Les plantes ont développé une forte capacité d'adaptation aux facteurs environnementaux comme les conditions nutritives. Les voies de signalisation qui perçoivent les signaux environnementaux et les intègrent au niveau du développement de la plante sont encore mal connues. La voie de signalisation TOR–S6Kinase qui est conservée au sein des eucaryotes, a été principalement étudié chez les animaux et la levure chez lesquels elle régule la croissance en réponse aux facteurs de l'environnement via le niveau de traduction, la synthèse des ribosomes et le cycle de division cellulaire. Chez l'angiosperme Arabidopsis thaliana, deux gènes codent pour des protéines S6Kinases mais les travaux publiés ne montrent pas une implication de ces deux gènes dans le développement de la plante. Notre travail a consisté à mettre en évidence l'implication des protéines S6Kinases chez les plantes en utilisant comme modèle la mousse Physcomitrella patens. Nous avons développé des conditions expérimentales pour étudier le développement du protonéma de mousse qui est constitué de deux types cellulaires, le chloronéma et le caulonéma. Par exemple, nous avons caractérisé un marqueur moléculaire du caulonéma, un type cellulaire induit en condition de carence. Nous avons identifié 3 gènes codants pour les protéines S6Kinases chez Physcomitrella patens puis, nous avons réalisé les trois simples mutants par transgénèse ciblée. Nos résultats indiquent que le gène PpS6K1 permet de réguler le développement du protonéma en fonction des conditions environnementales en jouant principalement sur le rythme de division des chloronémas en fonction des nutriments. / Plants have developed a strong capacity to adapt to environmental cues like nutritive conditions. However, the signalling pathways involved in the perception of environmental signals and their integration into plant development are still poorly understood. The TOR-S6kinase signalling pathway is conserved in all eukaryotes but has been mainly studied in yeast and animals where it is known to regulate growth in response to the environment via translation, ribosome synthesis and the cell cycle. In the angiosperm Arabidopsis thaliana, two genes encode S6 kinases but their functions during development are not known.The objective of this work was to characterise the function of S6 kinases in plants using the moss Physcomitrella patens as a model system. We have developed new methods to study the development of moss protonema, a filamentous tissue made of only two cell types: chloronema and caulonema. For example, we have characterized a molecular marker of caulonema, the cell type induced by starvation. We have characterized the three genes encoding P. patens S6 kinases and used gene targeting to generate knock-out mutants for each of them. Our results indicate that PpS6K1 regulates protonema development in response to nutrient conditions, mainly through the rate of chloronema cells proliferation. In the other hand, PpS6K2 is involved in the inhibition of the chloronema to caulonema transition and in nutrient sensing. PpS6K3 seems to be involved in the development of the gametophore and the sporophyte. Thus, our results show that the three S6Ks are involved at different levels in the regulation of growth and development in the moss P patens.

The calcium-sensing receptor (CaR) is a G-protein coupled receptor that controls mammalian extracellular calcium (Ca2+o) homeostasis. CaR downstream signalling involves intracellular calcium (Ca2+i) mobilisation which can be negatively modulated by protein kinase C (PKC)-mediated phosphorylation of CaR residue Thr-888 (CaRT888). The nature of this regulation was investigated here using siRNA-based knockdown of individual PKC isotypes. Knocking down PKCα expression increased CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly lowering the EC50 for Ca2+o relative to control siRNA-transfected cells. In accordance, PKCα knockdown also decreased CaRT888 phosphorylation which also permitted the triggering of Ca2+i mobilisation in CaR-HEK cells at sub-threshold Ca2+o concentrations. Interestingly, PKCε knockdown attenuated CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly increasing the EC50 for Ca2+o. However, this knockdown was also also found to inhibit CaRT888 phosphorylation and this is the first time that CaRT888 phosphorylation has been shown to be dissociate from Ca2+i mobilisation. The results show the complexity of the interactions that potentially underlie the CaR’s pleiotropic signalling and provides novel targets for examining signal bias. Classically an increase in cAMP is known to trigger PTH seceretion. The observation in this study shows that raising intracellular cAMP levels with forskolin also decreased CaRT888 phosphorylation permitting increased Ca2+i mobilisation. This suggests that cAMP may stimulate the phosphatase (most likely protein phosphatase 2A (PP2A)). Nevertheless, knocking down Gα12, which has been shown to activate PP2A, resulted in increased CaRT888 phosphorylation and lower Ca2+i mobilisation (increased EC50 for Ca2+o). This suggests the possibility of CaR as a cAMP sensor that can detect an increase in intracellular cAMP in order to stop PTH serection. Three novel CaR effectors, P70 ribosamal protein S6 kinase, insulin-like growth factor receptor-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, were identified in CaR-HEK cells. It was shown that a) high Ca2+o stimulated the activation of these effectors and b) each effector was inhibited by knockdown of PKCα and Gα12, which further confirmed the association of these signals with CaR. These data show that CaR also plays an important role outside Ca2+o homeostasis, such as growth and inflammation. Finally, five CaR mutations associated with autosomal dominant hypocalcaemia (ADH) were found to increase Ca2+o-induced Ca2+i mobilisation, as well as ERK and p38MAPK activation, when transfected stably in HEK-293 cells. Cotreatment with the calcilytic NPSP795 inhibited ERK and p38MAPK phosphorylation in all 5 gain-of-function mutants and in the wild type CaR cells, with IC50s for the compound in the nanomolar range. These data highlight the potential utility of CaR negative allosteric modulators in the treatment of gain-of-function CaR mutations. Together these data enhance our understanding of CaRT888 phosphorylation and CaR signalling.