La protéine kinase activée par AMP : Criblage de nouveaux substrats membranaires et phosphorylation de la créatine kinase liée à une compartimentation subcellulaire

Various stimuli such as ischemia, hypoxia and exercise can induce ATP depletion evoking impaired energy homeostasis which becomes physiologically relevant at high workloads. Interestingly, cells dispose of compensatory mechanisms to counteract and compensate for the deficit in cellular energy state. Two key enzymes take center stage in the regulation of cellular and whole body energy metabolism. Creatine kinase (CK) can reacts immediately as an energy buffer and transport system in the cell with fluctuating energy demands to maintain cellular energy homeostasis while AMP-activated protein kinase (AMPK) is acting via regulation of downstream targets affecting more medium and long term adaptations in the cell to relieve energy stress. In this thesis work, first, basic tools for screening AMPK substrates have been established, including multidimensional protocols for HPLC protein purification and in vitro screening of putative AMPK membrane targets, including subfractionation of membranes and blue native-SDS-PAGE. As a proof of principle, different putative novel substrate candidates were obtained, and two confirmed by in vitro phosphorylation. Second, a putative interplay of both enzymes, AMPK and CK, has been confirmed for the first time in vitro and in vivo. Cytosolic brain-type creatine kinase (BCK) was identified as an AMPK target phosphorylated at serine 6 in vitro and in vivo by using phosphorylation assays and a generated phospho-Ser6-specific antibody. Phosphorylation is accompanied by transient interaction specifically involving activated AMPK and non-phosphorylated BCK as shown with yeast two hybrid assays (Y2H). AMPK-mediated phosphorylation of BCK does not affect enzyme kinetics, but localizes the kinase to the endoplasmic reticulum (ER) in vivo. The work presented in this thesis confirms an interplay between key kinases in energy homeostasis. Taken together our results suggest that AMPK seems to regulate more probably the association of BCK to subcellular compartments and not enzyme activity. Although it is unknown yet, the physiological role of BCK in the endoplasmic reticulum (ER), we can hypothesis that ER-localization of BCK induced by AMPK under energy stress may support heavily ATP-dependent processes in this microenvironment like e.g. calcium pumping.Divers stimuli tels que l'ischémie, l'hypoxie et l'exercice peuvent induire la déplétion en ATP provoquant une déregulation de l'homéostasie énergétique. Cependant, les cellules disposant de mécanismes compensatoires pour contrebalancer et compenser le déficit de l'état énergétique cellulaire. Deux enzymes clés participent à la régulation du métabolisme énergétique cellulaire. La créatine kinase (CK) peut réagir immédiatement comme transporteur d'énergie. D'autre part la protéine kinase activée par l'AMP (AMPK), régule au niveau cellulaire et du corps en entier la livraison et la consommation d'énergie. Dans ce travail de thèse, les outils de base pour realiser un criblage des cibles de l'AMPK ont d'abord été mis en place. Ce sont les protocoles multidimensionnelles pour la purification de protéines par HPLC et le criblage in vitro de cibles membranaires de l'AMPK, dont le sous-fractionnement des membranes et la separation par électrophorèse de type blue native PAGE. Différents candidats potentiels de l'AMPK ont été obtenus, et deux ont été confirmés par phosphorylation in vitro. Ensuite, une interaction entre les deux enzymes, l'AMPK et la CK, a été confirmée pour la première fois in vitro et in vivo. L'AMPK phosphoryle la créatine kinase cytosolique du cerveau (BCK) sur la serine 6 in vitro et in vivo. Cette phosphorylation a été mis en evidance à l'aide de tests de phosphorylation et d'un anticorps généré spécifique de la phospho-Ser6. Le system de double hybride en levure a montré que cette phosphorylation est acompagné par une interaction transitoire impliquant spécifiquement l'AMPK active et BCK non phosphorylé. La phosphorylation médiée par AMPK n'affecte pas l'activité enzymatique de BCK mais localise la kinase dans le réticulum endoplasmique (RE) in vivo. Le travail presente dans ce manuscript confirme une interaction entre les deux enzymes clefs dans l'homeostasis energetique. La caracherization in vitro et in vivo de la phosphorylation de la BCK par l'AMPK suggère que l'AMPK regule probablement l'associattion de BCK aux compartements subcellulaires et pas l'activité enzymatique. Cette localisation est induite par l'AMPK en cas de stress énergetique et peut soutenir des processus fortement dépendant de l'ATP dans ce microenvironnement, comme par exemple le pompage de calcium

Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites

International audienceThere is an increasing body of evidence for local circuits of ATP generation and consumption that are largely independent of global cellular ATP levels. These are mostly based on the formation of multiprotein(-lipid) complexes and diffusion limitations existing in cells at different levels of organization, e.g., due to the viscosity of the cytosolic medium, macromolecular crowding, multiple and bulky intracellular structures, or controlled permeability across membranes. Enzymes generating ATP or GTP are found associated with ATPases and GTPases enabling the direct fueling of these energy-dependent processes, and thereby implying that it is the local and not the global concentration of high-energy metabolites that is functionally relevant. A paradigm for such microcompartmentation is creatine kinase (CK). Cytosolic and mitochondrial isoforms of CK constitute a well established energy buffering and shuttling system whose functions are very much based on local association of CK isoforms with ATP-providing and ATP-consuming processes. Here we review current knowledge on the subcellular localization and direct protein and lipid interactions of CK isoforms, in particular about cytosolic brain-type CK (BCK) much less is known compared to muscle-type CK (MCK). We further present novel data on BCK, based on three different experimental approaches: (1) co-purification experiments, suggesting association of BCK with membrane structures such as synaptic vesicles and mitochondria, involving hydrophobic and electrostatic interactions, respectively; (2) yeast-two-hybrid analysis using cytosolic split-protein assays and the identifying membrane proteins VAMP2, VAMP3 and JWA as putative BCK interaction partners; and (3) phosphorylation experiments, showing that the cellular energy sensor AMP-activated protein kinase (AMPK) is able to phosphorylate BCK at serine 6 to trigger BCK localization at the ER, in close vicinity of the highly energy-demanding Ca(2+) ATPase pump. Thus, membrane localization of BCK seems to be an important and regulated feature for the fueling of membrane-located, ATP-dependent processes, stressing again the importance of local rather than global ATP concentrations

