共查询到20条相似文献,搜索用时 15 毫秒
1.
Raaf J Bischoff N Klopffleisch K Brunstein E Olsen BB Vilk G Litchfield DW Issinger OG Niefind K 《Biochemistry》2011,50(4):512-522
The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design. 相似文献
2.
The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2β, noncatalytic subunit of CK2). CK2α belongs to the superfamily of eukaryotic protein kinases (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix αC and in the activation segment, leading to a characteristic arrangement of catalytic key elements. For CK2α, however, no strict physiological control of activity is known. Accordingly, CK2α was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N-terminal segment on one side, and helix αC and the activation segment on the other side. We report here the structure of a C-terminally truncated variant of human CK2α in which the enzyme adopts a decidedly inactive conformation for the first time. In this CK2α structure, those regulatory key regions still are in their active positions. Yet the glycine-rich ATP-binding loop, which is normally part of the canonical anti-parallel β-sheet, has collapsed into the ATP-binding site so that ATP is excluded from binding; specifically, the side chain of Arg47 occupies the ribose region of the ATP site and Tyr50, the space required by the triphospho moiety. We discuss some factors that may support or disfavor this inactive conformation, among them coordination of small molecules at a remote cavity at the CK2α/CK2β interaction region and binding of a CK2β dimer. The latter stabilizes the glycine-rich loop in the extended active conformation known from the majority of CK2α structures. Thus, the novel inactive conformation for the first time provides a structural basis for the stimulatory impact of CK2β on CK2α. 相似文献
3.
Waiwut P Shin MS Inujima A Zhou Y Koizumi K Saiki I Sakurai H 《Molecular and cellular biochemistry》2011,356(1-2):169-175
CK2 is a heterotetrameric ubiquitous kinase consisting of two catalytic subunits and two regulatory subunits. The two catalytic subunits, α and α', are highly homologous but differ in their C-terminal regions. It is not known whether CK2α and α' have distinctive substrate specificity, since no α- or α'-specific substrate has been identified. Thus, it is assumed that the two kinase isoforms overlap in their substrate specificity. CK2 protein levels and activity were found to be elevated in the brain when compared to other organs. Here we have studied the protein levels of CK2α and α' isoforms in nine major brain regions. We found that both, CK2α and α', are expressed in all brain regions tested. Whereas CK2α levels do not vary strongly across the regions, CK2α' levels are slightly higher in the cortex and hippocampus than in other regions. Furthermore, we show that CK2α protein levels in the striatum are relatively high when compared to CK2α'. The approximate stoichiometry ratio of CK2α:CK2α' is 8:1. Therefore, one can consider that CK2α levels are predominant in comparison to CK2α' levels throughout the mammalian brain. 相似文献
4.
5.
6.
Jennifer Hochscherf Dirk Lindenblatt Michaela Steinkrüger Eungyoung Yoo Özlem Ulucan Stefan Herzig Olaf-Georg Issinger Volkhard Helms Claudia Götz Ines Neundorf Karsten Niefind Markus Pietsch 《Analytical biochemistry》2015
Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2β in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α1–335 and the fluorescent probe CF-Ahx-Pc as a CK2β analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α1–335. Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2β. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α1–335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α1–335 were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240 nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2β, with an IC50 value of 92 μM. 相似文献
7.
Synthetic peptides reproducing the amino and carboxyl terminal region of CK2 subunit have been analyzed for their ability to mimic different properties of full length subunit. Peptide [1-77], containing both the autophosphorylation site and the down-regulatory domain 55-64, is readily phosphorylated by a subunit whose activity is concomitantly inhibited. Such inhibition is accompanied by a weak interaction detectable by BIAcore sensograms but not by far Western blots, and is not reversed by polylysine which conversely overcome inhibition of calmodulin phosphorylation by full length subunit. A strong interaction with is observed with [155-215] but not with its shorter derivative [170-215] as judged from far Western blotting and sucrose gradient ultracentrifugation analysis. Both peptides, however, affect the regular interaction between and subunits altering the autophosphorylation pattern and responsiveness to salt. [155-215], unlike [170-215] tends to aggregate more readily than full length subunit. This behaviour which is reminiscent of the homodimerization of full length subunit, would indicate that tight self-association of [155-215] crucially depends on residues in the 155-170 sequence. Failure of [1-77] fragment to mediate responsiveness to polybasic peptides and accentuated self-association propensity of [155-215] suggest that other structural elements between the sequences 1-77 and 155-215 are required in order to confer optimal functionality to the subunit. 相似文献
8.
