In this study, we describe the isolation and characterization of a functionally signaling-deficient Galpha16-negative somatic mutant (hereafter referred to clone 43) derived from the wildtype Jurkat T cell line by means of an activation-induced cell death resistance-based selection strategy. The catalytic activity of Lck in abrogated in clone 43 most likely due to a lack of autophosphorylation at the stimulatory site within the kinase domain of Lck, Y394. Based on Galpha16 cDNA sequencing data, the deficiency presented in clone 43 is most likely due to one or two point mutations located at amino acid residues 26 and 300 of the Galpha16 subunit. Importantly, these signaling defects were fully reversed by re-introduction of wildtype Galpha16 version into clone 43. Indeed, our findings are the first of their kind to validate the proposed model by which the Galpha subunit modulates the activity of Src family of protein tyrosine kinases. These studies underscore the integral role played by Galpha 16 in TCR-mediated signaling and offer a powerful genetic tool to strengthen our knowledge about the regulation and function of G proteins in T lymphocytes. We also describe the phenotype of two other signaling-deficient, Jurkat-derived clones, named clone 15 and clone 21. (Abstract shortened by UMI.)

Most eukaryotic mRNAs possess a 5' cap structure which is important for their translation. The eukaryotic translation initiation factor 4E (eIF4E) binds this structure directly and is repressed by the eIF4E-binbing proteins (4E-BPs), which in turn are regulated through the FRAP/mTOR pathway by a multitude of extracellular stimuli. The focus of this thesis is to investigate the function of the 4E-BPs in the cell by the use of mouse embryonic fibroblasts (MEFs) in which both the genes for 4E-BP1 (Eif4ebp1) and 4E-BP2 (Eif4ebp2) were disrupted. We first examined the role of 4E-BPs in encephalomyocarditis virus (EMCV) infection. 35S-methionine metabolic labeling of MEF proteins revealed that 4E-BPs are not essential for EMCV replication. We then decided to identify mRNAs regulated by 4E-BPs. This was achieved by polyribosomal analysis of specific mRNAs. Our results showed that translation efficiency of c-myc and ornithine decarboxylase (ODC) mRNAs is enhanced in double knockout cells. We also tested MEFs for their sensitivity to rapamycin, a drug known to inhibit FRAP/mTOR. Fluorescence-activated cell sorting (FACS) analysis demonstrated that 4E-BPs contribute to rapamycin-induced growth arrest. Finally, we studied Eif4ebpl-/- ;Eif4ebp2-/- mice in order to test whether they show a phenotype similar to Eif4ebp1 -/- which are known to be smaller and display a lower blood glucose compared with their wild type counterparts. Surprisingly, Eif4ebp1-/-;Eif4ebp2 -/- mice were neither small nor hypoglycemic. The above results emphasize the importance of the 4E-BPs in the cell, thus encouraging further investigations in order to better understand their function.

The activation of death receptor Fas (CD95/APO1) triggers apoptosis inducing caspase activation by direct (extrinsic) and mitochondria dependent (intrinsic) pathways. Mitogen activated protein kinases (MAPK), which include p38, JNK and ERK kinases, are also activated during Fas signaling and are known to either positively or negatively regulate apoptosis. Chemical inhibition of JNK (SP600125) and p38 (PD169316) sensitize tumor cells to Fas mediated apoptosis. I studied Fas mediated apoptosis in Jurkat cells and in some experiments HeLa cells. PD169316 is less potent than SP600125 and attenuates the effect of the later when present together. PD169316 inhibits two isoforms of p38 which either promote (p38alpha) or inhibit (p38beta) apoptosis; I investigated their relative regulatory influences on Fas signaling. I show that p38alpha is essential for Fas mediated caspase-8 activation: it promotes the dephosphorylation and exclusion of c-FLIPS but not c-FLIPL from the death inducing signal complex (DISC). Distally both p38 isoforms positively influenced the intrinsic pathway by common and selective effects on pro-apoptotic Bcl-2 proteins. The sensitizing effects of p38 and JNK inhibition were ablated by Bcl-2 localized at the endoplasmic reticulum. In HeLa cells, the proapoptotic effects of p38alpha are overridden by the anti-apoptotic effects of ERK.

