<p>The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines.<p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

L’inhibition de protéines kinases constitue une voie pleine de promesses pour la découverte de nouvelles thérapies ciblées contre le cancer. En 2003, le criblage de la chimiothèque de l’Institut Curie/CNRS a permis de mettre en évidence une famille de composés actifs sur deux de ces enzymes : CK2 et Pim-1. L’objectif de cette thèse était de synthétiser des analogues des « hits » de la chimiothèque possédant le squelette 5-azaindole afin d’en améliorer les propriétés biologiques. La préparation de tels composés étant peu décrite dans la littérature, trois voies de synthèse flexibles et efficaces ont été développées. L’élaboration de 5-azaindoles diarylés symétriques a tout d’abord été mise au point par hétéroannélation pallado-catalysée à partir de dérivés de la 4-aminopyridine. Les composés monosubstitués en position 2 ont ensuite été obtenus par réaction domino sila-Sonogashira/cyclisation 5-endo. Enfin, un procédé one-pot couplage de Sonogashira/aminopalladation/élimination réductrice a permis d’accéder aux molécules diarylées non symétriques avec une régiosélectivité contrôlée. L’application de ces méthodologies a conduit à la préparation de 70 composés fonctionnalisés dont la cytotoxicité et l’activité inhibitrice sur CK2 ont été évaluées. Une étude structure-activité a été réalisée et les fragments d’intérêt que doit posséder une molécule de type 5-azaindole pour inhiber efficacement la kinase ont ainsi été identifiés. / Protein kinases represent promising targets for anti-cancer drug design. In 2003, inhibitors of two of these enzymes, CK2 and Pim-1, were identified by the screening of the Curie Institute/CNRS small-molecule library. The aim of this thesis was to synthesize derivatives of these hits with a 5-azaindole scaffold in order to optimize their biological activity. As the synthesis of such molecules was not reported in the literature, efficient and flexible procedures were developed to access to these structures. Diarylated symmetrical 5-azaindoles were thus prepared by palladium-catalyzed heteroannulation from 4-aminopyridines derivatives. The methodology was subsequently extended to silylalkynes and led to monoarylated products through domino sila-Sonogashira/5-endo cyclization. Finally, a one-pot Sonogashira coupling/aminopalladation/reductive elimination afforded unsymmetrical compounds with a total control of the regioselectivity. Using these methodologies, 70 functionalized molecules were easily prepared. Their cytotoxicity and biological activity as CK2 inhibitors were then evaluated. A structure-activity relationship study was performed, which led to the identification of two key structural elements for the CK2 inhibitory potency of 5-azaindoles.

