human DAT (hDAT) and constructed a series of deletion mutants to test promoter activity . A comparison of promoter activity between non-neural and neuronal cell lines reveals an interesting difference in pattern . In the PC12 cell line, activity of the proximal promoter is strongly silenced by one or more unidentified elements spanning positions -395 to -2465 of the hDAT gene . Our studies focus on identifying and characterizing the activating factor for hDAT transcription in the sequence between -2511 and -2492 (5(')-CTA CCT GCA CAG TTC ACG GA-3('), termed HY1) . In this investigation, we cloned the zinc finger protein 161 (ZFP161) gene as a HY1-binding factor, using the yeast one-hybrid screen . Recombinant ZFP161 was produced to evaluate the DNA-binding properties of the protein . The ability of ZFP161 to directly bind HY1 was examined in an electrophoretic mobility shift assay . RT-PCR analyses revealed that transfection of ZFP161 induced hDAT mRNA expression in HEK293 cells . We additionally confirmed the expression and localization of the DAT protein, using a specific antibody . Both the HY1 sequence and the downstream region were necessary for activation of the hDAT promoter by ZFP161 . This finding suggests that the site of cofactor interaction with ZFP161 may exist downstream of HY1. Genomics, 2004 Feb, 83(2), 254 - 61 LETM1, a gene deleted in Wolf-Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein; Schlickum S et al.; The leucine zipper-, EF-hand-containing transmembrane protein 1 (LETM1) has recently been cloned in an attempt to identify genes deleted in Wolf-Hirschhorn syndrome (WHS), a microdeletion syndrome characterized by severe growth and mental retardation, hypotonia, seizures, and typical facial dysmorphic features . LETM1 is deleted in almost all patients with the full phenotype and has recently been suggested as an excellent candidate gene for the seizures in WHS patients . We have shown that LETM1 is evolutionarily conserved throughout the eukaryotic kingdom and exhibits homology to MDM38, a putative yeast protein involved in mitochondrial morphology . Using LETM1-EGFP fusion constructs and an anti-rat LetM1 polyclonal antibody we have demonstrated that LETM1 is located in the mitochondria . The present study presents information about a possible function for LETM1 and suggests that at least some (neuromuscular) features of WHS may be caused by mitochondrial dysfunction. Genomics, 2004 Feb, 83(2), 225 - 30 A physical map of the genomic region on mouse chromosome 3 containing the hindshaker (hsh) mutation; Karim SA et al.; Hindshaker (hsh), a spontaneous, autosomal recessive mouse mutation, displays a developmentally dependent tremor of the hindquarters due to hypomyelination in the CNS . This myelin deficit is followed by progressive, but incomplete, recovery by postnatal day 42 . Herein we describe the construction of a genomic contig spanning the interval between the markers D3Mit187 (42.4 cM) and D3Mit232 (45.2 cM) on mouse chromosome 3, which we have previously shown to contain the hsh mutation . A physical map, covering approximately 3.5 Mb, was constructed from a series of overlapping yeast and bacterial artificial chromosomes . A 1.2- to 1.4-Mb segment central to the contig was compared extensively with the syntenic regions in human (chromosome 1q21-q23) and rat (chromosome 2) . We present new data on 10 genes erroneously assigned to this area and on another 6 genes previously assigned elsewhere . For absent genes, our work suggests that they are telomeric to the region encompassed in our map . Accordingly, our findings both map the area surrounding the hsh mutation and present important corrections to the current maps in an area rich in genes related to the nervous system. Nat Biotechnol, 2004 Jan, 22(1), 78 - 85 Epub 2003 Dec 14. Gaining confidence in high-throughput protein interaction networks; Bader JS et al.; Although genome-scale technologies have benefited from statistical measures of data quality, extracting biologically relevant pathways from high-throughput proteomics data remains a challenge . Here we develop a quantitative method for evaluating proteomics data . We present a logistic regression approach that uses statistical and topological descriptors to predict the biological relevance of protein-protein interactions obtained from high-throughput screens for yeast . Other sources of information, including mRNA expression, genetic interactions and database annotations, are subsequently used to validate the model predictions without bias or cross-pollution . Novel topological statistics show hierarchical organization of the network of high-confidence interactions: protein complex interactions extend one to two links, and genetic interactions represent an even finer scale of organization . Knowledge of the maximum number of links that indicates a significant correlation between protein pairs (correlation distance) enables the integrated analysis of proteomics data with data from genetics and gene expression . The type of analysis presented will be essential for analyzing the growing amount of genomic and proteomics data in model organisms and humans. Science, 2004 Jan 30, 303(5658), 672 - 6 Epub 2004 Jan 02. RNAi-mediated targeting of heterochromatin by the RITS complex; Verdel A et al.; RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels . Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast . The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein) . In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production . These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains . The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci. Science, 2004 Jan 23, 303(5657), 540 - 3 Epub 2004 Jan 02. A map of the interactome network of the metazoan C . elegans; Li S et al.; To initiate studies on how protein-protein interaction (or "interactome") networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network . Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens . Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set . Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains approximately 5500 interactions . Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses. Nucleic Acids Res, 2004 Jan 02, 32(1), 169 - 78 Print 2004. Domain mapping of the Rad51 paralog protein complexes; Miller KA et al.; The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair . These proteins have been found to form two distinct complexes in vivo, Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and Rad51C-Xrcc3 (CX3) . Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs . The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region . Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1-75 interacts with the C-terminus and linker of Rad51C, residues 79-376, and this region of Rad51C also interacts with mRad51D and Xrcc3 . We have also determined that the N-terminal domain of mRad51D, residues 4-77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77-328, binds Rad51C . By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes. Nat Genet, 2004 Jan, 36(1), 94 - 9 Epub 2003 Dec 14. Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases; Garcia-Cao M et al.; Telomeres are capping structures at the ends of eukaryotic chromosomes composed of TTAGGG repeats bound to an array of specialized proteins . Telomeres are heterochromatic regions . Yeast and flies with defects in activities that modify the state of chromatin also have abnormal telomere function, but the putative role of chromatin-modifying activities in regulating telomeres in mammals is unknown . Here we report on telomere length and function in mice null with respect to both the histone methyltransferases (HMTases) Suv39h1 and Suv39h2 (called SUV39DN mice) . Suv39h1 and Suv39h2 govern methylation of histone H3 Lys9 (H3-Lys9) in heterochromatic regions . We show that primary cells derived from SUV39DN mice have abnormally long telomeres relative to wild-type controls . Using chromatin immunoprecipitation (ChIP) analysis, we found that telomeres were enriched in di- and trimethylated H3-Lys9 but that telomeres of SUV39DN cells had less dimethylated and trimethylated H3-Lys9 but more monomethylated H3-Lys9 . Concomitant with the decrease in H3-Lys9 methylation, telomeres in SUV39DN cells had reduced binding of the chromobox proteins Cbx1, Cbx3 and Cbx5, homologs of Drosophila melanogaster heterochromatin protein 1 (HP1) . These findings indicate substantial changes in the state of telomeric heterochromatin in SUV39DN cells, which are associated with abnormal telomere elongation . Taken together, the results indicate epigenetic regulation of telomere length in mammals by Suv39h1 and Suv39h2. Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 671 - 5 Epub 2003 Dec 30. Association of beta-catenin with the alpha-subunit of neuronal large-conductance Ca2+-activated K+ channels; Lesage F et al.; The association of Ca(2+)-activated K(+) channels with voltage-gated Ca(2+) channels at the presynaptic active zones of hair cells, photoreceptors, and neurons contributes to rapid repolarization of the membrane after excitation . Ca(2+) channels have been shown to bind to a large set of synaptic proteins, but the proteins interacting with Ca(2+)-activated K(+) channels remain unknown . Here, we report that the large-conductance Ca(2+)-activated K(+) channel of the chicken's cochlear hair cell interacts with beta-catenin . Yeast two-hybrid assays identified the S10 region of the K(+) channel's alpha-subunit and the ninth armadillo repeat and carboxyl terminus of beta-catenin as necessary for the interaction . An antiserum directed against the alpha-subunit specifically coprecipitated beta-catenin from brain synaptic proteins . beta-Catenin is known to associate with the synaptic protein Lin7/Velis/MALS, whose interaction partner Lin2/CASK also binds voltage-gated Ca(2+) channels . beta-Catenin may therefore provide a physical link between the two types of channels at the presynaptic active zone. Genes Dev, 2004 Jan 1, 18(1), 23 - 34 Epub 2003 Dec 30. hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region; Sanchez-Velar N et al.; Human immunodeficiency virus Rev facilitates