DNA binding activity of casein kinase II

Casein kinase II, an ubiquitous, oligomeric, messenger-independent protein kinase has previously been shown to concentrate in the nuclear compartment when cells are stimulated to proliferate. The present communication reports that purified mammalian CKII interacts with genomic DNA preparations in vitro. This interaction led to an apparent activation of the kinase, most likely explained by prevention of its aggregation and subsequent denaturation. Binding of CKII was optimum with double stranded DNA preparations; duplex lambda phage DNA exhibited at least two types of binding sites and the high affinity system (Kd approximately equal to 6 x 10(-13) M) represented a binding capacity of about 1 mol CKII per mol DNA. CKII-DNA interaction was stimulated in the presence of a polyamine and inhibited by heparin. Blotting experiments disclosed that DNA binds CKII through its alpha subunit. These observations are in line with the hypothesis that casein kinase II may be examined as a component in the transduction of the mitogenic signal from the cell membrane to the nucleus, in response to growth factors.     

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Effects of cationic polypeptides on the activity, substrate interaction, and autophosphorylation of casein kinase II: a study with calmodulin.

The effects of basic polypeptides on the ability of casein kinase II to phosphorylate an exogenous substrate (calmodulin) are correlated with steady-state autophosphorylation of the alpha- and beta-subunits of casein kinase II. Polylysine and polyarginine increase autophosphorylation of the alpha-subunit with a concomitant decrease in beta-subunit phosphorylation, while enhancing casein kinase II-stimulated phosphorylation of calmodulin over 100-fold. The highly basic carboxyl terminal segment of the endogenous p21c-Ki-ras has similar effects on the phosphorylation of calmodulin and the alpha- and beta-subunits of casein kinase II. Altering the concentration of cationic polypeptides produces a biphasic effect on the phosphorylation of both calmodulin and the alpha-subunit, which correlate positively with each other but do not correlate with beta-subunit phosphorylation. When the KCl concentration is changed, casein kinase II activity correlates positively only with alpha-subunit phosphorylation. In contrast, the biphasic response of calmodulin phosphorylation by casein kinase II at different Ca2+ concentrations correlates positively with both alpha- and beta-subunit phosphorylation. Therefore, in the presence of basic protein activators, the rate of phosphorylation of a substrate, calmodulin, correlates with steady-state phosphorylation of the alpha-subunit, but not with the beta-subunit under all conditions tested. Endogenous cationic factors may modulate the in vivo activity of casein kinase II and alter the interaction of the enzyme with specific intracellular substrates.     

Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.

Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.     

M-phase-specific cdc2 protein kinase phosphorylates the beta subunit of casein kinase II and increases casein kinase II activity

The M-phase-specific cdc2 (cell division control) protein kinase (a component of the M-phase-promoting factor) was found to activate casein kinase II in vitro. The increase in casein kinase II activity ranged over 1.5-5-fold. Increase in activity was prevented if ATP was replaced during the activation reaction by a non-hydrolysable analogue. Alkaline phosphatase treatment of the activated enzyme decreased the activity to the basal level. The beta subunit of casein kinase II was phosphorylated by cdc2 protein kinase at site(s) different from the autophosphorylation sites of the enzyme. Phosphoamino acid analysis showed that the beta subunit was phosphorylated by cdc2 protein kinase at threonine residues while autophosphorylation involved serine residues. Casein kinase II may be part of the cascade which leads to increased phosphorylation of many proteins at M-phase and therefore be involved in the pleiotropic effects of M-phase-promoting factor.     

Autoactivation of calmodulin-dependent protein kinase II by autophosphorylation

Autophosphorylation of calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) under limiting conditions (2 microM ATP) decreased progressively with increasing concentrations of a substrate, Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys (syntide-2), suggesting a competition between the substrate and the autophosphorylation site(s) of the enzyme. The rate and extent of the generation of Ca2+/CaM-independent activity of the enzyme by autophosphorylation were also decreased by the presence of syntide-2. The syntide-2 phosphorylation in the presence of Ca2+/CaM under the limiting conditions reached a steady state, after a lag, when the Ca2+/CaM-independent activity reached a plateau. A linear relationship was observed between the activities in the presence and absence of Ca2+/CaM of the enzyme which had undergone various degrees of autophosphorylation, and the extrapolation of activity in the absence of Ca2+/CaM to zero gave 15-20% of the maximum activity. The steady-state rate of syntide-2 phosphorylation in the presence of Ca2+/CaM by the enzyme that had not undergone prior autophosphorylation was decreased by high concentrations of syntide-2 which suppressed autophosphorylation as well as the generation of Ca2+/CaM-independent activity. These results suggest that although the nonautophosphorylated enzyme possesses a basal low level of Ca2+/CaM-dependent activity, autophosphorylation is required for full activation.     

Phosphorylation of RIG-I by casein kinase II inhibits its antiviral response

RIG-I is an intracellular RNA virus sensor that mediates a signaling pathway that triggers the alpha/beta interferon (IFN-α/β) immune defenses. However, the mechanism for regulation of RIG-I activity remains largely unknown. Here we show that RIG-I activity is regulated by phosphorylation and dephosphorylation in its repressor domain (RD). Threonine at amino acid (aa) 770 and serine at aa 854 to 855 of RIG-I are phosphorylated by casein kinase II (CK2) in the resting state of the cell and dephosphorylated when cells are infected by RNA virus. Mutation at aa position 770 or 854 to 855 of RIG-I renders it constitutively active. Pharmacological inhibition of CK2 enhances virus-induced expression of IFN-β and suppresses virus proliferation, while inhibition of phosphatase reduces virus-induced expression of IFN-β. Overexpression of CK2 suppresses RIG-I-mediated signaling, while silencing of CK2 results in the increased suppression of virus proliferation. Our results reveal a novel mechanism of the regulation of RIG-I activity during RNA virus infection.     