Ce travail de thèse a eu pour but la synthèse de nouveaux composés hétérocycliques (rhodanines et pyrazoles) potentiellement actifs sur des protéines kinases, des lignées cellulaires tumorales, les influx SOCE (Store Operated Calcium Entry) et a ciblé principalement comme pathologies la malaria, la leishmaniose et le cancer. La première partie de cette étude a permis la synthèse d'une nouvelle famille de dérivés 5-arylidène rhodanines dissymétriques comportant un bras espaceur diaminé et ce par l'intermédiaire de la technologie micro-onde. Sur les 7 protéines kinases testées avec ces composés, CK1δ/ε et CDK5/p25 ont été inhibées spécifiquement avec des CI50 comprises entre 1,1 et 10 µM. L'activité anticancéreuse enregistrée est moyenne avec des CI50 variant de 8 à 23 µM. Les travaux réalisés au cours de la seconde partie de cette étude se sont appuyés sur le SKF-96365 comme modèle structural et ont permis d'accéder à 3 librairies inédites d'analogues comportant les plateformes pyrazole et rhodanine. L'activité pharmacologique visée ici était une modulation des influx SOCE et diverses variations structurales ont été effectuées en vue de réaliser une étude Relation Structure-Activité (RSA). Plusieurs analogues "pyrazoles" ont montré une activité supérieure à celle du SKF-96365 et de la GSK-7975A sur les influx SOCE de la lignée HEK-293. Les 2 composés montrant la meilleure activité (30f et 30h), sont également plus actifs que la Synta 66 aux faibles concentrations. Ces analogues ont également une activité sélective des canaux SOCE concernés car totalement inactifs sur l'ensemble des protéines kinases testées. Les CI50 les plus significatives pour l'activité anticancéreuse varient entre 3 et 8 µM. / This thesis work has been aimed the synthesis of new heterocyclic compounds (rhodanines and pyrazoles) potentially active on kinase proteins, tumor cell lines, SOCE impulses (Store Operated Calcium Entry) and has mainly targeted pathologies such as malaria, leishmaniasis and cancer. The first part of this study allowed the synthesis of a new family of 5-arylidene rhodanine derivatives asymmetric having a diamino spacer arm and via microwave technology. Of the 7 protein kinases tested with these compounds, CK1δ/ε and CDK5/p25 have been specifically inhibited with IC50 between 1.1 and 10 µM. The recorded anticancer activity is average with IC50 ranging from 8 to 23 µM. The work carried out during the second part of this study was based on SKF-96365 as structural model and provided access to 3 unpublished libraries of analogs containing pyrazole and rhodanine platforms. The desired pharmacological activity was SOCE modulating and various structural changes were made to undertake a study Structure-Activity Relationship (SAR). Several "pyrazole" analogs have shown a higher activity than SKF-96365 and GSK-7975A on SOCE of HEK-293 line. The two compounds showing the best activity (30f and 30h) are also more active than Synta 66 at low concentrations. These analogs are completely inactive on all protein kinases tested, indicating selectivity for SOCE channels concerned. The most significant IC50 for the anticancer activity vary between 3 and 8 µM.

Cerebral ischemia is a highly debilitating condition where shortage of oxygen and glucose leads to profuse cell death. Insufficient blood supply to the brain leads to cerebral ischemia and increase in extracellular lactate concentrations. Rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischemia. Lactate is a neuroprotective metabolite whose concentrations increase to 15-30 mM during ischemia and TREK1 is a neuroprotective potassium channel which is upregulated during ischemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischemia, however the connecting link between the two is missing. We hypothesized that lactate might interact with TREK1 channels and mediate neuroprotection. The aim of this study was to investigate the effect of lactate on activity and expression of TREK1 channels and evaluate the role of lactate-TREK1 interaction in conferring neuroprotection in the ischemia-prone hippocampus Ischemic concentrations (15-30 mM) of lactate at pH 7.4 increased whole cell TREK1 current in CA1 stratum radiator astrocytes and caused membrane hyperpolarization. We confirmed the intracellular action of lactate on TREK1 in hippocampal slices using mono carboxylate transporter blockers. The intracellular effect of lactate on TREK1 channels is specific since other mono carboxylates such as pyruvate at pH 7.4 failed to increase TREK1 current. We used immunostaining, western blot and electrophysiology to show that 15-30 mM of lactate increased functional TREK1 protein expression by 1.5-3 fold in hippocampal astrocytes. Next, we performed quantitative PCR to investigate if the increase in TREK1 protein expression was due to increased transcription and found that lactate stimulated TREK1 mRNA transcription to increase TREK1 protein. Lactate mediated increase in TREK1 expression was dependent on protein kinase A as inhibitors of protein kinase A abolished the increase in TREK1 mRNA and protein. The role of lactate-TREK1 interaction in neuroprotection was subsequently investigated using an in vitro oxygen glucose deprivation model of ischemia. Addition of 30 mM lactate to oxygen glucose deprived slices reduced neuronal death in the hippocampal CA1 pyramidal layer. However, 30 mM lactate failed to reduce cell death in rat hippocampal slices treated with TREK1 channel blockers signifying the requirement of active TREK1 channels for lactate mediated neuroprotection. However, lactate in the presence of protein kinase inhibitor failed to reduce cell death. This might be related to the role of protein kinase A in upregulation of TREK1 channels. We also estimated CA1 pyramidal neuronal TREK1 channel expression and found both lactate and oxygen glucose deprivation to decrease TREK1 channel expression that was surprisingly opposite to the effects on astrocytes. As TREK1 channel activation and upregulation decreases neuronal excitability, a decrease in neuronal TREK1 channel expression in response to lactate is expected to cause higher neuronal death and fails to explain lactate mediated neuroprotection. Since, lactate upregulated TREK1 channel expression and functional activity in CA1 stratum radiate astrocytes, we reasoned that the lactate mediated neuroprotection might be via astrocytic TREK1 channels requiring viable functional astrocytes. This was tested by disrupting astrocyte function using gliotoxin, and estimating cell death in oxygen glucose deprived hippocampal slices. Lactate failed to reduce cell death in presence of gliotoxin signifying the importance of viable astrocytes for lactate mediated neuroprotection. The above effects were specific to lactate as pyruvate failed to increase TREK1 expression and reduce cell death. TREK1 channels contribute to neuroprotection by enhancing potassium buffering and glutamate clearance capacity of astrocytes. We propose that lactate promotes neuronal survival in hippocampus by increasing TREK1 channel expression and activity in astrocytes during ischemia. This pathway serves as an alternate mechanism of neuroprotection.

