蛋白激酶CK2的研究进展

蛋白激酶CK2是一种真核细胞中普遍存在的信使非依赖性丝／苏氨酸蛋白激酶。近年来,对蛋白激酶CK2的研究也取得了一些重要进展,尤其是蛋白激酶CK2的结构及其作用底物,蛋白激酶CK2与肿瘤及细胞凋亡的关系,越来越引起人们的关注。     

蛋白激酶CK2的结构及其生理功能研究进展

蛋白激酶CK2是一种常见的、进化保守的、普遍存在的蛋白激酶。近年来,越来越多的研究表明CK2具有多种磷酸化蛋白底物,这些底物在生长发育及各类疾病中都具有重要的作用,因此CK2可以通过调控这些底物的磷酸化参与这些生理过程。文中简要综述了蛋白激酶CK2的结构特征及其在生长发育、免疫、肿瘤等疾病中的生理功能,以期为进一步研究CK2的调控机制和应用提供理论依据。     

CK2 β的蛋白表达与纯化及其RNA沉默的研究

蛋白激酶CK2是一个丝氨酸/苏氨酸特异性的激酶,通常以异源四聚体形式存在,由两个催化亚基(CK2α和CK2α′)和两个调节亚基CK2β组成。CK2激酶α亚基含有和其它蛋白激酶的催化功能域同源的激酶功能域。CK2激酶β亚基在调节a亚基的基本活性方面起着很复杂的作用。通过CK2β的结合活性,CK2α改变它的活性和作用底物。本研究成功的表达和纯化了CK2β蛋白,并且构建了CK2β的S iRNA载体,为其进一步功能研究做好准备。     

山奈酚抑制蛋白激酶CK2活性

研究体外以及HL-60细胞内山奈酚对蛋白激酶CK2的抑制作用及机制. 通过测定药物作用后转移到CK2底物上的[γ-32P]ATP的32P的放射性活度, 探讨山奈酚对重组人CK2全酶以及细胞内CK2活性的影响; 采用多重RT-PCR检测CK2α、α' 和 β亚基的mRNA表达水平; 通过 Lineweaver-Burk作图法,分析CK2的酶动力学机制.山奈酚能显著抑制重组人CK2活性（IC50 = 1.88 μmol/L）和HL-60细胞内的CK2活性, 对细胞内CK2的作用效果强于阳性对照四溴-2-氮杂苯并咪唑(TBB). 山奈酚作用2h,对CK2各亚基的mRNA表达水平均没有影响. 山奈酚对重组人CK2的酶动力学分析表明, 山奈酚与ATP(Ki = 1.14 μmol/L)及酪蛋白(Ki = 1.03 μmol/L)均呈非竞争性抑制作用. 结果提示, 山奈酚是一种有效的蛋白激酶CK2的抑制剂, 其作用机制可能与其阻碍CK2与ATP以及底物的结合有关.     

蛋白激酶CK2-β在腺样囊性癌中的活性变化

探讨蛋白激酶CK2-β在涎腺腺样囊性癌及涎腺腺样囊性癌肺转移细胞中的活性及表达变化。2种细胞进行细胞培养,分别提取胞浆及胞核蛋白,然后利用激酶活性测定及Westernbloting方法进行活性及蛋白表达检测分析。与SACC-83细胞相比,SACC-LM细胞中蛋白激酶CK2-β具有较高的活性及表达;在2种细胞中,细胞核中的活性及表达都高于细胞质。蛋白激酶CK2-β在涎腺腺样囊性癌中活性与蛋白表达一致,并与涎腺腺样囊性癌的肺转移呈正相关。     

黄色黄素抑制人白血病HL-60细胞内蛋白激酶CK2的实验研究

蛋白激酶CK2在寻找抗肿瘤及抗病毒药物方面是一个具有吸引力的分子靶点,其特异性抑制剂具有潜在的临床应用价值.通过测定药物作用后转移到CK2底物上[γ-32P]ATP的放射性活度,探讨黄色黄素对重组人CK2全酶以及细胞内CK2活性的影响;采用多重RT-PCR检测CK2α、α'和β亚基的mRNA表达水平;Lineweaver-Burk作图法分析CK2的酶动力学机制.发现黄色黄素能显著抑制重组人CK2全酶(IC50=0.86μmol/L)以及HL-60细胞内的CK2活性,其作用效果均强于阳性对照TBB.另外,黄色黄素作用2h可使CK2α和α'亚基的mRNA表达下降,对β亚基则无明显的影响.酶动力学分析表明,黄色黄素与ATP呈竞争性抑制CK2的活性,与酪蛋白则呈混合性抑制CK2的活性.研究说明,黄色黄素是一种有效的细胞内蛋白激酶CK2抑制剂.     