the cytoplasmic accumulation of viral RNAs that contain a Rev binding site . A human Rev-interacting protein (hRIP) was originally identified based on its ability to interact with the Rev nuclear export signal (NES) in yeast two-hybrid assays . To date, however, the function of hRIP and a role for hRIP in Rev-directed RNA export have remained elusive . Here we ablate hRIP activity with a dominant-negative mutant or RNA interference and analyze Rev function by RNA in situ hybridization . We find, unexpectedly, that in the absence of functional hRIP, Rev-directed RNAs mislocalize and aberrantly accumulate at the nuclear periphery, where hRIP is localized . In contrast, in the absence of Rev or the Rev cofactor CRM1, Rev-directed RNAs remain nuclear . We further show that the RNA mislocalization pattern resulting from loss of hRIP activity is highly specific to Rev function: the intracellular distribution of cellular poly(A)(+) mRNA, nuclear proteins, and, most important, NES-containing proteins, are unaffected . Thus, hRIP is an essential cellular Rev cofactor, which acts at a previously unanticipated step in HIV-1 RNA export: movement of RNAs from the nuclear periphery to the cytoplasm. Mol Cell Biol, 2004 Jan, 24(2), 856 - 64 CHIP mediates degradation of Smad proteins and potentially regulates Smad-induced transcription; Li L et al.; Transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) family ligands interact with specific membrane receptor complexes that have serine/threonine kinase activities . The receptor phosphorylation and activation induced by the ligands leads to phosphorylation of the Smad proteins, which translocate to the nucleus, controlling gene expression . Thus, regulation of Smad proteins is a key step in TGF-beta/BMP-induced signal transduction . Here we report a novel mechanism of the regulation of SMAD-mediated signaling, by which the Smad1 protein level is controlled through expression of the CHIP protein . CHIP is a U-box-dependent E3 ubiquitin ligase, previously identified as a cochaperon protein . However, we have isolated CHIP as a Smad-interacting protein in a yeast two-hybrid screen using Smad1 as bait . Furthermore we have shown CHIP-Smad interaction using the (35)S-labeled CHIP protein, which can interact with glutathione S-transferase (GST)-Smad1 and GST-Smad4 in an in vitro protein-binding assay . The CHIP-Smad interaction has been confirmed in vivo in mammalian cells through coimmunoprecipitation . Interestingly, we demonstrate that the coexpression of Smad1 and Smad4 with the CHIP protein results in the degradation of the Smad proteins through a ubiquitin-mediated process . Consistent with the observation that CHIP induces Smad1 degradation, we further show that the expression of CHIP can inhibit the transcriptional activities of the Smad1/Smad4 complex induced by BMP signals . Intriguingly, pBS/U6/CHIPi, which diminishes CHIP expression, significantly enhanced Smad1/Smad4- or BMPRIB(QD)-induced gene transcription . These results suggest that CHIP can interact with the Smad1/Smad4 proteins and block BMP signal transduction through the ubiquitin-mediated degradation of Smad proteins. Mol Cell Biol, 2004 Jan, 24(2), 809 - 22 Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions; Li S et al.; Foxp1, Foxp2, and Foxp4 are large multidomain transcriptional regulators belonging to the family of winged-helix DNA binding proteins known as the Fox family . Foxp1 and Foxp2 have been shown to act as transcriptional repressors, while regulatory activity of the recently identified Foxp4 has not been determined . Given the importance of this Fox gene subfamily in neural and lung development, we sought to elucidate the mechanisms by which Foxp1, Foxp2, and Foxp4 repress gene transcription . We show that like Foxp1 and Foxp2, Foxp4 represses transcription . Analysis of the N-terminal repression domain in Foxp1, Foxp2, and Foxp4 shows that this region contains two separate and distinct repression subdomains that are highly homologous termed subdomain 1 and subdomain 2 . However, subdomain 2 is not functional in Foxp4 . Screening for proteins that interact with subdomains 1 and 2 of Foxp2 using yeast two-hybrid analysis revealed that subdomain 2 binds to C-terminal binding protein 1, which can synergistically repress transcription with Foxp1 and Foxp2, but not Foxp4 . Subdomain 1 contains a highly conserved leucine zipper similar to that found in N-myc and confers homo- and heterodimerization to the Foxp1/2/4 family members . These interactions are dependent on the conserved leucine zipper motif . Finally, we show that the integrity of this subdomain is essential for DNA binding, making Foxp1, Foxp2, and Foxp4 the first Fox proteins that require dimerization for DNA binding . These data reveal a complex regulatory mechanism underlying Foxp1, Foxp2, and Foxp4 activity, demonstrating that Foxp1, Foxp2, and Foxp4 are the first Fox proteins reported whose activity is regulated by homo- and heterodimerization. Mol Cell Biol, 2004 Jan, 24(2), 765 - 73 Negative regulation of histone deacetylase 8 activity by cyclic AMP-dep