With an increased popularity for systems-based approaches in biology, a wide spectrum of techniques has been applied to the simulation and analysis of biochemical systems which involves uncertainty and stochasticity. It is particularly concerned with modelling and analysis of metabolic pathways, regulatory and signal transduction networks for understanding intra-cellular pathway behaviour. Typically, parameter estimation in ordinary differential equations(ODEs) models is used for this purpose when there is large number of molecules involved in the reaction system. However this approach is correct when the system is large enough to be deterministic in nature. But there are uncertainty involved in the system and the processes are stochastic in nature due to smaller population molecules participating in the pathway reactions. In this thesis the common theme is the study of uncertainties in the chemical kinetics of biochemical reaction systems associated with various intra-cellular pathways and channels. The study is at the mesoscale of the system, i.e., we study systems that do not have too few molecules disallowing any higher scale system level approximation nor too many where a non-stochastic (mesoscale) system approximation will be valid. In our first study we estimate the parameters in the mitogen activated protein kinase (MAPK) signal transduction pathway involved in the departure from the normal Epithelial Growth Factor(EGF) dose-dependency in prostate cancer cells. A model-based pathway analysis is performed. The pathway is mathematically modelled with 28 rate equations yielding those many ordinary differential equations(ODE) with kinetic rate constants that have been reported to take random values in the existing literature. This has led to us treating the ODE model of the pathways kinetics as a random differential equations(RDE) system in which the parameters are random variables. The most likely set of values of the kinetic rate constants obtained from fitting the RDE model into the experimental data is then used in a direct transcription based dynamic optimization method for computing the changes needed in these kinetic rate constant values for the restoration of the normal EGF dose response. It identifies the parameters, i.e., the kinetic rate constants in the RDE model, that are the most sensitive to the change in the EGF dose response behaviour in the PC3 prostate cancer cells. Biochemical pathways involving chemical kinetics equations in terms of low concen-trations of the model variables can be represented as chemical Langevin equations(CLE) as there is stochasticity involved in the processes. Most CLE systems come with the implicit constraint that the concentration state cannot be negative at any time over the sample path. Due to the inherent stiffness(especially in diffusion coefficient) of the CLE system, it has been difficult for numerical schemes to meet this positivity constraint during numerical simulations. Most available methods resort to heuristics by dropping selective noise terms from the original CLE inconsistent with the mesoscale physics involved in forming the CLE. Other methods take very small time steps thus making the simulation inefficient. In our second study we preserve positivity by using a physically consistent numerical scheme which is a modified form of fully stochastic α method for stiff stochastic differential equation. Ion channels are fundamental molecules in the nervous system that catalyse the flux of ions across the cell membrane. Single ion channel flux activity is comparable to the catalytic activity of single enzyme molecules. Saturating concentrations of substrate induce dynamic disorder in the kinetic rate processes of single enzyme molecules and consequently, develop correlative memory of the previous history of activities. Conversely, binding of substrate ion is known to alter the catalytic turnover of single ion channels. Here, we investigated the possible existence of dynamic disorder and molecular memory in single human TREK1 channel due to binding of substrate/agonist using the excised inside-out patch-clamp technique. Our results suggest that single hTREK1 channel behaves as a typical Michaelis-Menten enzyme molecule with a single high-affinity binding site for substrate K+ ion but with uncertainty in reaction rates.