During HIV-1 assembly tRNALys isoacceptors are selectively packaged into HIV-1. One of the tRNALys isoacceptors, tRNA Lys3 serves as a primer for the reverse transcriptase-catalyzed synthesis of minus strand, strong stop cDNA. Lysyl-tRNA synthetase (LysRS) a tRNA Lys-binding protein is also selectively package into HIV-1, making it a candidate for being the signal which targets tRNALys for incorporation into viruses. We have tested whether the cytoplasmic tRNA Lys/LysRS interaction is required for selective packaging of tRNA Lys into HIV-1 by constructing anticodon mutations within the anticodon domain of tRNALys3. The anticodon is the major binding domain for tRNALys/LysRS interaction. We found that the ability of tRNALys3 to bind to LysRS, and be aminoacylated was directly correlated with its ability to be incorporated into HIV-1. / However it was not clear from these results if in addition to the tRNALys/LysRS interaction, tRNA aminoacylation was required for selective packaging of tRNA Lys3. To investigate this, we constructed and expressed two mutant LysRS species. One has lost its aminoacylation activity because of a deletion within the catalytic core. The other mutant LysRS has a reduced ability to bind to tRNALys, because it contains a deletion within tRNA Lys binding site. This mutant LysRS does not have the ability to facilitate tRNALys packaging into the virus, while the mutant LysRS which binds to tRNALys, but fails to aminoacylate it, does facilitate tRNALys packaging, indicating that tRNALys aminoacylation is not required for its packaging into viruses. / LysRS is carried into the viruses by its interaction with Gag. We have mapped the sites of interaction between HIV-1 Gag and LysRS using in vivo and in vitro techniques. We find that Gag sequences within the C-terminal domain of CA which contains the CA dimerization site, interact with LysRS sequences which include motif 1, which contains the LysRS dimerization site.

The initiation of protein synthesis consists in the recruitment of a ribosome·initiator tRNA complex to the initiation codon of a messenger RNA. In eukaryotes, this process is facilitated by eukaryotic translation initiation factor 4E (eIF4E), which recognizes the cap structure present at the 5' end of all nuclear-transcribed mRNAs. The eIF4E-binding proteins (4E-BPs) inhibit cap-dependent translation by binding to eIF4E, and preventing ribosomal recruitment to the mRNA. The interaction of 4E-BPs with eIF4E is reversible, and it is regulated by the 4E-BPs phosphorylation state. Here I report the isolation of a third member of the 4E-BP family, 4E-BP3, and I demonstrate that it shares all the basic structural and functional characteristics of 4E-BP1 and 4E-BP2. I also analyzed the genomic locus for human 4E-BP3 (EIF4EBP3). I established that EIF4EBP3 can be fused with the gene located immediately upstream, and which encodes the human homologue of Drosophila MASK (Multiple Ankyrin Repeats Single KH domain). Interestingly, the open reading frame of the MASK-BP3 fusion transcript is different from that of 4E-BP3, indicating that the EIF4EBP3 locus encodes two different proteins. Finally, I investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice are remarkably lean, and the males' white adipose tissue contains cells exhibiting the distinctive multilocular appearance of brown adipocytes, and it expresses the uncoupling protein 1. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1) is increased in white adipose tissue from Eif4ebp1-/- mice. PGC1 is a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis. These findings demonstrate that 4E-BP1 is a novel regulator of metabolism in mammals.

