Das Eukaryotische-Initiations faktor-4E Bindungsprotein (4E-BP) ist ein Inhibitor der Translationsinitiation. Nicht-phosphoryliertes 4E-BP bindet an den eukaryotischen Initiationsfaktor 4E (eIF4E). Diese Bindung blockiert die Rekrutierung des Initiationskomplexes eIF4F an die Cap-Struktur des 5´Endes von eukaryotischen zellulären mRNAs, was die Initiation der Translation verhindert. Phosphorylierung von 4E-BP durch die mTOR Kinase führt zur Dissoziation des 4E-BP/eIF4E Komplexes und erhöht die Verfügbarkeit von eIF4E, dies wird mit Zellproliferation assoziiert. Die Aktivität von eIF4E wird nicht nur von 4E-BP, sondern auch durch Phosporylierung reguliert, welche wiederum durch die "MAP-Kinase-Interacting-Protein-Kinase" (MNK) reguliert wird. Drei Isoformen von 4E-BP sind bekannt: 4E-BP1, 4E-BP2 and 4E-BP3. 4E-BP1 und 4E-BP2 sind an oxidativem und adipogenetischen Stress beteiligt. Beide Proteine werden im h?matopoetischen System gleich exprimiert, wohingegen 4E-BP3 nicht detektiert wird. 4E-BP1 wird während der Erythroblasten-Proliferation phosphoryliert. Aus diesem Grund habe ich die Hämatopoese und die durch Phenylhydrazine (PHZ) induzierte Stress-Erythropoese in 4E-BP1 und 4E-BP2 Knock-Out Mäusen und 4E-BP1,2 Doppel-Knock-Out Mäusen analysiert. Ich konnte zeigen, dass die Hämatopoese in 4E-BPs defizienten Mäusen nicht beeinflusst wird. Allerdings zeigten 4E-BP1,2-/- und 4E-BP2-/- Mäuse eine verspätete Antwort auf Phenylhydrazin (PHZ) induzierten erythropoetischen Stress. Gleichzeitig war die mRNA Translation von GATA-1, ein essentieller erythropoetischer Transkriptionsfaktor in Erythroblasten runterreguliert. Die Signaltransduktionswege mTOR und MNK1 waren bei erythropoetischen Stress aktiviert. Diese Daten zeigen, dass 4E-BP2, aber nicht 4E-BP1, notwendig ist um auf erythropoetischen Stress zu reagieren und deuten an, dass die 4E-BP gesteuerte translations-regulierende Maschinerie eine Rolle in der Stress-Erythropoese spielt. / Translational regulation allows an organism to generate fast responses to environmental changes quickly. Eukaryotic initiation factor 4E binding protein (4E-BP) is an inhibitor of translation initiation. Unphosphorylated 4E-BP binds to eukaryotic initiation factor 4E (eIF4E) blocking recruitment of the initiation complex eIF4F to the cap structure at the 5´ terminus of eukaryotic cellular mRNAs. Thus initiation of translation is blocked. Phosphorylation of 4E-BP by the mTOR kinase causes disassociation of the 4E-BP/eIF4E complex and increases the availability of eIF4E. EIF4E activity is not only regulated by 4E-BP, but also phosphorylation which is regulated by MAP kinase - interacting protein kinase (MNK). Three isoforms of 4E-BP are known, termed 4E-BP1, 4E-BP2 and 4E-BP3. 4E-BP1 and 4E-BP2 are involved in oxidative and adipogenetic stresses in vivo. They are equally expressed in hematopoietic system, whereas 4E-BP3 is not detected. 4E-BP1 is phosphorylated during erythroblast proliferation. Erythroid differentiation is blocked by overexpresssion of eIF4E in tissue culture. These studies implied that 4E-BPs might play role in response to erythropoietic stress. I examined hematopoiesis and phenylhydrazine (PHZ) induced stress erythropoiesis in 4E-BP1 and 4E-BP2 individual knock out mice and 4E-BP1,2 compound knock out mice. I found that the hematopoiesis of 4E-BPs deficient mice were unaffected. However, 4E-BP1,2-/- and 4E-BP2-/- mice showed delayed response to phenylhydrazine (PHZ) induced erythropoietic stress. Simultaneously, the mRNA translation of GATA-1, which is the essential erythroid transcription factor, was downregulated in their erythroblasts. The signaling pathways through the mTOR and MNK1 were activated in erythropoietic stress. These data showed that 4E-BP2 but not 4E-BP1 was required for the response to erythropoietic stress and suggested that 4E-BP related translation regulatory machinery played a role in stress erythropoiesis.

