It has been proposed that the various 14-3-3 isotypes and isoforms present in all eukaryotes are largely functionally equivalent. However, this is not consistent with the conservation of multiple isoforms and isotypes, especially in vertebrates with seven 14-3-3 encoding genes and nine isotypes. The hypothesis tested in this thesis is that both isoform-specific and overlapping functions are likely mediated through tissue specific expression, colocalization and dimerization of 14-3-3 proteins occur in vivo. Drosophila melanogaster was selected because it offers a simple, but representative system to study these proteins functionally. This thesis focuses primarily on D14-3-3?, although the expression pattern and phenotypes associated with all three Drosophila 14-3-3s were determined. I first determined the expression pattern of the three different 14-3-3 isotypes (leoI, leoII and D14-3-3?) and described developmental phenotypes associated with mutations in 14-3-3 isotypes in Drosophila. I found that there is partial redundancy with respect to lethality. Both LEO and D14-3-3? appear required for normal germ-line and somatic gonadal development. However, they do not appear to be functionally equivalent with respect to this phenotype since LEO is unable to compensate for the loss of D14-3-3?. I also determined that D14-3-3? mutants have unique phenotypes including deficits in adult cross-vein formation and rapid habituation to olfactory and footshock stimuli. To further understand the unique role that D14-3-3? plays in the adult CNS, I mapped the areas in the brain involved in olfactory and footshock habituation. I found that although the mushroom bodies (MBs) are necessary to inhibit premature habituation such as that exhibited by D14-3-3? mutants, D14-3-3? expression specifically in the MBs is not sufficient to rescue premature habituation. Although the loss of either LEO or D14-3-3? appears to cause a deficit in olfactory associative learning, premature habituation is the cause of the deficit seen in D14-3-3? mutants. As leo mutants do not exhibit a premature habituation phenotype, it appears that within the MBs LEO and D14-3-3? are not functionally equivalent. Therefore, the data supports the hypothesis that 14-3-3s have functional specificity and redundancy likely to represent use of homo and heterodimers in different processes within the tissues of an organism.

It has been proposed that the various 14-3-3 isotypes and isoforms present in all eukaryotes are largely functionally equivalent. However, this is not consistent with the conservation of multiple isoforms and isotypes, especially in vertebrates with seven 14-3-3 encoding genes and nine isotypes. The hypothesis tested in this thesis is that both isoform-specific and overlapping functions are likely mediated through tissue specific expression, colocalization and dimerization of 14-3-3 proteins occur in vivo. Drosophila melanogaster was selected because it offers a simple, but representative system to study these proteins functionally. This thesis focuses primarily on D14-3-3?, although the expression pattern and phenotypes associated with all three Drosophila 14-3-3s were determined. I first determined the expression pattern of the three different 14-3-3 isotypes (leoI, leoII and D14-3-3?) and described developmental phenotypes associated with mutations in 14-3-3 isotypes in Drosophila. I found that there is partial redundancy with respect to lethality. Both LEO and D14-3-3? appear required for normal germ-line and somatic gonadal development. However, they do not appear to be functionally equivalent with respect to this phenotype since LEO is unable to compensate for the loss of D14-3-3?. I also determined that D14-3-3? mutants have unique phenotypes including deficits in adult cross-vein formation and rapid habituation to olfactory and footshock stimuli. To further understand the unique role that D14-3-3? plays in the adult CNS, I mapped the areas in the brain involved in olfactory and footshock habituation. I found that although the mushroom bodies (MBs) are necessary to inhibit premature habituation such as that exhibited by D14-3-3? mutants, D14-3-3? expression specifically in the MBs is not sufficient to rescue premature habituation. Although the loss of either LEO or D14-3-3? appears to cause a deficit in olfactory associative learning, premature habituation is the cause of the deficit seen in D14-3-3? mutants. As leo mutants do not exhibit a premature habituation phenotype, it appears that within the MBs LEO and D14-3-3? are not functionally equivalent. Therefore, the data supports the hypothesis that 14-3-3s have functional specificity and redundancy likely to represent use of homo and heterodimers in different processes within the tissues of an organism.

