Claudia Götz

The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation.

The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro 0.5 – 1 mol of phosphate were incorporated per mol MDM2 protein. The catalytic subunit of protein kinase CK2 (α-subunit) catalyzed the incorporation of twice as much phosphate into the MDM2 protein as it was obtained with the holoenzyme. Polylysine stimulated MDM2 phosphorylation by CK2 holoenzyme threefold in contrast to the α-subunit-catalyzed MDM2 phosphorylation which was reduced by about 66% when polylysine was added. Full length p53, but also a peptide representing a C-terminal fragment of the tumor suppressor gene product p53 (amino acids 264 – 393 which also harbors the CK2β interaction site at amino acids 287 – 340) mimicked the polylysine effect in all respects, ie. stimulation of phosphate incorporation by CK2 holoenzyme and inhibition in the presence of the catalytic CK2 α-subunit. Stimulation by p53264 – 393 was on the average close to twofold and inhibition in the case of the α-subunit-catalyzed MDM2 phosphorylation was about 40%. Phosphorylation of MDM2 by CK2 holoenzyme in the presence of the p21WAF1/CIP1, known to be a potent inhibitor of cyclin-dependent protein kinases, also led to a significant reduction of phosphate incorporation into MDM2 indicating that p21WAF1/CIP1 does not exclusively inhibit cell cycle kinases. Furthermore, these data add new insight into the autoregulatory loop which include p21WAF1/CIP1, MDM2 protein, CK2 and p53.

Wild‐type p53 inhibits protein kinase CK2 activity

The growth suppressor protein p53 and the protein kinase CK2 are both implicated in cellular growth regulation. We previously found that p53 binds to protein kinase CK2 via its regulatory β‐subunit. In the present study, we analyzed the consequences of the binding of p53 to CK2 for the enzymatic activity of CK2 in vitro and in vivo. We found that the carboxy‐terminus of p53 which is a potent transforming agent stimulated CK2 activity whereas full length wild‐type p53 which is a growth suppressor inhibited the activity of protein kinase CK2. Inhibition of protein kinase CK2 by p53 was dose‐dependent and was seen for various CK2 substrates. Experiments with heat‐denatured p53 and the conformational mutant p53R175H revealed that an intact conformation of p53 seemed to be necessary. Transfection of wild‐type and of mutant p53 into p53−/− cells showed that the inhibition of p53 on CK2 activity was also detectable in intact cells and specific for wild‐type p53 indicating that the growth suppressing function of p53 might at least be partially achieved by down‐regulation of protein kinase CK2. J. Cell. Biochem. 81:172–183, 2001.

Indeno(1,2-b)indole derivatives as a novel class of potent human protein kinase CK2 inhibitors

Herein we describe the synthesis and properties of indeno[1,2-b]indole derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 19 compounds was obtained using a convenient and straightforward synthesis protocol. The compounds were tested for inhibition of human protein kinase CK2, which was recombinantly expressed in Escherichia coli. New inhibitors with IC(50) in the micro- and sub-micromolar range were identified. Compound 4b (5-isopropyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-dione) inhibited human CK2 with an IC(50) of 0.11 μM and did not significantly inhibit 22 other human protein kinases, suggesting selectivity towards CK2. ATP-competitive inhibition by compound 4b was shown and a K(i) of 0.06 μM was determined. Our findings indicate that indeno[1,2-b]indoles are a promising starting point for further development and optimization of human protein kinase CK2 inhibitors.

A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening

A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5‐[(2‐aminoethyl)amino]naphthalene‐1‐sulfonic acid at the C‐terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non‐phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6‐methyl‐1,3,8‐trihydroxyanthraquinone and 4,5,6,7‐tetrabromobenzotriazole resulted in IC50 values of 1.33 and 0.27 μM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.

