CK2ß Regulates Hematopoietic Stem Cell Biology and Erythropoiesis.

The Ser-Thr kinase CK2 plays important roles in sustaining cell survival and resistance to stress and these functions are exploited by different types of blood tumors. Yet, the physiological involvement of CK2 in normal blood cell development is poorly known. Here, we discovered that the ß regulatory subunit of CK2 is critical for normal hematopoiesis in the mouse. Fetal livers of conditional CK2ß knockout embryos showed increased numbers of hematopoietic stem cells associated to a higher proliferation rate compared to control animals. Both hematopoietic stem and progenitor cells (HSPCs) displayed alterations in the expression of transcription factors involved in cell quiescence, self-renewal, and lineage commitment. HSPCs lacking CK2ß were functionally impaired in supporting both in vitro and in vivo hematopoiesis as demonstrated by transplantation assays. Furthermore, KO mice developed anemia due to a reduced number of mature erythroid cells. This compartment was characterized by dysplasia, proliferative defects at early precursor stage, and apoptosis at late-stage erythroblasts. Erythroid cells exhibited a marked compromise of signaling cascades downstream of the cKit and erythropoietin receptor, with a defective activation of ERK/JNK, JAK/STAT5, and PI3K/AKT pathways and perturbations of several transcriptional programs as demonstrated by RNA-Seq analysis. Moreover, we unraveled an unforeseen molecular mechanism whereby CK2 sustains GATA1 stability and transcriptional proficiency. Thus, our work demonstrates new and crucial functions of CK2 in HSPC biology and in erythropoiesis.

Regulation of sclerostin by the SIRT1 stabilization pathway in osteocytes.

Osteocytes play a critical role in bone remodeling through the secretion of paracrine factors regulating the differentiation and activity of osteoblasts and osteoclasts. Sclerostin is a key osteocyte-derived factor that suppresses bone formation and promotes bone resorption, therefore regulators of sclerostin secretion are a likely source of new therapeutic strategies for treatment of skeletal disorders. Here, we demonstrate that protein kinase CK2 (casein kinase 2) controls sclerostin expression in osteocytes via the deubiquitinase ubiquitin-specific peptidase 4 (USP4)-mediated stabilization of Sirtuin1 (SIRT1). Deletion of CK2 regulatory subunit, Csnk2b, in osteocytes (Csnk2bDmp1) results in low bone mass due to elevated levels of sclerostin. This phenotype in Csnk2bDmp1 mice was partly reversed when sclerostin expression was downregulated by a single intravenous injection with bone-targeting adeno-associated virus 9 (AAV9) carrying an artificial-microRNA that targets Sost. Mechanistically, CK2-induced phosphorylation of USP4 is important for stabilization of SIRT1 by suppressing ubiquitin-dependent proteasomal degradation. Upregulated expression of SIRT1 inhibits sclerostin transcription in osteocytes. Collectively, the CK2-USP4-SIRT1 pathway is crucial for the regulation of sclerostin expression in osteocytes to maintain bone homeostasis.

CK2ß-regulated signaling controls B cell differentiation and function.

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the ß regulatory subunit of CK2. CK2ßKO mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2ßKO animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2ßKO mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2ß have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2ßKO mice suggesting the importance of the ß subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.

A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation.

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.

Protein kinase CK2 inhibition is associated with the destabilization of HIF-1α in human cancer cells.

Screening for protein kinase CK2 inhibitors of the structural diversity compound library (DTP NCI/NIH) led to the discovery of 4-[(E)-(fluoren-9-ylidenehydrazinylidene)-methyl]benzoic acid (E9). E9 induces apoptotic cell death in various cancer cell lines and upon hypoxia, the compound suppresses CK2-catalyzed HSP90/Cdc37 phosphorylation and induces HIF-1α degradation. Furthermore, E9 exerts a strong anti-tumour activity by inducing necrosis in murine xenograft models underlining its potential to be used for cancer treatment in future clinical studies. Crystal structure analysis of human and maize CK2α in complex with E9 reveals unique binding properties of the inhibitor to the enzyme, accounting for its affinity and selectivity.

Expression and purification of PI3 kinase alpha and development of an ATP depletion and an alphascreen PI3 kinase activity assay.

Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal His-tag and the p85alpha regulatory domain in Sf9 insect cells. The complex consisting of p110alpha and p85alpha was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110alpha/p85alpha. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110alpha/p85alpha and determined the IC(50) for wortmannin, a known PI3 kinase inhibitor. The IC(50) for wortmannin was determined to be 7 nM. From a selection of substrates, phosphatidylinositol-4, 5-biphosphate turned out to be the best substrate at a concentration of 50 microM. p110alpha/p85alpha underwent autophosphorylation most prominently at the p85alpha subunit. However, in the presence of lipid substrate, the autophosphorylation was negligible. In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors.

Exploring the intramolecular phosphorylation sites in human Chk2.

A comparative biochemical analysis was performed using recombinant human protein kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a concentration-dependent manner. Despite distinct time-dependent autophosphorylation kinetics by monitoring the phosphorylation of amino acid residues T68, S19, S33/35, T432, in Chk2 wildtype and Chk2 mutants (T68A, T68D and Q69E) they gave identical specific activities. However, upon gel filtration of Chk2 wildtype and the mutants, only Chk2 wildtype and the T68D mutant led to the formation of a 'pure' dimer; dephosphorylated wildtype Chk2 eluted as a monomer. Transfection of HEK293 cells with Chk2 wildtype and Chk2 mutants in the absence or presence of DNA damage showed significant T68 phosphorylation already in the absence of DNA damaging reagents. Upon DNA damage, phosphorylation of additional Chk2 sites was observed (S19, S33/35). A comparison of ATM+/+ and ATM-/- cells with respect to phosphorylation of residues T68, S19, S33/35 in the absence and presence of DNA damage showed in all cases phosphorylation of T68, although signal intensity was increased ca. three-fold after DNA damage. Mass spectrometric analyses of human recombinant Chk2 isolated from bacteria and insect cells showed distinct differences. The number of phosphorylated residues in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of the recombinant human Chk2 from insect cells it was 8. Except for phosphorylated amino acid T378 which was not found in the Chk2 isolated from bacteria, all other phosphorylated residues identified in human Chk2 from insect cells were present also in Chk2 from bacteria.

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

Knocking 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.

Mineralocorticoid receptor antagonists do not block rapid ERK activation by aldosterone.

Aldosterone can elicit rapid nongenomic effects both in vivo and in vitro, often mediated by signal transduction cascades. However, it is not understood how these rapid effects are initiated. In this study we show that aldosterone leads to rapid activation of mitogen activated protein kinases ERK1/2 in the cortical collecting duct cell line M-1. Inhibitors of transcription and translation could not block this activation, which suggests an extranuclear (nongenomic) mechanism. Although it is known that M-1 cells do not contain a transcriptionally functional MR, it is not known whether a closely related protein still could mediate the effects, or an unrelated nonclassic receptor. To test this hypothesis, the effects of four classical mineralocorticoid receptor antagonists were studied. None of the compounds could block the response to aldosterone. Altogether, the data suggest that rapid aldosterone effects in M-1 cells are initiated by a receptor different from the classical mineralocorticoid receptor.

Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit.

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2beta dimer modulates the activity of CK2alpha. The beta4/beta5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2alpha subunits of the CK2 holoenzyme. CK2alpha monomers with this closed beta4/beta5 loop conformation are unable to bind CK2beta dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2alpha from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2alpha monomers and CK2beta dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.