Les plantes dans leur milieu naturel, sont constamment soumises à de multiples contraintes environnementales de nature biotique ou abiotique qui sont à l'origine de nombreuses pertes de rendement. Afin de répondre à ces stress, les plantes mettent en place des réponses adaptées qui reposent sur l'activation de voies de signalisations. L'une des voies importantes est celle impliquant la phosphorylation des protéines par les Mitogen Activated Protein Kinase (MAPKs). La régulation de l'activité des MAPKs est indispensable et dépend en partie de protéines phosphatases telles que les MAPK Phosphatases (MKPs). Ce travail a consisté à étudier les mécanismes de régulation de l'activité phosphatase de TMKP1, la seule MKP connue de blé dur, par les calmodulines et les protéines 14-3-3. Dans un premier temps, nous avons montré que l'activité phosphatase de TMKP1 est stimulée en présence des ions K+, Li+, Mg 2+ et surtout par le Mn2+. Des expériences de GST pull dawn ont permis de mettre en évidence une interaction, calcium dépendante, entre TMKP1 et la calmoduline (CaM) et nous avons démontré que l'activité de TMKP1 est inhibée par le complexe CaM/Ca2+. En revanche, ce même complexe stimule l'activité de TMKP1 en présence des ions Mn 2+. Ce mode de régulation d'une MKP par CaM/Ca2+ dépendant des ions Mo2+ est décrit ici pour la première fois. Dans un second temps, la présence au niveau de la séquence de TMKP1 d'un domaine de liaison aux protéines 14-3-3s, nous a incité à mener des essais d'immunoprécipitation à l'issue desquelles nous avons montré que TMKP1 pourrait interagir avec les 14-3-3s chez le Blé et que celles-ci stimulent l'activité phosphatase de TMKP1 in vitro. Enfin, l'implication de TMKP1 dans la réponse des plantes aux stress biotiques et abiotiques a été évaluée. Les tests de gennination ont révélé que la sur-expression de TMKP1 chez Arabidopsis permet une meilleure tolérance à la salinité. Ces mêmes plantes semblent également être plus résistantes à une infection bactérienne causée par Pseudomonas syringae que des plantes sauvages. Ces données suggèrent que TMKP1 agirait comme régulateur positif de la réponse d'Arabidopsis au stress salin et à l'infection P. syringae. L'ensemble des résultats obtenus ici a permis de dévoiler de nouvelles propriétés jamais décrites chez une MKP végétale et offre une nouvelle vision sur le rôle de ces phosphatases dans le contrôle des voies de signalisations MAPKs impliquées dans la réponse des plantes aux stress de l'environnement. / Due to their sessile lifestyle, plants are constantly subjected to a variety of biotic and abiotic stresses causing tremendous yield losses. To survive under these conditions, plants have evolved different sigualing pathways allowing efficient stress responses. The Mitogen Activated Protein Kinase (MAPKs) are key sigual transduction molecules, which respond to varions external stimuli. However, the activity of MAPKs has to be strictly regulated by protein phosphatase such as MAPK Phosphatase (MKPs). ln the present study we describe the regulation ofTMKPI, a wheat MKP, by Calmodulins (CaM) and 14-3-3 proteins. We fust showed that phosphatase activity oTMKP1 is stimulated by K\ Li\ Mg2+ and especially by Mo2+. Using GST pull dawn assays, we demonstrated that TMKP1 binds to CaM in a Ca2+-dependent manner. Moreover, the CaM/Ca2+ complex inhibits the catalytic activity of TMKP1 in a CaM-dose dependent marmer. However, in the presence of Mo2+, this activity is enhanced by CaM/Ca2+ complex. Such effects were not reported so far, and raise a possible role for CaM and Mo 2+ in the regulation of plant MKPs during cellular response to extemal siguals. Moreover, a fine sequence analysis ofTMKPl revealed the presence of a conserved 14-3-3 mode 1 binding site. This finding incited us to perfonn co-immunoprecipitation assays on wheat protein extracts through which we provide proof-of-concept evidence for interaction of TMKP1 with 14-3-3 proteins. Interestingly, the phosphatase activity of TMKP1 was shawn to be enhanced by several plant and yeast 14-3-3 isoforms. Finally, the involvement of TMKP1 in plant stress responses was evaluated. Our data showed that the overexpression of TMKP1 in Arabidopsis resulted in a higher tolerance to salt stresses and to bacterial infection caused by P. syringae. Such findings suggest thal TMKP1 may act as a positive regulator of Arabidopsis responses to salt stresses and P. syringae infection. All together, our results provide novel functional properties for plant MKPs, and should add new knowledge to our understanding of the raie of these phosphatases in the control of MAPK signaling pathways controlling plant responses to various environmental stresses.