Chez les mammifères l’initiation de la traduction et, plus particulièrement, la formation du complexe eIF4F, est principalement régulée par la protéine kinase TOR (Target of rapamycin). Cette voie de régulation fait intervenir les protéines 4E-BP (eIF4E-binding proteins) dont l’activité est modulée par la phosphorylation par TOR. Sous leur forme non-phosphorylée, les 4E-BP se lient au facteur d’initiation eIF4E, empêchent son recrutement dans le complexe eIF4F et inhibent ainsi l’initiation de la traduction. Phosphorylées par TOR, les 4E-BP perdent leur affinité pour eIF4E et sont remplacées par eIF4G ce qui active la traduction. La régulation de l’initiation de la traduction par TOR via 4E-BP a été bien décrite dans plusieurs modèles eucaryotes, tels que la levure, les insectes et les mammifères, mais reste encore obscure chez les plantes. Les recherches réalisées au cours de ma thèse ont permis l’identification de deux protéines homologues de 4E-BP chez Arabidopsis. Ces protéines, que nous avons appelées ToRP1 et ToRP2 (TOR Regulatory Proteins), sont caractérisées par la présence d’un motif consensus indispensable pour la liaison à eIF4E, et qui existe chez les protéines 4E-BP des mammifères ainsi que chez eIF4G et eIFiso4G d’Arabidopsis. La protéine ToRP1 est capable d’interagir spécifiquement avec eIF4E, mais aussi avec TOR via son extrémité N-terminale en système double-hybride de levure. ToRP1 et ToRP2 ont également été caractérisées comme étant des cibles directement phosphorylées par TOR chez Arabidopsis. Deux sérines, en position 49 et 89 dans la protéine ToRP1, ont été identifiées comme des sites potentiels de cette phosphorylation. De plus, l’état de phosphorylation de ces sites affecte l’interaction avec eIF4E en système double-hybride de levure. Par ailleurs, des plants d’Arabidopsis déficients en ToRP1 et ToRP2 renforcent la traduction strictement coiffe-dépendante de l’ARNm CYCB1;1, alors que la surexpression de ToRP1 ou de ToRP2 réprime sa traduction. Ces résultats suggèrent donc que les protéines ToRP, identifiées chez Arabidopsis, sont de nouvelles cibles directes de TOR, qui, par leur phosphorylation, régule l’initiation de la traduction coiffe-dépendante. / The target of rapamycin (TOR) is an evolutionarily conserved kinase that is a critical sensor of nutritional and cellular energy and a major regulator of cell growth. TOR controls cap-dependent translation initiation, in particular the assembly of the eIF4F complex, by modulating the activity of eIF4E-binding proteins (4E-BPs). In their unphosphorylated state 4E-BP proteins sequester eIF4E and repress translation. Upon phosphorylation by TOR, 4E-BPs have a low affinity binding to eIF4E and are replaced by eIF4G thus activating translation initiation. 4E-BPs have been discovered in yeast and mammals but remain to be obscure in plants. Here, we identified and characterized two Arabidopsis proteins termed TOR Regulatory Proteins (ToRPs 1 and 2) that display some characteristics of mammalian 4E-BPs. ToRP1 and ToRP2 contain a canonical eIF4E-binding motif (4E-BM) found in mammalian 4E-BPs and Arabidopsis eIF4G and eIFiso4G. ToRP1 interacts with eIF4E, and, surprisingly, the N-terminal HEAT domain of TOR in the yeast two-hybrid system. ToRP1 and ToRP2 are highly phosphorylated at several phosphorylation sites in TOR-dependent manner in planta. Two of these phosphorylation sites have been identified as—S49 and S89—their phosphorylation status modulates ToRP1 binding to eIF4E in the yeast two-hybrid system. In plant protoplasts, ToRP2 can function as translation repressor of mRNAs that are strictly cap-dependent. Our results suggest that ToRPs can specifically bind the Arabidopsis cap-binding proteins (eIF4E/eIFiso4E) and regulate translation initiation under the control of TOR

The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...

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