Influence of Casein kinase II inhibitor CX-4945 on BCL6-mediated apoptotic signaling in B-ALL in vitro and in vivo

A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.

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Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.899 ◽

1996 ◽

Vol 16 (3) ◽

pp. 899-906 ◽

Cited By ~ 130

Author(s):

J A McElhinny ◽

S A Trushin ◽

G D Bren ◽

N Chester ◽

C V Paya

Keyword(s):

Casein Kinase Ii ◽

Casein Kinase ◽

Kinase Assay ◽

Resting Cells ◽

Nf Kappa B ◽

I Kappa B Alpha ◽

Cell Extracts ◽

Protein Kinase Ckii

The phosphoprotein I kappa B alpha exists in the cytoplasm of resting cells bound to the ubiquitous transcription factor NF-kappa B (p50-p65). In response to specific cellular stimulation, I kappa B alpha is further phosphorylated and subsequently degraded, allowing NF-kappa B to translocate to the nucleus and transactivate target genes. To identify the kinase(s) involved in I kappa B alpha phosphorylation, we first performed an I kappa B alpha in-gel kinase assay. Two kinase activities of 35 and 42 kDa were identified in cellular extracts from Jurkat T and U937 promonocytic cell lines. Specific inhibitors and immunodepletion studies identified the I kappa B alpha kinase activities as those of the alpha and alpha' subunits of casein kinase II (CKII). Immunoprecipitation studies demonstrated that CKII and I kappa B alpha physically associate in vivo. Moreover, phosphopeptide maps of I kappa B alpha phosphorylated in vitro by cellular extracts and in vivo in resting Jurkat T cells contained the same pattern of phosphopeptides as observed in maps of I kappa B alpha phosphorylated in vitro by purified CKII. Sequence analysis revealed that purified CKII and the kinase activity within cell extracts phosphorylated I kappa B alpha at its C terminus at S-283, S-288, S-293, and T-291. The functional role of CKII was tested in an in vitro I kappa B alpha degradation assay with extracts from uninfected and human immunodeficiency virus (HIV)-infected U937 cells. Immunodepletion of CKII from these extracts abrogated both the basal and enhanced HIV-induced degradation of I kappa B alpha. These studies provide new evidence that the protein kinase CKII physically associates with I kappa B alpha in vivo, induces multisite (serine/threonine) phosphorylation, and is required for the basal and HIV-induced degradation of I kappa B alpha in vitro.

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Tubulin phosphorylation by casein kinase II is similar to that found in vivo.

The Journal of Cell Biology ◽

10.1083/jcb.105.4.1731 ◽

1987 ◽

Vol 105 (4) ◽

pp. 1731-1739 ◽

Cited By ~ 80

Author(s):

L Serrano ◽

J Díaz-Nido ◽

F Wandosell ◽

J Avila

Keyword(s):

Neuroblastoma Cells ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Carboxy Terminal Domain ◽

Phosphatase Treatment ◽

Microtubule Protein ◽

Carboxy Terminal ◽

Brain Tubulin

Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.

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Actin Dynamics Is Controlled by a Casein Kinase II and Phosphatase 2C Interplay on Toxoplasma gondii Toxofilin

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0462 ◽

2003 ◽

Vol 14 (5) ◽

pp. 1900-1912 ◽

Cited By ~ 44

Author(s):

Violaine Delorme ◽

Xavier Cayla ◽

Grazyna Faure ◽

Alphonse Garcia ◽

Isabelle Tardieux

Keyword(s):

Toxoplasma Gondii ◽

Actin Polymerization ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Gliding Motility ◽

Actin Dynamics ◽

Central Feature ◽

Parasite Motility

Actin polymerization in Apicomplexa protozoa is central to parasite motility and host cell invasion. Toxofilin has been characterized as a protein that sequesters actin monomers and caps actin filaments in Toxoplasma gondii. Herein, we show that Toxofilin properties in vivo as in vitro depend on its phosphorylation. We identify a novel parasitic type 2C phosphatase that binds the Toxofilin/G-actin complex and a casein kinase II-like activity in the cytosol, both of which modulate the phosphorylation status of Toxofilin serine53. The interplay of these two molecules controls Toxofilin binding of G-actin as well as actin dynamics in vivo. Such functional interactions should play a major role in actin sequestration, a central feature of actin dynamics in Apicomplexa that underlies the spectacular speed and nature of parasite gliding motility.