9.
Primary and secondary interactions between CK2α and CK2β lead to ring-like structures in the crystals of the CK2 holoenzyme 总被引:1,自引:0,他引:1
Protein kinase CK2 predominantly exists as a heterotetrameric holoenyzme consisting of two catalytic subunits (CK2α) and two non-catalytic subunits (CK2β). Early investigations which we review here had revealed the presence of two types of contacts between CK2α and CK2β: a primary interaction responsible for the stability of the CK2 holoenzyme and stimulatory for the catalytic activity, and a secondary interaction which is inhibitory and in which the acidic loop of CK2β associates with the basic stretch and the (p+1)-loop of CK2α. At the end of the last decade both types of interactions were assumed to occur within the same tetrameric complex. The CK2 holoenyzme structure, however, suggested that the secondary interactions must happen between different CK2 tetramers. Such a behaviour should lead to higher-ordered aggregates consistent with several previous reports about a distinct aggregation propensity of CK2. We demonstrate here that in the CK2 holoenzyme crystals contacts between different CK2 tetramers exists which provide structural details of the secondary CK2α/CK2β interactions. These mainly ionic interactions lead to trimeric rings of CK2 holoenzymes in the crystal. In these rings each CK2 tetramer possesses one CK2α subunit open for substrate binding and another one whose active site is blocked by a secondary contact with CK2β from a neighbouring tetramer. This observation fits to previous findings that salt-sensitive ring-like aggregates of CK2 holoenzymes can exist which possess significant catalytic activity. Furthermore it suggests that earlier ideas about a regulatory role of the enzyme’s aggregation propensity may be worth to be revitalised. 相似文献
10.
Bischoff N Olsen B Raaf J Bretner M Issinger OG Niefind K 《Journal of molecular biology》2011,407(1):1-2348
Protein kinase CK2 (formerly “casein kinase 2”) is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement the limited biochemical knowledge about the second paralog (CK2α′), we developed a well-soluble catalytically active full-length mutant of human CK2α′, characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity of CK2α′ for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α′, while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop in CK2α′ is stabilized by two elements that are absent in CK2α: (1) the extension of the N-terminal β-sheet by an additional β-strand, and (2) the filling of a conserved hydrophobic cavity between the β4/β5 loop and helix αC by a tryptophan residue. Moreover, the interdomain hinge region of CK2α′ adopts a fully functional conformation, while unbound CK2α is often found with a nonproductive hinge conformation that is overcome only by CK2β binding. Taken together, CK2α′ exhibits a significantly lower affinity for CK2β than CK2α; moreover, in functionally critical regions, it is less dependent on CK2β to obtain a fully functional conformation. 相似文献
11.
Dong Y Wang H Cao J Ren J Fan R He X Smith GW Dong C 《Molecular and cellular biochemistry》2011,356(1-2):255-260
In all eukaryotes, the typical CK2 holoenzyme is an heterotetramer composed of two catalytic (CK2α and CK2α') and two regulatory (CK2β) subunits. One of the distinctive traits of plant CK2 is that they present a greater number of genes encoding for CK2α/β subunits than animals or yeasts, for instance, in Arabidopsis and maize both CK2α/β subunits belong to multigenic families composed by up to four genes. Here, we conducted a genome-wide survey examining 34 different plant genomes in order to investigate if the multigenic property of CK2β genes is a common feature through the entire plant kingdom. Also, at the level of structure, the plant CK2β regulatory subunits present distinctive features as (i) they lack about 20 aminoacids in the C-terminal domain, (ii) they present a specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain, and (iii) the acidic loop region is poorly conserved at the aminoacid level. Since there is no data about CK2β or holoenzyme structure in plants, in this study, we use human CK2β as a template to predict a structure for Zea mays CK2β1 by homology modeling and we discuss about possible structural changes in the acidic loop region that could affect the enzyme regulation. 相似文献
12.
