CK2b regulates thrombopoiesis and Ca21-Triggered platelet activation in arterial thrombosis

© 2017 by The American Society of Hematology. Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-) physiology has remained elusive. The present study explored the impact of the CK2 regulatory b-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β-/-) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-Triphosphate receptor-dependent intracellular calcium (Ca21) release. Presumably due to a blunted increase in the concentration of cytosolic Ca21, activation-dependent increases of a and dense-granule secretion and integrin aIIbb3 activation, and aggregation were abrogated in ck2β-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo wassignificantly blunted in ck2β-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2b-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2b as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo

Etude des voies de signalisation et de l'expression genique liees a la transition GO/Gl du cycle de division cellulaire dans les fibroblastes murins Balb/c 3T3 transformes chimiquement

SIGLEINIST T 74867 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Étude fonctionnelle de la sous-unité régulatrice de la protéine kinase CK2 dans les cellules souches embryonnaires murines : survie cellulaire, oligodendrogenèse et cancérogenèse

La protéine kinase CK2 est un complexe moléculaire dynamique composé de 2 sous-unités catalytiques a et d'un dimère de sous-unités régulatrices b. La structure cristallographique du dimère de CK2b suggère qu il représente une plate-forme moléculaire, plaçant la protéine au cœur d un réseau de voies de signalisation. L'invalidation du gène CK2b chez la souris conduit à un défaut de viabilité des cellules souches embryonnaires (ES). Nous avons pu étudier la fonction de différents domaines fonctionnels in vitro dans un modèle cellulaire reposant sur une stratégie d invalidation conditionnelle du gène CK2b dans les cellules ES murines. L abolition de l expression du gène endogène est létale pour les cellules ES et leur viabilité est restaurée par l expression exogène de la protéine CK2b sauvage. Ce modèle nous permet d étudier l implication de divers domaines de CK2b dans la survie, la prolifération et la différenciation des cellules souches embryonnaires, en remplaçant la protéine endogène par diverses formes mutées de la protéine. Parallèlement, l invalidation conditionnelle du gène chez la souris ciblée dans les précurseurs neuraux a montré que CK2b est un élément clef dans les voies de signalisation qui contrôlent l oligodendrogenèse. La différenciation des cellules ES nullizygotes exprimant la protéine exogène sauvage nous a permis de montrer que celle-ci restaure la lignée oligodendrocytaire. Nous avons aussi montré que certains domaines de CK2b, impliqués dans la fonction régulatrice du dimère, sont des déterminants structuraux cruciaux pour la prolifération des cellules souches neurales et leur progression dans la lignée oligodendrocytaire.The protein kinase CK2 is a dynamic molecular complex of 2 catalytic subunits a and of a dimer of regulatory subunit b. Cristallographic structure of the CK2b dimer suggests the existence of functional domains involved in the regulating function of the dimer towards the catalytic subunits, as well as in interactions with various protein partners. The CK2b dimer would represent a molecular platform, linking the protein to the center of signalling pathway networks. The knockout of the murin CK2b gene results in a defect of embryonic stem cell viability. We studied the in vitro function of different functional domains with the use of a death-rescue cellular model of by additional transgenesis of mutants CK2b. This cellular system is based on a conditional CK2b gene knockout strategy using murin embryonic stem cells (ES cells). The abolition of the endogenous gene expression is lethal for ES cells and the exogenous expression of the wild-type protein CK2b rescues their viability. This death-rescue model allows us to study the involvement of various domains of CK2b in the survival, the proliferation and the differentiation of embryonic stem cells. In parallel, the conditional targeted knockout in murin neural precursors demonstrated that CK2b is a key element in signalling pathways that control oligodendrogenesis. The differentiation of nullizygotes ES cells expressing the wild-type exogenous protein reveals that wild-type CK2b rescues the oligodendrocyte lineage. We also showed that some CK2b domains, linked to dimer regulatory functions, are crucial structural determinants for the proliferation of neural stem cells and their progression into the oligodendrocyte lineage.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

La protéine kinase CK2, une enzyme qui cultive la différence

La perte des mécanismes de contrôle de la progression du cycle de division cellulaire ou du déclenchement des processus d’apoptose permet aux cellules d’acquérir des propriétés décisives pour leur transformation tumorale. Plusieurs protéine kinases participent à la transduction de signaux qui neutralisent des composants de la machinerie apoptotique. Dans ce contexte, la protéine kinase CK2 (caséine kinase 2), dont la structure vient d’être élucidée, apparaît comme un régulateur déterminant pour la viabilité cellulaire. Il est concevable que la surexpression de la CK2 observée dans les cancers puisse conduire à la formation de signaux de survie contribuant à la tumorigenèse.Protein kinase CK2 (formerly known as casein kinase 2) was among the first protein kinases to be identified and characterized. Surprisingly, in spite of intense efforts, the regulation and cellular functions of CK2 remain obscure. However, recent data on its molecular structure, its signal-mediated intracellular dynamic localization and its unexpected function in cell survival have raised new interest in this enzyme. These studies reveal unique features of CK2 and highlight its importance in the transduction of survival signals

Knocking out the regulatory beta subunit of protein kinase CK2 in mice: gene dosage effects in ES cells and embryos.

