ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing , Osteocytes , Sirtuin 1 , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Mice , Osteoblasts/metabolism , Osteocytes/metabolism , Osteogenesis , Sirtuin 1/metabolismABSTRACT
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.
Subject(s)
Casein Kinase II , Lymphocyte Activation , Animals , Mice , Sheep , Casein Kinase II/genetics , Signal Transduction , Protein Serine-Threonine Kinases/metabolism , Mice, Knockout , Receptors, Antigen, B-Cell/genetics , Cell DifferentiationABSTRACT
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.
Subject(s)
Core Binding Factor Alpha 1 Subunit/metabolism , Ossification, Heterotopic/metabolism , Osteogenesis , Adult , Aged , Animals , Casein Kinase II/genetics , Casein Kinase II/metabolism , Cell Differentiation , Cleidocranial Dysplasia/genetics , Cleidocranial Dysplasia/pathology , Female , Gene Deletion , Haploinsufficiency/genetics , Hindlimb/metabolism , Humans , Male , Mice, Inbred C57BL , Middle Aged , Ossification, Heterotopic/genetics , Ossification, Heterotopic/pathology , Osteoblasts/metabolism , Phosphorylation , Protein Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ubiquitin-Specific Peptidase 7/metabolismABSTRACT
OBJECTIVES: In humans, non-obstructive azoospermia (NOA) is a major cause of male infertility. However, the aetiology of NOA is largely unknown. Previous studies reported that protein CK2ß was abundantly and broadly expressed in spermatogenic cells. Here, we investigate whether protein CK2ß participates in spermatogenesis. MATERIALS AND METHODS: In this study, we separated spermatogenic cells using STA-PUT velocity sedimentation, analysed the expression pattern of protein CK2ß by immunoblotting, specifically deleted Ck2ß gene in early-stage spermatogenic cells by crossing Ck2ßfl mice with Stra8-Cre+ mice and validated the knockout efficiency by quantitative RT-PCR and immunoblotting. The phenotypes of Ck2ßfl/Δ ;SCre+ mice were studied by immunohistochemistry and immunofluorescence. The molecular mechanisms of male germ cell development arrest were elucidated by immunoblotting and TUNEL assay. RESULTS: Ablation of Ck2ß gene triggered excessive germ cell apoptosis, germ cell development arrest, azoospermia and male infertility. Inactivation of Ck2ß gene caused distinctly reduced expression of Ck2α' gene and CK2α' protein. CONCLUSIONS: Ck2ß is a vital gene for germ cell survival and male fertility in mice.
Subject(s)
Apoptosis/genetics , Azoospermia , Casein Kinase II/deficiency , Germ Cells , Animals , Azoospermia/enzymology , Azoospermia/genetics , Azoospermia/pathology , Casein Kinase II/metabolism , Gene Deletion , Germ Cells/enzymology , Germ Cells/pathology , Male , Mice , Mice, KnockoutABSTRACT
The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.
Subject(s)
Casein Kinase II/immunology , T-Lymphocytes, Regulatory/immunology , Th2 Cells/immunology , Animals , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Growth Processes/immunology , Cell Line , Dendritic Cells/enzymology , Dendritic Cells/immunology , Forkhead Transcription Factors/immunology , Humans , Hypersensitivity/blood , Hypersensitivity/immunology , Interferon Regulatory Factors/immunology , Leukocytes, Mononuclear/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Cell Surface/immunology , T-Lymphocytes, Regulatory/enzymology , Th2 Cells/enzymologyABSTRACT
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.
Subject(s)
Benzoates/pharmacology , Casein Kinase II/antagonists & inhibitors , Cell Proliferation/drug effects , Fluorenes/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Neoplasms/drug therapy , Protein Conformation/drug effects , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Animals , Blotting, Western , Cell Cycle/drug effects , Combinatorial Chemistry Techniques , Female , Fluorescent Antibody Technique , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunoenzyme Techniques , Luciferases/metabolism , Mice , Mice, Nude , Neoplasms/enzymology , Neoplasms/pathology , Phosphorylation/drug effects , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Adenosine Triphosphate/deficiency , Biological Assay/methods , Phosphatidylinositol 3-Kinases/isolation & purification , Phosphatidylinositol 3-Kinases/metabolism , Androstadienes/pharmacology , Class I Phosphatidylinositol 3-Kinases , Humans , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Time Factors , Titrimetry , WortmanninABSTRACT
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.
