Protein kinase CK1alpha regulates mRNA binding by heterogeneous nuclear ribonucleoprotein C in response to physiologic levels of hydrogen peroxide

At low concentrations, hydrogen peroxide (H(2)O(2)) is a positive endogenous regulator of mammalian cell proliferation and survival; however, the signal transduction pathways involved in these processes are poorly understood. In primary human endothelial cells, low concentrations of H(2)O(2) stimulated the rapid phosphorylation of the acidic C-terminal domain (ACD) of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear restricted pre-mRNA-binding protein, at Ser(240) and at Ser(225)-Ser(228). A kinase activity was identified in mouse liver that phosphorylates the ACD of hnRNP-C at Ser(240) and at two sites at Ser(225)-Ser(228). The kinase was purified and identified by tandem mass spectrometry as protein kinase CK1alpha (formerly casein kinase 1alpha). Protein kinase CK1alpha immunoprecipitated from primary human endothelial cell nuclei also phosphorylated the ACD of hnRNP-C at these positions. Pretreatment of endothelial cells with the protein kinase CK1-specific inhibitor IC261 prevented the H(2)O(2)-stimulated phosphorylation of hnRNP-C. Utilizing phosphoserine-mimicking Ser-to-Glu point mutations, the effects of phosphorylation on hnRNP-C function were investigated by quantitative equilibrium fluorescence RNA binding analyses. Wild-type hnRNP-C1 and hnRNP-C1 modified at the basal sites of phosphorylation (S247E and S286E) both avidly bound RNA with similar binding constants. In contrast, hnRNP-C1 that was also modified at the CK1alpha phosphorylation sites exhibited a 14-500-fold decrease in binding affinity, demonstrating that CK1alpha-mediated phosphorylation modulates the mRNA binding ability of hnRNP-C.     

Basal and hydrogen peroxide stimulated sites of phosphorylation in heterogeneous nuclear ribonucleoprotein C1/C2

Hydrogen peroxide (H2O2) is a recently recognized second messenger, which regulates mammalian cell proliferation and migration. The biochemical mechanisms by which mammalian cells sense and respond to low concentrations of H2O2 are poorly understood. Recently, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP-C1/C2) was found to be rapidly phosphorylated in response to the application of low concentrations of H2O2 to human endothelial cells. Here, using tandem mass spectrometry, four sites of phosphorylation are identified in hnRNP-C1/C2, all of which are in the acidic C-terminal domain of the protein. Under resting conditions, the protein is phosphorylated at S247 and S286. In response to low concentrations of H2O2, there is increased phosphorylation at S240 and at one of the four contiguous serine residues from S225-S228. Studies using a recombinant acidic C-terminal domain of hnRNP-C overexpressed in Escherichia coli demonstrate that protein kinase CK2 phosphorylates hnRNP-C1/C2 at S247, while protein kinase A and several protein kinase C isoforms fail to phosphorylate the isolated domain. These findings demonstrate that the acidic C-terminal domain of hnRNP-C1/C2 serves as the site for both basal and stimulated phosphorylation, indicating that this domain may play an important role in the regulation of mRNA binding by hnRNP-C1/C2.     

Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells

Oxidative inactivation of protein tyrosine phosphatases and calcineurin is a well established mechanism; however, little information with regard to the effect of oxidants on PP1 and PP2A activity is available. Herein, we show that PP1 activity is inhibited by H(2)O(2) treatment in differentiated PC12 cells both in vitro and in vivo experiments. Thiol-antioxidant N-acetyl-cysteine (NAC) and reduced glutathione (GSH), when added in vitro to lysates from H(2)O(2)-treated cells, reversed PP1 inhibition. H(2)O(2) treatment increased eIF2 alpha phosphorylated levels (eIF2 alpha P) in a time- and dose-dependent fashion and promoted protein synthesis inhibition. Interestingly, NAC pretreatment protected cells from H(2)O(2)-induced PP1 inactivation and, consequently, it abolished increased H(2)O(2)-induced eIF2 alpha phosphorylation and protein synthesis inhibition. In addition, PP1 inhibitor tautomycin prevented both NAC-induced PP1 reactivation and eIF2 alpha P dephosphorylation in H(2)O(2)-treated cells. Taken together, our findings support a role for PP1 in eIF2 alpha phosphorylation and oxidative stress-triggered translation down regulation.     

Biochemical and cellular characteristics of the four splice variants of protein kinase CK1alpha from zebrafish (Danio rerio)

Protein kinase CK1 (previously known as casein kinase I) conforms to a subgroup of the great protein kinase family found in eukaryotic organisms. The CK1 subgroup of vertebrates contains seven members known as alpha, beta, gamma1, gamma2, gamma3, delta, and epsilon. The CK1alpha gene can generate four variants (CK1alpha, CK1alphaS, CK1alphaL, and CK1alphaLS) through alternate splicing, characterized by the presence or absence of two additional coding sequences. Exon "L" encodes a 28-amino acid stretch that is inserted after lysine 152, in the center of the catalytic domain. The "S" insert encodes 12 amino acid residues and is located close to the carboxyl terminus of the protein. This work reports some biochemical and cellular properties of the four CK1alpha variants found to be expressed in zebrafish (Danio rerio). The results obtained indicate that the presence of the "L" insert affects several biochemical properties of CK1alpha: (a) it increases the apparent Km for ATP twofold, from approximately 30 to approximately 60 microM; (b) it decreases the sensitivity to the CKI-7 inhibitor, raising the I50 values from 113 to approximately 230 microM; (c) it greatly decreases the heat stability of the enzyme at 40 degrees C. In addition, the insertion of the "L" fragment exerts very important effects on some cellular properties of the enzyme. CK1alphaL concentrates in the cell nucleus, excluding nucleoli, while the CK1alpha variant is predominantly cytoplasmic, although some presence is observed in the nucleus. This finding supports the thesis that the basic-rich region found in the "L" insert acts as a nuclear localization signal. The "L" insert-containing variant was also found to be more rapidly degraded (half-life of 100 min) than the CK1alpha variant (half-life of 400 min) in transfected Cos-7 cells.     