植物蛋白激酶CK2的调控与功能研究进展

蛋白激酶CK2(Casein Kinase 2,CK2)是最早发现的蛋白激酶之一,属于丝氨酸/苏氨酸蛋白激酶家族.CK2在真核生物中广泛存在,结构高度保守,参与调节生物多种生命代谢活动,参与植物生长发育中光信号转导、抗逆境生存、昼夜节律调节等过程,且与肿瘤的发生、炎症反应及神经功能障碍性疾病等密切相关.该文对近年来有关CK2的结构特征、理化特性、活性调控、在植物中的功能等的研究进展进行综述,为进一步研究其在代谢及信号转导中的调控作用,明确其在植物生长发育调控网络中的地位及在作物品质提高中的应用提供参考.     

蛋白激酶CK2与DNA断裂修复

蛋白激酶CK2（酪蛋白激酶Ⅱ）是真核细胞中普遍存在的一种信使非依赖的丝氨酸／苏氨酸蛋白激酶,它底物众多,功能广泛。DNA断裂修复是一个涉及很多种酶和蛋白的过程,CK2在其中起着很重要的作用。     

蛋白激酶CK2α在人喉癌细胞Hep-2中的表达及意义

目的研究蛋白激酶CK2α(Protein kinase CK2α,PK-CK2α)在人喉癌细胞Hep-2中的表达,探讨其与喉癌之间的关系。方法体外培养Hep-2细胞和Hacat细胞,应用半定量逆转录-聚合酶链反应(RT-PCR)技术分别检测蛋白激酶CK2αmRNA在Hep-2细胞和Hacat细胞中的表达;应用免疫细胞化学技术分别检测蛋白激酶CK2α蛋白在Hep-2细胞和Hacat细胞中的表达。结果蛋白激酶CK2αmRNA在Hep-2细胞中高表达,在Hacat细胞中低表达,差异有统计学意义(P<0.01)。蛋白激酶CK2α蛋白在Hep-2细胞中高表达,在Hacat细胞中低表达,差异有统计学意义(P<0.01)。结论蛋白激酶CK2α过表达可能与喉癌的发生和发展有关。     

重组人蛋白激酶CK 2α亚基的原核表达、纯化与鉴定

将构建成功的人蛋白激酶 CK2 α亚基 c DNA的重组质粒 ,转化大肠杆菌 BL2 1 ( DE3) ,IPTG诱导后获特异高效表达 ,表达蛋白占菌体总蛋白的 30 % ,但大多数重组蛋白以不溶形式存在 .表达产物依次进行 DE- 5 2、P1 1磷酸纤维素和肝素 - Sepharose柱层析分离 ,最后从 30 9mg可溶性蛋白质中得到 6.1 mg纯化蛋白 . SDS- PAGE显示纯化的蛋白质为一分子量 4 2 k D的单一蛋白带 .Western- blot的结果证明 :纯化的表达产物与抗人 CK2α抗体可发生特异性免疫反应 . CK2α和β亚基等摩尔分子混合可组成有完全活性的全酶 .重组的 CK2全酶的性质和功能与该酶的已知特性一致 .这些结果证明重组蛋白是人蛋白激酶 CK2 α亚基     

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2

Protein kinase CK2 is a highly pleiotropic enzyme whose high constitutive activity is suspected to be instrumental to the enhancement of the tumour phenotype and to the propagation of infectious diseases. Here we describe a novel compound, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), which is superior to the commonly used specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in several respects. DMAT displays the lowest K(i) value ever reported for a CK2 inhibitor (40 nM); it is cell permeable and its efficacy on cultured cells, both in terms of endogenous CK2 inhibition and induction of apoptosis, is several fold higher than that of TBB. The selectivity of DMAT assayed on a panel of >30 protein kinases is comparable to that of TBB, with the additional advantage of being ineffective on protein kinase CK1 up to 200 microM. These properties make DMAT the first choice CK2 inhibitor for in vivo studies available to date.     