Un grand nombre de 3,4-bisindolylmaléimides possède une activité inhibitrice des protéine-kinases. Les 3-isothiazolone-1,(1)-(di)oxydes pouvant être considérées comme des analogues de la maléimide, la fonctionalisation des 5-chloro- et 4,5-dichloro-3-isothiazolone 1,(1)-(di)oxydes par des réactions de couplage palladocatalysé de Suzuki-Miyaura, permet d'accéder aux analogues “thia” des 3,4-bisindolylmaléimides. La synthèse de sulfonamides primaires tels que les dérivés 4-(hétéro)aryl substitués du benzènesulfonamide comme inhibiteurs potentiels de l'anhydrase carbonique, a également été envisagée dans ce travail.Une collection de matériaux hybrides constitués de nanoparticules de palladium adsorbées sur des nanostructures de carbone a été préparée et testée dans des réactions de couplages palladocatalysés, comme catalyseurs en milieu hétérogène. Le plus efficace d'entre eux, associant des nanoparticules de palladium stabilisées par le dodécanethiol et adsorbées sur des nanotubes de carbones “multi-walled”, a été employé afin de préparer vingt-quatre nouveaux dérivés 4-(hétéro)aryl substitués du benzènesulfonamide. L'échec de l'utilisation de ce catalyseur dans les reactions de fonctionalisation des isothiazolone-1,(1)-(di)oxydes nous a contraints à employer un catalyseur plus conventionnel, le PdCl2(dppf)•CH2Cl2.Le dernier volet de ce projet visait à concevoir des catalyseurs à base de nanoparticules de palladium encapsulées dans des nanofibres de carbone “grafitisées” (nanoréacteurs). Une série de nanoréacteurs a pu être préparée et nous avons étudié l'effet du confinement généré à l'intérieur de la nanofibre, sur la réaction palladocatalysée de Suzuki-Miyaura. / 3,4-bisindolylmaleimides possess an inhibitory activity against protein kinase. Because 3-isothiazolone-1,(1)-(di)oxide can be considered as maleimide analog, 5-chloro and 4,5-dichloro-3-isothiazolone-1,(1)-(di)oxide were functionalised using a palladium-catalysed Suzuki-Miyaura cross-coupling reaction achieving the “thia” analogs of 3,4-bisindolylmaleimides. We were also interested in the preparation primary sulfonamides such as 4-(hetero)aryl substituted benzenesulfonamides as carbonic anhydrases inhibitors.A series of hybrid materials comprising palladium nanoparticles adsorbed on carbon nanostructures has been prepared and tested as heterogeneous catalysts of palladium-mediated cross-coupling reactions. The best catalyst, resulting in palladium nanoparticles stabilised by dodecanethiol adsorbed on multi-walled carbon nanotubes, was employed in Suzuki-Miyaura reactions for the preparation of twenty-four new 4-(hetero)aryl substituted benzenesulfonamides. As this catalyst failed in the functionalisation of isothiazolone-1,(1)-(di)oxides, this latter was realised using a more conventional catalyst, PdCl2(dppf)•CH2Cl2.A last part of the project aimed to the conception of catalysts made of palladium nanoparticles encapsulated in graphitised carbon nanofibres (nanoreactors). We prepared a series of nanoreactors and we studied the effect of the confinement inside the nanofibre channel on the Suzuki-Miyaura cross-coupling reaction.