Onsory K Sobti RC Al-Badran AI Watanabe M Shiraishi T Krishan A Mohan H Kaur P 《Molecular and cellular biochemistry》2008,314(1-2):25-35
The purpose of this study was to analyse the frequency and type of mutations in the coding region of androgen receptor (AR) and to determine the role of polymorphisms in the intron 1 of ERalpha, exon 5 of ERbeta, intron 7 of progesterone, exon 7 of the aromatase (CYP19) and exon 9 of VDR genes in the risk of prostate cancer. PCR-RFLP analysis of all above the genes was on 100 prostate cancer patients and an equal number of matching controls. The study also included PCR-SSCP analyses of exons 2-8 of AR gene. The genotype containing -/- allele of ERalpha gene was statistically significant for the risk of prostate cancer pose (OR, 2.70; 95% CI, 1.08-6.70, P = 0.032) Rr genotype of ERbeta gene also have a higher risk (OR, 1.65; 95% CI, 0.52-5.23) for prostate cancer. The Cys allele of CYP19 gene was also associated with statistically significant increased risk of prostate cancer (OR; 2.28, 95% CI, 1.20-4.35, P = 0.012). tt genotype of codon 352 of VDR gene showed an OR of 0.43 for (95% CI, 0.13-1.39) and an OR for Tt genotype was 0.65 (95% CI, 0.36-1.16). Taken together, the results showed that in North Indian population, ERalpha and CYP19 genes may be playing a role in the risk of prostate cancer. 相似文献
13.
Multiple myeloma (MM) is a malignant tumor of transformed plasma cells. MM pathogenesis is a multistep process. This cancer can occur de novo (rarely) or it can develop from monoclonal gammopathy of undetermined significance (most of the cases). MM can be asymptomatic (smoldering myeloma) or clinically active. Malignant plasma cells exploit intrinsic and extrinsic bone marrow microenvironment-derived growth signals. Upregulation of stress-coping pathways is also instrumental to maintain MM cell growth. The phylogenetically related Ser/Thr kinases CSNK1A1 (CK1α) and CSNK2 (CK2) have recently gained a growing importance in hematologic malignancies arising both from precursors and from mature blood cells. In multiple myeloma, CK1α or CK2 sustain oncogenic cascades, such as the PI3K/AKT, JAK/STAT, and NF-κB, as well as propel stress-related signaling that help in coping with different noxae. Data also suggest that these kinases modulate the delivery of growth factors and cytokines from the bone marrow stroma. The “non-oncogene addiction” phenotype generated by the increased activity of CK1α and CK2 in multiple myeloma contributes to malignant plasma cell proliferation and survival and represents an Achilles’ heel for the activity of small ATP competitive CK1α or CK2 inhibitors. 相似文献
14.
15.
The Wee1 protein kinase plays a prominent role in keeping cyclin dependent kinase 1 (CDK1) inactive during the G2 phase of the cell cycle. At the onset of mitosis, Wee1 is ubiquitinated by the E3 ubiquitin ligase SCF(beta-TrCP) and subsequently degraded by the proteasome machinery. Previously, it has been reported that although Wee1 lacks the conserved binding motif recognised by beta-TrCP, the CDK-catalysed phosphorylation of Wee1 at Ser123 creates a phosphodegron and primes phosphorylation of two other protein kinases, polo-like kinase 1 (PLK1) and protein kinase CK2, which create two additional phosphodegrons recognised by beta-TrCP. These events contribute to destabilise Wee1 at the onset of mitosis (Watanabe et al. Proc Natl Acad Sci USA 101:4419-4424, 2004). We show here that in addition to the ability of CK2 to phosphorylate Wee1 as reported earlier, the regulatory beta-subunit of protein kinase CK2 can interact with Wee1 in high molecular mass complexes. Indirect immunofluorescence microscopy revealled subcellular co-localisation of CK2beta and Wee1 in the nucleus. Moreover, in vitro phosphorylation assays showed that CK2beta indirectly up-regulates the activity of CDK1 with respect to histone H1 phosphorylation by inhibiting Wee1 kinase. These findings support the view that CK2beta regulates various intracellular processes by modulating the activity of protein kinases that are distinct from CK2 and that protein kinase CK2 plays an important role in events related to the regulation of cell cycle progression as a tetrameric enzyme but also through the individual subunits. 相似文献
16.