Members of the zinc cluster (or binuclear cluster) protein family are characterized by a Zn(II)Cys6 zinc finger involved in DNA recognition and binding. These fungal proteins are transcriptional regulators of genes involved in a wide variety of cellular processes. One member, Gal4p, is involved in galactose metabolism, while others play a major role in primary and secondary metabolism, control of meiosis, and multidrug resistance. Sequencing of the Saccharomyces cerevisiae genome has revealed that 55 genes encoding putative zinc cluster proteins are present in budding yeast. However, the roles of many of these zinc cluster proteins are unknown. In order to better understand their functions, we have performed a phenotypic analysis of these putative zinc cluster proteins. We have implicated a number of them in a variety of processes in the cell, including multidrug resistance. Stb5p has been shown to be a major player in regulating the expression of multidrug resistance genes. Other zinc cluster activators of multidrug resistance genes include Pdr1p, Pdr3p, and Yrr1p. These regulators of multidrug resistance appear to interact with each other, forming many different sub-populations of homo- and heterodimers. Stb5p is found predominantly as a heterodimer with Pdr1p. It also appears that Pdr1p is a master regulator able to interact with many different partners, enabling it to mediate control over multidrug resistance genes.

Introduction. Recent lines of evidence suggest that retinoids1,2 and RARbeta23 may have both beneficial and harmful effects in cancer. Little is known regarding the specifics of RARbeta2 promoter silencing in cancer, or about the role that RARbeta2 plays in RARbeta2-expressing cancer cells. Objectives. To analyze the patterns and heritability of RARbeta2 promoter methylation in cancer and determine whether it is subject to allelic bias; and to analyze the effects of RARbeta2 knockdown on growth and mRNA expression profiles in cancer. Methods. RARbeta2 promoter methylation was analyzed in 20 parental cancer cell lines and 5 subcloned lines using bisulfate genomic sequencing (>150 sequencings); the proportion of methylated alleles was estimated using methylation-sensitive restriction enzyme digestion followed by PCR and single nucleotide polymorphism identification; 18 antisense oligonucleotides against RARbeta2 were tested in various cancer cell lines using RT-PCR, cell counting and annexin V staining; and gene expression profiles were compared following knockdown versus all trans retinoic acid (ATRA) stimulation using cDNA microarray technology (>14,000 genes), SOURCE and GOMINER databases. Results. Hypo- and hypermethylated alleles frequently co-exist in lines in which RARbeta2 is inactivated (5111, 45%); divergent methylation is heritable in the majority of subclones analyzed (618, 75%); and methylation is subject to allelic bias at a ratio of ~2:1 (3l3 CpG sites, 100%). Cellular proliferation is correlated with RARbeta2 expression levels (p=0.0003); the most effective oligo reduces cellular proliferation by up to 80% in cancer cell lines in which RARbeta2 expression has been retained (313, 100%), but has no apparent effects in lines in which it has been lost (313, 100%); reduction in cell growth following oligo treatment is at least partially due to activation of programmed cell death; and over a dozen pro-carcinogenesis genes are downr / 1Omenn GS, Goodman GE, Thornquist MO, Balmes J, Cullen MR, Glass A, et al. Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial. J Natl Cancer Inst 1996;88:15509. 2Anon., The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. N Engl J Med 1994;330:1029-35. 3Khuri FR, Wu H, Lee JJ, Kemp BL, Lotan R, Lippman SM, et al. Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I non-small cell lung cancer. Olin Cancer Res 2001;7:861-7