Subject(s)
Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Line , Checkpoint Kinase 2 , DNA-Binding Proteins/genetics , Enzyme Activation , Escherichia coli/genetics , Fibroblasts/metabolism , Humans , Kidney/cytology , Kinetics , Mass Spectrometry , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Peptides/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spodoptera/cytology , Spodoptera/metabolism , Transfection , Tumor Suppressor Proteins/geneticsABSTRACT
The release of Agrin by motoneurons activates the muscle-specific receptor tyrosine kinase (MuSK) as the main organizer of subsynaptic specializations at the neuromuscular junction. MuSK downstream signaling is largely undefined. Here we show that protein kinase CK2 interacts and colocalizes with MuSK at post-synaptic specializations. We observed CK2-mediated phosphorylation of serine residues within the kinase insert (KI) of MuSK. Inhibition or knockdown of CK2, or exchange of phosphorylatable serines by alanines within the KI of MuSK, impaired acetylcholine receptor (AChR) clustering, whereas their substitution by residues that imitate constitutive phosphorylation led to aggregation of AChRs even in the presence of CK2 inhibitors. Impairment of AChR cluster formation after replacement of MuSK KI with KIs of other receptor tyrosine kinases correlates with potential CK2-dependent serine phosphorylation within KIs. MuSK activity was unchanged but AChR stability decreased in the presence of CK2 inhibitors. Muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function. This is the first description of a regulatory cross-talk between MuSK and CK2 and of a role for the KI of the receptor tyrosine kinase MuSK for the development of subsynaptic specializations.
Subject(s)
Casein Kinase II/metabolism , Neuromuscular Junction/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cholinergic/metabolism , Serine/metabolism , Amino Acid Sequence , Animals , Casein Kinase II/genetics , Cell Line , Humans , In Vitro Techniques , Mice , Mice, Knockout , Molecular Sequence Data , Muscle Fibers, Skeletal/physiology , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Rats , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cholinergic/genetics , Two-Hybrid System TechniquesABSTRACT
Protein kinase CK2 (former name: "casein kinase 2") is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2alpha) and two regulatory subunits (CK2beta). In higher animals two paralog catalytic chains-abbreviated CK2alpha and CK2alpha'--exist which can combine with CK2beta to three isoforms of the holoenzyme: CK2alpha(2)beta(2), CK2alpha(2)(')beta(2), and CK2alphaalpha(')beta(2). While CK2alpha and the "normal" holoenzyme CK2alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2alpha' and the "alternative" holoenzyme CK2alpha(2)(')beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2alpha' rather than CK2alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2alpha(2)(')beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2alpha'-MBP in the presence of human CK2beta so that CK2alpha' subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2alpha'-based and the CK2alpha-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.
Subject(s)
Casein Kinase II/isolation & purification , Carrier Proteins , Casein Kinase II/genetics , Holoenzymes/genetics , Holoenzymes/isolation & purification , Humans , Isoenzymes/genetics , Isoenzymes/isolation & purification , Maltose-Binding Proteins , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
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.
Subject(s)
Casein Kinase II/metabolism , Embryo, Mammalian/abnormalities , Gene Dosage , Protein Subunits/metabolism , Stem Cells/physiology , Animals , Casein Kinase II/genetics , Cell Cycle , Embryo, Mammalian/enzymology , Heterozygote , Mice , Mice, Knockout , Phenotype , Protein Subunits/genetics , Stem Cells/enzymology , Teratocarcinoma/enzymology , Teratocarcinoma/pathologyABSTRACT
The Drosophila clock proteins timekeeper (CK2a(Tik)) and andante (CK2beta(And)) are mutated CK2alpha and CK2beta subunits, respectively. In order to revisit the hypothesis concerning a perturbation of the beta/beta and/or alpha/beta subunit association, involving the andante mutant we have cloned, expressed and purified the recombinant andante mutant CK2beta(And) and a CK2 holoenzyme composed of CK2beta(And) and the wildtype CK2alpha subunit. Biochemical analyses using gel filtration analysis, inhibitor and heat treatment, as well as urea denaturation studies did not yield significant differences between the wildtype holoenzyme (alpha2beta2) and a holoenzyme containing wildtype CK2alpha and andante CK2beta(And). The timekeeper mutant, CK2alpha(Tik) has been reported to show a significant reduction in enzyme activity. In order to closely investigate the reason for this reduction in activity, we have also cloned and expressed the human homologue of Drosophila timekeeper. Using a CK2 holoenzyme containing the human timekeeper mutant and the wildtype CK2beta subunit we could confirm a strongly reduced activity towards CK2 substrates, but also a significant reduction in the autophosphorylation of the CK2beta in the absence of any substrate. Based on a structure-based model we postulate that the mutation M161K in Drosophila (i.e. M163K in human) is responsible for the drastic loss of activity, where the lysine residue may cause improper binding of the tri-nucleotide.