Redox regulation of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3) is a protein tyrosine phosphatase (PTP) for which in vivo evidence suggests that regulation can occur by oxidation and/or reduction of the active site cysteine. Using kinetics and mass spectrometry, we have probed the biochemical details of oxidation of the active site cysteine in MKP3, with particular focus on the mechanism of protection from irreversible inactivation to the sulfinic or sulfonic acid species. Like other PTPs, MKP3 was found to be rapidly and reversibly inactivated by mild treatment with hydrogen peroxide. We demonstrate that unlike the case for some PTPs, the sulfenic acid of the active site cysteine in MKP3 is not stabilized in the active site but instead is rapidly trapped in a re-reducible form. Unlike the case for other PTPs, the sulfenic acid in MKP3 does not form a sulfenyl-amide species with its neighboring residue or a disulfide with a single proximate cysteine. Instead, multiple cysteines distributed in both the N-terminal substrate-binding domain (Cys147 in particular) and the C-terminal catalytic domain (Cys218) are capable of rapidly and efficiently trapping the sulfenic acid as a disulfide. Our results extend the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of their active sites and expand the role of the N-terminal substrate recognition domain in MKP3 to include redox regulation.     

Redox regulation of the AMP-activated protein kinase

Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.

Objectives

The aim of this study is to determine if AMP-activated protein kinase (AMPK), a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC).

Methods

Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.

Results

In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC) at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N^G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor) blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol) pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.

Conclusion

Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.     

Myc oncoproteins are phosphorylated by casein kinase II.

Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, E1a and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-II, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus.     

Nuclear extracellular signal-regulated kinase 1 and 2 translocation is mediated by casein kinase 2 and accelerated by autophosphorylation

The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase [MAPK] family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. These results provide an important role of CK2 in regulating nuclear ERK activities.     

Phosphorylation of type II (beta) protein kinase C by casein kinase II.

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.     

Association of casein kinase II with microtubules

A magnesium-dependent heparin-inhibited protein kinase activity associated with brain microtubule preparations has been identified as casein kinase II using a monospecific polyclonal antibody. This enzyme appears enriched in cold-stable microtubule fractions. By immunofluorescence microscopy using an antiserum against casein kinase II, the in situ immunolabeling of some microtubule assays has been observed. Thus, mitotic spindles are stained by the anti-casein kinase II antibody in fibroblast cells. In neuroblastoma cells induced to differentiate, the labeling of microtubule arrays inside developing axon-like processes is also seen. These results support the view that casein kinase II can modulate cytoskeletal assembly and dynamics through phosphorylation of microtubule proteins.     

Inhibition of casein kinase II by heparin

Casein kinase II, a cyclic nucleotide-independent protein kinase from rabbit reticulocytes, was shown to be inhibited by heparin. Heparin specifically inhibited the enzyme and had no effect on other protein kinases, including casein kinase I, the type I and II cAMP-dependent protein kinases, protease-activated kinase I, and the hemin-controlled repressor. Heparan sulfate was found to be 40-fold less effective than heparin towards casein kinase II; other acid mucopolysaccharides had little or no effect on the enzymatic activity. Steady state studies revealed that heparin acted as a competitive inhibitor with respect to the substrate, casein. A value of 20 ng/ml or about 1.4 nM was obtained for the apparent Ki. The inhibition was not reversed by ATP and varying the ATP and heparin concentrations in the assay only altered the maximum velocity.     

Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitrite

Accumulating evidence suggests that enhanced peroxynitrite formation occurs during diabetes. This report describes the effect of peroxynitrite on insulin receptor (IR) function. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, peroxynitrite dramatically reduced ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity of IR preparations. Moreover, SIN-1 administration decreased blood glucose levels in normal mice via upregulation of IR/IRS-1 tyrosine phosphorylation. In contrast, SIN-1 markedly increased blood glucose levels in diabetic mice concomitant with downregulation of IR/IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity, and SIN-1 has hyperglycemic effect in diabetic mice.     

The phosphotransferase activity of casein kinase II is required for its physiological function in vivo.

We have previously shown that the inviability associated with disruption of both catalytic subunits of casein kinase II in Saccharomyces cerevisiae can be rescued by plasmids expressing the catalytic subunit of the Drosophila enzyme (Padmanabha et al., 1990, Mol. Cell. Biol. 10, 4089). Here we describe the construction of mutant forms of the Drosophila catalytic subunit in which residues known to be crucial for catalytic activity in other protein kinases have been altered by site-directed mutagenesis. Mutation of either Lys66 or Asp173, which correspond to Lys72 and Asp184 of cAMP-dependent protein kinase, respectively, yields a casein kinase II catalytic subunit which fails to rescue a yeast strain lacking both endogenous catalytic subunit genes. The data indicate that the phosphotransferase activity of casein kinase II is required for its physiological function in vivo.