Initiation is the rate-limiting step in the process of translation, which is a complex process that requires the participation of numerous translation initiation factors (eIFs). The poly (A)-binding protein (PABP) interacts simultaneously with the poly(A) tail of mRNAs and the scaffolding protein eIF4G to mediate mRNA circularization resulting in the stimulation of protein translation. PABP is regulated by the PABP-interacting protein, Paip1. Paip1 is thought to act as a translational activator in 5' cap dependent translation by interacting with PABP and the initiation factors eIF4A and eIF3. In this study, the X-ray crystal structure of the middle domain of Paip1 (MPaip1; spanning 157-371) has been determined to a 1.7 Å resolution, revealing a crescent-shaped domain consisting of ten alpha helices arranged as five α-HEAT repeats. I show that the interaction of full-length Paip1 with eIF4A is very weak, suggesting that it is merely a stabilizing interaction in the translation initiation complex. Binding analysis between MPaip1 and eIF4A utilizing pull-down experiments, isothermal titration calorimetry and surface plasmon resonance, show that unlike MIF4G (the middle of domain of eIF4G), MPaip1 does not bind eIF4A. This suggests that the weak interaction between Paip1 and eIF4A is mediated by residues upstream or downstream of the middle domain. / L'initiation est l'étape déterminante de vitesse de réaction dans la traduction, un processus complexe qui requiert la participation d'un grand nombre de facteurs d'initiation. PABP (la protéine qui se lie à la queue poly(A) de l'ARNm) interagit simultanément avec l'ARNm et la protéine d'échaffaudage eIF4G, ce qui déclenche la circularization de l'ARNm et stimule la synthèse de protéines. PABP est régulée par la protéine Paip1. Paip1 est suggérée être activatrice du processus de traduction medié par la coiffe 5' de l'ARNm, par son interaction avec PABP et les facteurs d'initiation eIF4A et eIF3. Dans cette étude, la structure du domaine du milieu de Paip1 (Mpaip1, comprenant 157-371) a été résolué à travers la cristallographie par rayons X, à une résolution de 1.7 Å. Cette structure a révélé un domaine en forme de croissant, contenant 10 hélices alpha formant cinq HEAT repeats. Je montre que l'interaction du domaine complet de Paip1 avec eIF4A est très faible, suggérant qu'elle constitute simplement une interaction stabilizante dans le complexe d'initiation de traduction. Une analyse de l'interaction entre Mpaip1 et eIF4A en utilisant des expériences de pull-down, ITC et SPR, montre qu'au contraire de MIF4G (le domaine du milieu de eIF4G), Mpaip1 ne se lie pas à eIF4A. Cela suggère que l'interaction faible entre Paip1 et eIF4A est médiée par des résidues en montée ou en aval du domain du milieu.

Recent work published from our lab showed that modulation of the dynein motor complex affected HIV-1 viral production but not viral infectivity and that trafficking of viral components played an important role in assembly. It was further demonstrated that viral genomic RNA and the HIV-1 structural protein pr55Gag co-localized on LAMP-1 - a transmembrane glycoprotein found on endosomal and lysosomal membranes - late endosomal vesicles. Using the Lumio™ dyes fluorescein arsenical helix binder (FlAsH) and resorufin arsenical helix binder (ReAsH) and TC-tagged Gag-TC and pNL4-3TC constructs, we show the localization of newly synthesized pr55Gag through dual biarsenical labeling in the perinuclear region of HeLa cells. By overexpressing the p50/dynamitin-HA and RILP-myc proteins, we also show the physical interaction of newly synthesized pr55Gag and LAMP-1 late endosomal membranes. / Les récents travaux de notre laboratoire ont montré que la modulation du complexe moteur dynein (Dynein motor complex) avait un impact sur la production virale du VIH-1 mais pas sur son infectivité. Ainsi, le trafic de composantes virales joue un rôle important dans l'assemblage du virus. De plus, l'ARN génomique viral et la protéine structurelle pr55Gag du VIH-1 sont co-localisés sur les vésicules d'endosome tardif portant le marqueur LAMP-1, une glycoprotéine transmembranaire que l'on retrouve sur les membranes endosomales et lysosomales. En utilisant les molécules fluorescentes Lumio™ fluorescein arsenical helix binder (FlAsH) et resorufin arsenical helix binder (ReAsH) et des construits viraux portant une étiquette TC (Gag-TC et pNL4-3-TC), nous montrons que l'emplacement de pr55Gag nouvellement synthétisé se trouve dans la région péri-nucléique de cellules HeLa. En sur-exprimant les protéines p50/dynamitin-HA et RILP-myc, nous montrons également l'interaction physique entre les protéines pr55Gag nouvellement synthétisées et les vésicules d'endosomes tardifs marqués par LAMP-1.