This thesis contains three main sections. In the first section, we examine methodologies to discriminate Type II protein kinase inhibitors from the Type I inhibitors. We have studied the structure of 55 Type II kinase inhibitors and have notice specific descriptive geometric features. Using this information we have developed a pharmacophore and a shape based screening approach. We have found that these methods did not effectively discriminate between the two inhibitor types used independently, but when combined in a consecutive way – pharmacophore search first, then shape based screening, we have found a method that successfully filtered out all Type I molecules. The effect of protonation states and using different conformer generators were studied as well. This method was then tested on a freely available database of decoy molecules and again shown to be discriminative. In the second section of the thesis, we implement and assess swarm-based docking methods. We implement a repulsive particle swarm optimization (RPSO) based conformational search approach into Autodock 3.05. The performance of this approach with different parameters was then tested on a set of 51 protein ligand complexes. The effect of using different factoring for the cognitive, social and repulsive terms and the importance of the inertia weight were explored. We found that the RPSO method gives similar performance to the particle swarm optimization method. Compared to the genetic algorithm approach used in Autodock 3.05, our RPSO method gives better results in terms of finding lower energy conformations. In the final, third section we have implemented a Monte Carlo (MC) based conformer searching approach into Gaussian03. This enables high level quantum mechanics/molecular mechanics (QM/MM) potentials to be used in docking molecules in a protein active site. This program was tested on two Zn2+ ion-containing complexes, carbonic anhydrase II and cytidine deaminase. The effects of different QM region definitions were explored in both systems. A consecutive and a parallel docking approach were used to study the volume of the active site explored by the MC search algorithm. In case of the carbonic anhydrase II complex, we have used 1,2-difluorobenzene as a ligand to explore the favourable interactions within the binding site. With the cytidine deaminase complex, we have evaluated the ability of the approach to discriminate the native pose from other higher energy conformations during the exploration of the active site of the protein. We find from our initial calculations, that our program is able to perform a conformational search in both cases, and the effect of QM region definition is noticeable, especially in the description of the hydrophobic interactions within the carbonic anhydrase II system. Our approach is also able to find poses of the cytidine deaminase ligand within 1 Å of the native pose.

A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKC&#946;I. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínas / The protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKC&#946;I. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins.

Dopamine (DA) governs movement, sleep, reward, and cognition. The presynaptic dopamine transporter (DAT), clears released DA, controlling DA signaling and homeostasis. Genetic DAT ablation causes hyperactivity, sleep reduction, and altered psychostimulant response. DAT surface expression is dynamic; DAT constitutively internalizes and recycles to and from the plasma membrane, and acute PKC activation stimulates DAT endocytosis. Cell line experiments demonstrated that PKC-stimulated DAT endocytosis requires Ack1 inactivation and the GTPase, Rit2. How Rit2 controls PKC-dependent DAT internalization, or whether regulated DAT endocytosis impacts behavior, is unknown. Here, I present data supporting that PKC activation stimulates Rit2/DAT dissociation, mediated by the DAT N-terminus. Further, Ack1 and Rit2 function independently to facilitate PKC-stimulated DAT internalization. Moreover, PKC-stimulated DAT endocytosis was limited to ventral striatum in ex vivo slice preparations, and required Rit2. Our lab previously demonstrated that certain DA-dependent behaviors required DAergic Rit2 in mice, however whether this was due to perturbed PKC-stimulated DAT internalization, or DAT-independent Rit2 function(s) remains untested. To address this, I turned to Drosophila and its Rit2 homolog Ric. I found that Ric and dDAT proteins interact in cell lines, and that constitutively active Ric (RicQ117L) increased dDAT function in cultured cells and ex vivo whole fly brains. However, neither DAergic Ric knockdown nor RicQ117L altered overall locomotion or sleep, suggesting that these fundamental behaviors do not require DAergic Ric. Together, these results expand our understanding of intrinsic mechanisms controlling DAT endocytosis, and their impact on behavior.

5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...