Environmental reprogramming of the expression of protein kinase CK2β subunit in fish 总被引:2,自引:0,他引:2
Abul H Abul A Khan I Mathew TC Ayed A Al-Athary E 《Molecular and cellular biochemistry》2001,227(1-2):107-112
Interleukin-8 (IL-8) is considered as the major polymorphonuclear neutrophils (PMNs) chemoattractant cytokine in lung diseases such as asthma and adult respiratory distress syndrome (ARDS). However, controversial results were obtained regarding the involvement of IL-8 in the pathogenesis of pneumonia. This study examines the role of IL-8 in the recruitment and activation of PMNs in the lung of pneumonia patients. The interesting aspect of this study is that it is a site- specific analysis of the infected and uninfected lungs of the same patient. The level of IL-8 mRNA, protein and myeloperoxidase present in the cells of the bronchioalveolar lavages (BALs) taken from the areas of known pneumonic consolidations on chest X-ray (infected lung) are compared with the BALs obtained from areas of no obvious infiltrate (non-infected lung). The results obtained from the infected and non-infected lungs of pneumonic patients were further compared with that of a control group of non-smoking patients. The level of IL-8 mRNA and protein were determined by RT-PCR and ELISA respectively. There was a significant increase in the level of IL-8 mRNA in the infected lung as compared to its level in the non-infected lung (p < 0.001). In correlation with the increase in mRNA, IL-8 protein concentrations in BAL fluids from the infected lung were 6 fold higher than those taken from the non-infected lung (p < 0.0001). This pattern was also consistent with MPO activity in the BALs (4.5 fold more MPO activity in the infected lung as compared to that of the non-infected lung), indicating that IL-8 is directly implicated in neutrophil accumulation that follows acute respiratory infection. The results of the present study, therefore, indicate the involvement of IL-8 in the pathogenesis of pneumonia. 相似文献
17.
18.
Leroy D Filhol O Quintaine N Sarrouilhe D Loue-Mackenbach P Chambaz EM Cochet C 《Molecular and cellular biochemistry》1999,191(1-2):43-50
We have characterized several subdomains of the subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2 subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane.The C-terminal domain of the CK2 subunit plays a role in the oligomerization of the kinase since it was observed that a truncated form of this subunit lacking its 33-last amino acids was incompetent for the assembly of polymeric forms of CK2. Altogether, our results support the notion that the subunit of CK2 is a modular protein made by the association of interdependent domains that are involved in its multiple functions. 相似文献
19.
Protein kinase CK2 participates in the regulation of fundamental cellular processes. Among these processes, cell polarity and cell morphology are controlled by this enzyme probably through the phosphorylation of key proteins. To further study the involvement of CK2 in these processes, we showed that in epithelial cells, the regulatory CK2β subunit was required for LKB1-dependent polarization and cell adhesion. Moreover, CK2β silencing in MCF10A mammary epithelial cells triggered changes in their morphology correlated with the acquisition of mesenchymal phenotype, which were reminiscent to TGFβ-induced epithelial-to-mesenchymal-transition (EMT). TGFβ has emerged as a major inducer of EMT both in vitro and in vivo. We found that among the TGFβ isoforms, TGFβ2 expression was strongly induced in CK2β-knockdown cells. However, the EMT phenotype induced in response to CK2β silencing was not abolished by blocking the TGFβ signaling pathway at TGFβ receptor level, suggesting that alternative pathways might be involved. Given the importance of CK2 in tumorigenesis, a dysregulation of CK2β expression might contribute to EMT induction during cancer progression. 相似文献
20.