Plant cells are encased within a complex polysaccharide wall that not only strengthens the plant, but also has key roles in plant growth, cell differentiation, and defence. The plant cell wall is comprised of a network of cellulose microfibrils interconnected by hemicelluloses; this framework is embedded in a more soluble pectin matrix. This dynamic structure is under continual modification during plant growth and development, and its synthesis and modification requires the activity of a myriad of enzymes. Recent research has provided insight into how plants manufacture and regulate the cell wall during development, but much remains unknown. The mucilage secretory cells (MSCs) of Arabidopsis thaliana are used as a model to discover novel genes involved in the synthesis, secretion and modification of cell wall components, particularly pectin. These cells synthesize copious amounts of pectinaceous mucilage during development and, upon hydration of the desiccated seed, this mucilage rapidly swells, bursts from the MSCs and surrounds the seed in a gelatinous capsule. The patchy (pty)/beta-xylosidase1(bxl1) mutant has a peculiar phenotype where mucilage release is patchy and slow, and mutant seeds are delayed in germination. Cloning of the mutant locus revealed a lesion in an encoded bifunctional β-xylosidase/α-arabinofuranosidase. Chemical and immunological analyses indicate an increase in 1,5-linked arabinans, suggesting the action of AtBXL1 is required for the trimming of these chains to allow correct mucilage release. In addition to the study of AtBXL1, an enhancer/suppressor screen of the mum4 reduced mucilage mutant was performed in order to identify novel genes involved in mucilage secretory cell differentiation. The screen identified six novel mutants named mum enhancer (men) 1-6. Characterization of men mum4 double mutants revealed two distinct groups, those that produced similar amounts of mucilage to mum4 but failed to release it (men2, 6), and / Les cellules végétales sont encastrées dans une paroi de polysaccharide complexe qui non seulement renforce la plante mais aussi agit de façon cruciale dans les mécanismes de croissance, de différentiation cellulaire et de défense. La paroi des cellules végétales consiste en un réseau de microfibrilles de cellulose connectées par des hemicelluloses. Ce réseau est encastré dans une matrice de pectine plus solubilisable. Cette structure dynamique est en modification perpétuelle pendant le développement et la croissance de la plante. Ces changements et sa synthèse engage l'action d'une myriade d'enzymes. Des études récentes ont données de nouvelles perspectives sur comment les plantes produisent et régulent leur paroi cellulaire pendant le développement cependant beaucoup reste à découvrir. Les cellules sécréteuses de mucilage (MSCs) d'Arabidopsis thaliana sont utilisées comme modèles pour la découverte de nouveaux gène impliqués dans la synthèse, sécrétion et la modification des composants de la paroi cellulaire, particulièrement la pectine. Ces cellules synthétisent de grandes quantité de mucilage pectiné durant le développement et, après hydration de la graine disséquée, celui-ci gonfle rapidement, jaillit des MSCs entourant la graine d'une capsule gélatineuse. Le mutant patchy (pty)/beta-xylosidase1(bxl1) présente un phénotype particulier où le relargage est épars et lent, ces graines présentent aussi un retard dans la germination. Le clonage du locus muté a révélé une lésion dans la β-xylosidase/α-arabinofuranosidase bifonctionelle transcrite. Les analyses chimique et immunologique ont indiquées une augmentation des 1,5-linked arabinans suggérant que l'action de BXL1 est requise pour l'hydrolyse de ces chaines permettant un bon relargage du mucilage. En parallèle de cette étude, un screen des enhancers/suppresseurs du mutant au mucilage réduit mum4 dans l'intention d'identifier des nouveaux gènes imp