International audienceKnocking out the regulatory beta subunit of protein kinase CK2 in mice leads to early embryonic lethality. Heterozygous CK2beta (CK2beta+/-) knockout mice do not show an obvious phenotype. However, the number of heterozygous offsprings from CK2B+/- inter-crossings is lower than expected, meaning that some heterozygous embryos do not survive. Interestingly, CK2beta+/- ES (Embryonic Stem) cells express a considerably lower level of CK2beta than wild-type ES cells, whereas the level of CK2beta in organs from heterozygous adult mice does not significantly differ from those of wild-type mice. The data suggest a compensatory mechanism that adjusts CK2beta levels during development in the majority of, but not in all, cases (Mol Cell Biol 23: 908-915, 2003). In order to find an explanation for the gene dosage effect observed for heterozygous offsprings, we analysed embryos at mid-gestation (E10.5) as well as wild-type and CK2beta+/- ES cells for differences in growth rate and response to different stress agents. Analysis of E10.5 embryos generated from heterozygous matings revealed about 20% of smaller retarded CK2beta+/- embryos. No correlation between CK2beta levels in normal looking and retarded CK2beta+/- embryos were found. However, a different post-translational form of CK2beta protein has been detected in these retarded embryos. Cellular parameters such as growth rate and G1-, G2-checkpoints in ES cells were identical in both wild-type and CK2beta+/- cells. When ES cells were injected to induce differentiated teratocarcinoma in syngenic mice, the size of the tumours correlated with the level of CK2beta

RNA-Guided Genomic Localization of H2A.L.2 Histone Variant

International audienceThe molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin

Disruption of CK2β in Embryonic Neural Stem Cells Compromises Proliferation and Oligodendrogenesis in the Mouse Telencephalon▿ †

Genetic programs that govern neural stem/progenitor cell (NSC) proliferation and differentiation are dependent on extracellular cues and a network of transcription factors, which can be regulated posttranslationally by phosphorylation. However, little is known about the kinase-dependent pathways regulating NSC maintenance and oligodendrocyte development. We used a conditional knockout approach to target the murine regulatory subunit (beta) of protein kinase casein kinase 2 (CK2β) in embryonic neural progenitors. Loss of CK2β leads to defects in proliferation and differentiation of embryonic NSCs. We establish CK2β as a key positive regulator for the development of oligodendrocyte precursor cells (OPCs), both in vivo and in vitro. We show that CK2β directly interacts with the basic helix-loop-helix (bHLH) transcription factor Olig2, a critical modulator of OPC development, and activates the CK2-dependent phosphorylation of its serine-threonine-rich (STR) domain. Finally, we reveal that the CK2-targeted STR domain is required for the oligodendroglial function of Olig2. These findings suggest that CK2 may control oligodendrogenesis, in part, by regulating the activity of the lineage-specific transcription factor Olig2. Thus, CK2β appears to play an essential and uncompensated role in central nervous system development

HDAC6 controls the kinetics of platelet activation.

International audienceHDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology

Disruption of the Regulatory β Subunit of Protein Kinase CK2 in Mice Leads to a Cell-Autonomous Defect and Early Embryonic Lethality

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory β subunit, CK2β, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2β gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2β(−/−) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2β is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2β. Thus, our study demonstrates that in mammals, CK2β is essential for viability at the cellular level, possibly because it acquired new functions during evolution

Marginal band microtubules are acetylated by αTAT1

International audienceThe discoid shape of resting platelets is maintained by a peripheral, circular bundle of microtubules called marginal band. Marginal band microtubules are acetylated on lysine 40 of the alpha-tubulin subunits. We have previously shown that the deacetylase HDAC6 is responsible for tubulin deacetylation in platelets and that the hyperacetylated state of the microtubules in HDAC6KO platelets correlates with faster activation/spreading kinetics, pointing to a regulatory role of this modification. So far, the question about the reverse enzyme, responsible for tubulin acetylation in platelets, has remained unanswered. Several enzymes have been described as having tubulin acetylation activity. Here we identify αTAT1 as the enzyme responsible for the acetylation of marginal band microtubules. We show that αTAT1 deficiency has only minor consequences for platelet production and function. A residual tubulin acetylation level in αTAT1 deficient platelet lysates suggests the presence of an additional tubulin-acetylating enzyme that is unable to acetylate marginal band microtubules