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.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

Casein kinase 2 is the major enzyme in brain that phosphorylates Ser129 of human alpha-synuclein: Implication for alpha-synucleinopathies

In Lewy body diseases and multiple system atrophy, α-synuclein is hyperphosphorylated at Ser129, suggesting a role in pathogenesis. Here, we report purification of the protein kinase in rat brain that phosphorylates Ser129 and its identification as casein kinase-2 (CK2). We show that most of the activity can be inhibited by heparin, an inhibitor of CK2. Phosphorylated Ser129 was detected in primary cultured neurons and inhibited by CK2 inhibitors. In some cases of Lewy body disease, CK2-like immunoreactivity was recovered in the sarkosyl-insoluble fraction, which was enriched in phosphorylated α-synuclein. Taken together, these findings suggest that CK2 may be involved in the hyperphosphorylation of α-synuclein in α-synucleinopathies.     

Validation of protein kinase CK2 as oncological target

Protein kinase CK2 is a highly conserved enzyme composed of two catalytic subunits α and/or α′ and two regulatory subunits β whose activity is elevated in diverse tumour types as well as in highly proliferating tissues. Several results suggest that the overexpression of either CK2 catalytic subunits or the CK2 holoenzyme contributes to cellular transformation. In a similar vein, experiments performed compromising the intracellular expression of CK2 has led to somehow contradictory results with respect to the ability of this enzyme to control survival and apoptosis. To better elucidate the role of CK2 in programmed cell death, we have depleted cells of CK2 catalytic subunits by the application of antisense oligodeoxynucleotides and siRNAs techniques, respectively. Our results indicate that protein kinase CK2 is characterized by an extremely high stability that might be due to its association with other intracellular proteins, enhanced half-life or lower vulnerability towards proteolytic degradation. In addition, we show that despite the effectiveness of the methods applied in lowering CK2 kinase activity in all cells investigated, CK2 might not by itself be sufficient to trigger enhanced drug-induced apoptosis in cells.     

Modulation of death receptor-mediated apoptosis by CK2

Protein kinase CK2 has long been known to be involved in cell growth and proliferation. Recent work has also implicated its role in the suppression of apoptosis. We originally documented that removal of survival or growth stimuli resulted in rapid loss of CK2 from the nuclear matrix and chromatin which preceded induction of apoptosis. Further, we demonstrated that overexpression of CK2 in cells promotes suppression of drug-mediated apoptosis. In the present work, we have extended these observations to demonstrate that CK2 can influence apoptosis mediated via the death receptors. Overexpression of CK2 resulted in suppression of apoptosis mediated by TNF-α, TRAIL, and Fas-L in cells responsive to these ligands, whereas downregulation of CK2 resulted in augmentation of apoptosis mediated by these ligands. To our knowledge, this is the first report to show that receptor-mediated apoptosis can be modulated by changes in CK2 in prostate cancer cells. Based on our previous observations together with the evidence presented here, we propose that CK2 has an impact on the process of apoptosis mediated by diverse type of mechanisms thus playing a global role in regulation of apoptotic activity in cells.     

Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits

The acronym CK2 denotes a highly pleiotropic Ser/Thr protein kinase whose over-expression correlates with neoplastic growth. A vexed question about the enigmatic regulation of CK2 concerns the actual existence in living cells of the catalytic (alpha and/or alpha') and regulatory beta-subunits of CK2 not assembled into the regular heterotetrameric holoenzyme. Here we take advantage of novel reagents, namely a peptide substrate and an inhibitor which discriminate between the holoenzyme and the catalytic subunits, to show that CK2 activity in CHO cells is entirely accounted for by the holoenzyme. Transfection with individual subunits moreover does not give rise to holoenzyme formation unless the catalytic and regulatory subunits are co-transfected together, arguing against the existence of free subunits in CHO cells.     

Expression and regulation of protein kinase CK2 during the cell cycle

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2, CK2 and CK2 on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2 and CK2 following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2 to CK2 during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2 relative to CK2 in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2 increase in mitotic cells, that CK2 and CK2 are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.