The detyrosination/re-tyrosination cycle of tubulin and its role and dysfunction in neurons and cardiomyocytes

Among the variety of post-translational modifications to which microtubules are subjected, the detyrosination/re-tyrosination cycle is specific to tubulin. It is conserved by evolution and characterized by the enzymatic removal and re-addition of a gene-encoded tyrosine residue at the C-terminus of α-tubulin. Detyrosinated tubulin can be further converted to Δ2 tubulin by the removal of an additional C terminal glutamate resi¬due. Detyrosinated and Δ2 tubulin are carried by stable microtubules whereas tyrosinated microtubules are present on dynamic polymers. The cycle regulates trafficking of many cargo transporting molecular motors and is linked to the microtubule dynamics via regulation of microtubule interactions with specific cellular effectors such as kinesin-13. Here, we give an historical overview of the general features discovered for the cycle. We highlight the recent progress toward structure and functioning of the enzymes that keep the levels of tyrosinated and detyrosinated tubulin in cells, the long-known tubulin tyrosine ligase and the recently discovered vasohibin-SVBP complexes. We further describe how the cycle controls microtubule functions in healthy neurons and cardiomyocytes and how deregulations of the cycle are involved in dysfunctions of these highly differentiated cells, leading to neurodegeneration and heart failure in humans

Déchiffrage du code tubuline: Le voile se lève sur le rôle de l’acétylation et de la détyrosination

International audienceLes microtubules sont des fibres du cytosquelette formées par l’assemblage d’hétérodimères d’α- et de β-tubuline. Ils contribuent à l’établissement de la forme des cellules et de leur polarité, ainsi qu’à leur mobilité. Ils jouent aussi un rôle important dans le transport intracellulaire et dans la division cellulaire. Le réseau microtubulaire s’adapte constamment aux besoins de la cellule. Il peut être constitué de microtubules très dynamiques ou d’autres plus stables. Pour moduler dans l’espace et le temps les différentes fonctions de ces fibres, de nombreuses modifications post-traductionnelles réversibles de la tubuline sont mises en jeu, à l’origine de ce qui est maintenant appelé le « code tubuline ». Dans cette revue, nous nous intéresserons au rôle de deux modifications caractéristiques des microtubules stables : l’acétylation et la détyrosination de l’α-tubuline. Nous discuterons également de l’implication de leur dérégulation dans certaines pathologies

A New Quantitative Cell-Based Assay Reveals Unexpected Microtubule Stabilizing Activity of Certain Kinase Inhibitors, Clinically Approved or in the Process of Approval

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Tau co-organizes dynamic microtubule and actin networks.

International audienceThe crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction, phosphorylation and ER localization

AbstractAMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca2+-pumping

Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation- dependent mechanism

International audienceProper regulation of microtubule dynamics is essential for cell functions and involves various microtubule-associated proteins (MAPs). Among them, end-binding proteins (EBs) accumulate at microtubule plus ends, whereas structural MAPs bind along the microtu-bule lattice. Recent data indicate that the structural MAP tau modulates EB subcellular local-ization in neurons. However, the molecular determinants of EB/tau interaction remain unknown , as is the effect of this interplay on microtubule dynamics. Here we investigate the mechanisms governing EB/tau interaction in cell-free systems and cellular models. We find that tau inhibits EB tracking at microtubule ends. Tau and EBs form a complex via the C-terminal region of EBs and the microtubule-binding sites of tau. These two domains are required for the inhibitory activity of tau on EB localization to microtubule ends. Moreover, the phos-phomimetic mutation S262E within tau microtubule-binding sites impairs EB/tau interaction and prevents the inhibitory effect of tau on EB comets. We further show that microtubule dynamic parameters vary, depending on the combined activities of EBs and tau proteins. Overall our results demonstrate that tau directly antagonizes EB function through a phos-phorylation-dependent mechanism. This study highlights a novel role for tau in EB regulation, which might be impaired in neurodegenerative disorders

VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules

ABSTRACT The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzyme complexes involved in microtubule detyrosination. However, little is known about their mode of action. Here, we show in reconstituted systems and in cells that VASH1-SVBP and VASH2-SVBP drive global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of human VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We further show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH domain is critical for microtubule-detachment and diffusion of VASH-SVBP enzymes on the lattice. Together, our results suggest a mechanism by which these enzymes could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions. SUMMARY VASH1-SVBP and VASH2-SVBP produce global and local detyrosination patterns of microtubule lattices, respectively. These activities rely on the interplay between the N- and C-terminal disordered regions of the enzymes, which determine their differential molecular mechanism of action. GRAPHICAL ABSTRACT Schematic representation of divergent molecular mechanisms of action of VASH-SVBP detyrosination complexes