Intracellular hydrogen peroxide production is an upstream event in apoptosis induced by down-regulation of casein kinase 2 in prostate cancer cells

We have shown previously that down-regulation of CK2 activity (protein kinase CK2, formerly casein kinase 2) by employing its inhibitors apigenin or 4,5,6,7-tetrabromobenzotriazole promotes apoptosis in prostatic carcinoma cells. In an effort to define the downstream mediators of this action, we show that cell apoptosis observed on down-regulation of CK2 is preceded by intracellular generation of hydrogen hydroxide (H2O2) in various normal and cancer cells. In this regard, both androgen-dependent ALVA-41 and androgen-independent PC-3 cells treated with 80 micromol/L apigenin or 4,5,6,7-tetrabromobenzotriazole or with antisense CK2alpha oligonucleotide or small interfering RNA respond similarly to down-regulation of CK2. Interestingly, whereas chemical inhibitors of CK2 elicited H2O2 production in both cancer and noncancer cells, the antisense CK2alpha-mediated down-regulation of CK2 showed significant H2O2 production in cancer cells but had minimal effect in noncancer cells. The basis of this key difference is unclear at present, but this observation may have implications for the therapeutic potential of antisense CK2 oligonucleotide in cancer therapy. The H2O2 production induced by antisense CK2alpha was associated with robust caspase-3 activity, nuclear factor-kappaB nuclear translocation, cytochrome c release, and subsequent DNA fragmentation in prostate cancer cells (ALVA-41 and PC-3). These findings describe, for the first time, a relationship between CK2 and reactive oxygen species, such that CK2 inhibition leads to production of intracellular H2O2, which may serve as a downstream mediator of apoptosis in cancer cells.     

Concomitant Transitory Up-Regulation of X-Linked Inhibitor of Apoptosis Protein (XIAP) and the Heterogeneous Nuclear Ribonucleoprotein C1–C2 in Surviving Cells During Neuronal Apoptosis

Although cap-dependent translation initiation is the prevalent mode of ribosome binding to mRNAs in eukaryotes, some mRNAs exhibit the ability to bypass the requirement for the cap structure. The translation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA is controlled by an internal ribosome entry site (IRES) element, which requires the interaction of the heterogeneous nuclear ribonucleoprotein C1–C2 (hnRNP-C1/C2). We analyze, at the protein level, the time course and distribution of XIAP and hnRNP-C1/C2 upon ischemia in mice or staurosporine (STP)-induced apoptosis in HT22 cells. Both ischemia and STP induced a parallel upregulation of XIAP and hnRNP-C1/C2 protein levels in the penumbra and in HT22 cells. These results suggest that the increased levels of hnRNP C1/C2 may modulate XIAP translation, probably by interacting with the XIAP-IRES. The up-regulation of hnRNP-C1/C2 may foster the synthesis of XIAP as a protective pathway by which neurons try to counteract the initial deleterious effects of apoptosis.     

Purification and characterization of the CK2alpha'-based holoenzyme, an isozyme of CK2alpha: a comparative analysis

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.     

The protein of a new gene,Tctex4, interacts with protein kinase CK2beta subunit and is highly expressed in mouse testis

Casein kinase 2 (CK2) is a ubiquitous, multifunctional eukaryotic serine/threonine kinase that phosphorylates an array of proteins. CK2 is a heterotetramer composed of two catalytic (alpha,alpha(')) and two regulatory (beta) subunits. CK2 plays an essential role in regulatory pathways in cell transformation and proliferation. But the role and function of the individual subunits of CK2, which are not in the holoenzyme, are not yet clear. Northern blot analysis reveals the highest CK2beta activity in mouse testicles and brain. By employing a yeast two-hybrid screen to identify the proteins that interact with CK2beta, we have isolated a cDNA clone encoding a 14-kDa protein with homology to dynein light chains and have designated it as Tctex4. CK2beta interacts specifically with Tctex4 both in a yeast two-hybrid system and in an in vitro interaction assay. Northern blot and in situ hybridization showed that Tctex4 is a novel gene that is expressed in mouse testis.     

Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid

The non-beta-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10T1/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase C alpha (PKC alpha), and PKC beta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

Role of the carboxyl terminus on the catalytic activity of protein kinase CK2alpha subunit

Protein kinase CK2 (also known as casein kinase 2) has catalytic (alpha, alpha') and regulatory (beta) subunits. The role of carboxyl amino acids in positions from 324 to 328 was studied for Xenopus laevis CK2alpha. Deletions and mutations of these residues were produced in recombinant CK2alpha, which was assayed for kinase activity. Activity dropped 7000-fold upon deletion of amino acids 324-328. The key residues are isoleucine 327 and phenylalanine 324. A three dimensional model of CK2alpha indicates that these hydrophobic residues of helix alphaN may interact with hydrophobic residues in helix alphaE which is linked to the catalytic center.     

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.     

Casein kinase 2 regulates the mRNA-destabilizing activity of tristetraprolin

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.