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Functions of the C-terminal domain of CTP: phosphocholine cytidylyltransferase. Effects of C-terminal deletions on enzyme activity, intracellular localization and phosphorylation potential

Biochemical Journal ◽

10.1042/bj3100699 ◽

1995 ◽

Vol 310 (2) ◽

pp. 699-708 ◽

Cited By ~ 49

Author(s):

R B Cornell ◽

G B Kalmar ◽

R J Kay ◽

M A Johnson ◽

J S Sanghera ◽

...

Keyword(s):

Amino Acids ◽

Lipid Vesicles ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Regulatory Domain ◽

Cdc2 Kinase ◽

Phosphocholine Cytidylyltransferase ◽

Membrane Bound

The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored by the creation of a series of deletion mutations in rat liver cDNA, which were expressed in COS cells as a major protein component. Deletion of up to 55 amino acids from the C-terminus had no effect on the activity of the enzyme, its stimulation by lipid vesicles or on its intracellular distribution between soluble and membrane-bound forms. However, deletion of the C-terminal 139 amino acids resulted in a 90% decrease in activity, loss of response to lipid vesicles and a significant decrease in the fraction of membrane-bound enzyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of 32P-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was 32P-labelled in vivo. Phosphorylation was restricted to the C-terminal 52 amino acids (domain P) and occurred on multiple sites. CT phosphorylation in vitro was catalysed by casein kinase II, cell division control 2 kinase (cdc2 kinase), protein kinases C alpha and beta II, and glycogen synthase kinase-3 (GSK-3), but not by mitogen-activated kinase (MAP kinase). Casein kinase II phosphorylation was directed exclusively to Ser-362. The sites phosphorylated by cdc2 kinase and GSK-3 were restricted to several serines within three proline-rich motifs of domain P. Sites phosphorylated in vitro by protein kinase C, on the other hand, were distributed over the N-terminal catalytic as well as the C-terminal regulatory domain. The stoichiometry of phosphorylation catalysed by any of these kinases was less than 0.2 mol P/mol CT, and no effects on enzyme activity were detected. This study supports a tripartite structure for CT with an N-terminal catalytic domain and a C-terminal regulatory domain comprised of a membrane-binding domain (domain M) and a phosphorylation domain (domain P). It also identifies three kinases as potential regulators in vivo of CT, casein kinase II, cyclin-dependent kinase and GSK-3.

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Preclinical In Vitro and In Vivo Evidence of an Antitumor Effect of CX-4945, a Casein Kinase II Inhibitor, in Cholangiocarcinoma

Translational Oncology ◽

10.1016/j.tranon.2018.09.005 ◽

2019 ◽

Vol 12 (1) ◽

pp. 143-153 ◽

Cited By ~ 11

Author(s):

Kais Zakharia ◽

Katsuyuki Miyabe ◽

Yu Wang ◽

Dehai Wu ◽

Catherine D. Moser ◽

...

Keyword(s):

Antitumor Effect ◽

Casein Kinase Ii ◽

Casein Kinase

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Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.5966 ◽

1995 ◽

Vol 15 (11) ◽

pp. 5966-5974 ◽

Cited By ~ 50

Author(s):

M Oelgeschläger ◽

J Krieg ◽

J M Lüscher-Firzlaff ◽

B Lüscher

Keyword(s):

Dna Binding ◽

Phosphorylation Site ◽

Direct Interaction ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Wild Type ◽

Type C ◽

A Site

Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.

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An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

Molecular Biology of the Cell ◽

10.1091/mbc.8.1.97 ◽

1997 ◽

Vol 8 (1) ◽

pp. 97-108 ◽

Cited By ~ 16

Author(s):

K E McGrath ◽

J F Smothers ◽

C A Dadd ◽

M T Madireddi ◽

M A Gorovsky ◽

...