Subject(s)
Casein Kinase II/metabolism , Circadian Rhythm , Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Models, Molecular , Amino Acid Sequence , Animals , Casein Kinase II/chemistry , Casein Kinase II/genetics , Drosophila Proteins/genetics , Holoenzymes/genetics , Holoenzymes/metabolism , Humans , Hypothalamus/metabolism , Molecular Sequence Data , Mutation , Phosphorylation , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Aldosterone has attracted considerable interest as an independent cardiovascular risk marker, which has been demonstrated in a number of studies. Furthermore, recent studies revealed the prevalence of hyperaldosteronism to be about tenfold higher than previously assumed, which underlines its clinical importance. Aldosterone affects virtually any part of the cardiovascular system, namely cardiac fibroblasts and myocytes, and vascular endothelial and smooth muscle cells. In the latter cells, our laboratory has demonstrated a variety of rapid effects of the steroid, e.g. on intracellular calcium, inositol trisphosphate, and cAMP. There is also evidence for a modulation of genomic events by rapid aldosterone effects that occur via phosphorylation of transcription factors such as CREB. Furthermore, rapid tyrosine phosphorylation has been observed in vascular cells. The majority of rapid responses reported to date are insensitive towards the classic mineralocorticoid receptor (MR) antagonist, spironolactone. The in vitro experiments are complemented by a series of clinical studies in healthy volunteers, which could demonstrate rapid modulation of cardiovascular parameters after aldosterone administration, e.g. of systemic vascular resistance. In addition, an interaction of aldosterone with the adrenergic system has been observed. Most recently, rapid aldosterone induced contraction of resistance arteries has been reported. In general, the rapid in vivo effects of aldosterone are likely to participate in the pathogenesis of cardiovascular disorders. As many rapid and thus nonclassic aldosterone responses cannot be blocked by spironolactone, further research is required in order to provide adequate inhibitors to interfere with these pathways.
Subject(s)
Aldosterone/pharmacology , Cardiovascular System/drug effects , Aldosterone/physiology , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/physiopathology , Cardiovascular System/cytology , Cardiovascular System/metabolism , Clinical Trials as Topic , Cyclic AMP Response Element-Binding Protein/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , Mineralocorticoid Receptor Antagonists/pharmacology , Phosphorylation , Receptors, Mineralocorticoid/metabolism , Spironolactone/pharmacology , Vascular Resistance/drug effects , Vascular Resistance/physiologyABSTRACT
We investigated the effect of aldosterone on Src kinase. In the kidney cell line, M-1 aldosterone leads to a >2-fold transient activation of Src kinase seen as early as 2 min after aldosterone administration. Maximal Src kinase activation was measured at an aldosterone concentration of 1 nM. In parallel to activation, autophosphorylation at Tyr-416 of Src kinase increased. Src kinase activation was blocked by spironolactone. Aldosterone led to increased association of Src with HSP84. Furthermore, rapamycin blocked aldosterone-induced Src activation. We conclude that Src activation by aldosterone is mediated through the mineralocorticoid receptor and HSP84.
Subject(s)
Aldosterone/pharmacology , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Receptors, Mineralocorticoid/metabolism , src-Family Kinases/metabolism , Aldosterone/metabolism , Animals , Blotting, Western , Cell Line , Dose-Response Relationship, Drug , Enzyme Activation , Kidney/metabolism , Mice , Phosphorylation , Precipitin Tests , Sirolimus/pharmacology , Spironolactone/metabolism , Time Factors , Tyrosine/chemistryABSTRACT
Aldosterone elicits not only genomic effects with physiological consequences within hours or days but also elicits rapid nongenomic effects, such as activation of sodium transport in target cells, within seconds or minutes. Rapid aldosterone effects, which have also been shown in several in vivo studies in humans (e.g., increase in peripheral vascular resistance and blood pressure), are of potential clinical importance.
Subject(s)
Aldosterone/physiology , Blood Pressure/physiology , Sodium/metabolism , Vascular Resistance/physiology , Humans , Time FactorsABSTRACT
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.
Subject(s)
Aldosterone/pharmacology , Mineralocorticoid Receptor Antagonists , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Aldosterone/metabolism , Animals , Butadienes/pharmacology , Cell Line , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Hydrocortisone/pharmacology , Kidney Cortex/cytology , Kidney Cortex/metabolism , Kidney Tubules, Collecting/cytology , Mice , Mineralocorticoid Receptor Antagonists/pharmacology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Nitriles/pharmacology , Phosphorylation , Proto-Oncogene Proteins c-raf/metabolismABSTRACT
Nongenomic actions of aldosterone have been described in a number of cell culture and in vivo systems. They occur, in contrast to the classical genomic effects on gene transcription, rapidly within seconds to minutes after aldosterone administration. The primary effector is still unknown. Whether it is a so far unidentified membrane bound aldosterone receptor or the classical mineralocorticoid receptor or both is under debate. The downstream signaling cascade involved in such rapid actions begins to be elucidated. In this work, we discuss the nature of the putative membrane receptor for aldosterone and summarize observed rapid aldosterone effects in different in vitro and in vivo systems.
Subject(s)
Aldosterone/physiology , Signal Transduction/physiology , Transcription, Genetic , Animals , Cell Membrane/physiology , Humans , Receptor Cross-Talk/physiologyABSTRACT
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.