Keyword(s):

Casein Kinase Ii ◽

Ribosomal Gene ◽

Casein Kinase ◽

Electron Microscopic ◽

Rna Recognition Motifs ◽

Amino Terminal ◽

Carboxyl Terminal ◽

Recognition Motifs

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

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The Human Cytomegalovirus Virion Possesses an Activated Casein Kinase II That Allows for the Rapid Phosphorylation of the Inhibitor of NF-κB, IκBα

Journal of Virology ◽

10.1128/jvi.02382-06 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5305-5314 ◽

Cited By ~ 24

Author(s):

Maciej T. Nogalski ◽

Jagat P. Podduturi ◽

Ian B. DeMeritt ◽

Liesl E. Milford ◽

Andrew D. Yurochko

Keyword(s):

Human Cytomegalovirus ◽

Molecular Mechanisms ◽

Kinase Activity ◽

Human Fibroblasts ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Temporal Regulation ◽

Protein Kinase Ckii

ABSTRACT We documented that the NF-κB signaling pathway was rapidly induced following human cytomegalovirus (HCMV) infection of human fibroblasts and that this induced NF-κB activity promoted efficient transactivation of the major immediate-early promoter (MIEP). Previously, we showed that the major HCMV envelope glycoproteins, gB and gH, initiated this NF-κB signaling event. However, we also hypothesized that there were additional mechanisms utilized by the virus to rapidly upregulate NF-κB. In this light, we specifically hypothesized that the HCMV virion contained IκBα kinase activity, allowing for direct phosphorylation of IκBα following virion entry into infected cells. In vitro kinase assays performed on purified HCMV virion extract identified bona fide IκBα kinase activity in the virion. The enzyme responsible for this kinase activity was identified as casein kinase II (CKII), a cellular serine-threonine protein kinase. CKII activity was necessary for efficient transactivation of the MIEP and IE gene expression. CKII is generally considered to be a constitutively active kinase. We suggest that this molecular characteristic of CKII represents the biologic rationale for the viral capture and utilization of this kinase early after infection. The packaging of CKII into the HCMV virion identifies that diverse molecular mechanisms are utilized by HCMV for rapid NF-κB activation. We propose that HCMV possesses multiple pathways to increase NF-κB activity to ensure that the correct temporal regulation of NF-κB occurs following infection and that sufficient threshold levels of NF-κB are reached in the diverse array of cells, including monocytes and endothelial cells, infected in vivo.

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Insulin-stimulated phosphorylation of calmodulin

Biochemical Journal ◽

10.1042/bj2860211 ◽

1992 ◽

Vol 286 (1) ◽

pp. 211-216 ◽

Cited By ~ 34

Author(s):

D B Sacks ◽

H W Davis ◽

D L Crimmins ◽

J M McDonald

Keyword(s):

Insulin Receptor ◽

Rat Hepatocytes ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Intact Cells ◽

Insulin Receptor Tyrosine Kinase ◽

Phosphate Incorporation ◽

Recent Demonstration

Calmodulin is phosphorylated in vitro by the insulin-receptor tyrosine kinase and a variety of serine/threonine kinases. Here we report that insulin stimulates the phosphorylation of calmodulin on average 3-fold in intact rat hepatocytes. Although calmodulin is constitutively phosphorylated, insulin increases phosphate incorporation into serine, threonine and tyrosine residues. We demonstrate that casein kinase II, an insulin-sensitive kinase, phosphorylates calmodulin in vitro on serine/thyronine residues (Thr-79, Ser-81, Ser-101 and Thr-117). The ability of the insulin receptor to phosphorylate calmodulin that has been pre-phosphorylated by casein kinase II is enhanced up to 35-fold, and the sites of phosphorylation on calmodulin are shifted from tyrosine to threonine and serine. These observations, obtained with a new specific monoclonal antibody to calmodulin, confirm that insulin stimulates calmodulin phosphorylation in intact cells. The observation that calmodulin is phosphorylated in vivo, coupled with the recent demonstration that phosphocalmodulin exhibits altered biological activity, strongly suggests that phosphorylation of calmodulin is a critical component of intracellular signalling.

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