Protein kinase CK2 interacts with a multi-protein binding domain of p53

p53 is one of the most powerful negative regulators of growth. To manage this in an efficient way it has to interact with a set of different cellular proteins. Most contacts with the cellular environment occur in the N- or the C-terminal domain of the protein. Since we previously found that p53 binds to the regulatory β-subunit of CK2 we now analyzed N- and C-terminal domains of p53 separately for the binding of protein kinase CK2, an enzyme which seems to have a certain importance for proliferation processes. With different overlay assays we could map the binding domain of protein kinase CK2 to a sequence between amino acids 325–344, a region which coincides with the interaction domain of some other p53 binding proteins. We also found that the regulatory β-subunit of protein kinase CK2 binds independent of the catalytic α-subunit to this C-terminal domain of p53. (Mol Cell Biochem 191: 111–120, 1999)

Functional homology between yeast piD261/Bud32 and human PRPK: both phosphorylate p53 and PRPK partially complements piD261/Bud32 deficiency.

Yeast piD261/Bud32 belongs to the piD261 family of atypical protein kinases structurally conserved, from Archaea to human. The disruption of its gene is causative of severely defective growth. Its human homologue, PRPK, interacts with and phosphorylates the oncosuppressor p53 protein, which is lacking in yeast. Here we show that on one hand piD261/Bud32 interacts with and phosphorylates human p53 in vitro, on the other hand PRPK can partially complement the phenotype of yeast lacking the gene encoding piD261/Bud32. These data indicate that, despite considerable structural divergence, members of the piD261 family from distantly related organisms display a remarkable functional conservation.

Mutation of a CK2 phosphorylation site in cdc25C impairs importin α / β binding and results in cytoplasmic retention

cdc25C is a phosphatase, which activates the mitosis-promoting factor cyclin B1/cdc2 by dephosphorylation, and thus triggers G2/M transition. The activity of cdc25C itself is controlled by phosphorylation of certain amino-acid residues, which among other things determines the subcellular localization of the enzyme. Here, we describe a new phosphorylation site at threonine 236 of cdc25C, which is phosphorylated by protein kinase CK2. This phosphorylation site is located near the nuclear localization signal (amino acids 239–245). We demonstrate that cdc25C interacts with importin β and the importin α/β heterodimer but not with importin α. We further found that a cdc25C phosphorylation mutant where threonine 236 was replaced by aspartic acid as well as cdc25C phosphorylated by CK2 binds importin β or the importin α/β heterodimer less efficiently than wild type or the corresponding alanine mutant. Furthermore, the cdc25CT236D shows a retarded uptake into the nucleus in a cell import assay. Inhibition of protein kinase CK2 enzyme activity in vivo resulted in an enhanced nuclear localization of cdc25C. Thus, phosphorylation of cdc25C at threonine 236 is an important signal for the retention of cdc25C in the cytoplasm.

Fine mapping and regulation of the association of p53 with p34cdc2.

In vivo p53 is multiply phosphorylated by different protein kinases suggesting a central role for phosphorylation in modulating p53 function. In addition, p53 was found to be associated with two protein kinases, p34cdc2 and protein kinase CK2. Here we report the precise mapping of the interaction sites of p53 – p34cdc2 complexes. The p34cdc2 binding site on human p53 maps to one distinct C-terminal site LQIRGRERFE (aa 330 – 339) close to the corresponding phosphorylation site at serine 315. In order to test whether phosphorylation of p53 might influence the binding of p53 to p34cdc2 phosphorylation mutants of the C-terminus of p53, which mimick permanent phosphorylation, were tested on their ability to bind to p34cdc2 in vitro. Substitution of serine 315 (the p34cdc2 phosphorylation site) with aspartic acid had only little effect on complex formation whereas an exchange of serine 392 (the protein kinase CK2 phosphorylation site) to aspartic acid resulted in a significant reduced relative binding affinity of p53 to p34cdc2. The same result was obtained when the C-terminus of p53 was phosphorylated by purified protein kinase CK2 prior to examination of complex formation. In addition, the specificity of the complex formation has been checked by competition experiments with full length p53 proteins and the influence of cyclin B on complex formation was examined.

CK2 phosphorylation of Pdx-1 regulates its transcription factor activity.

The duodenal homeobox-1 protein Pdx-1 is one of the regulators for the transcription of the insulin gene. Pdx-1 is a phosphoprotein, and there is increasing evidence for the regulation of some of its functions by phosphorylation. Here, we asked whether protein kinase CK2 might phosphorylate Pdx-1 and how this phosphorylation could be implicated in the functional regulation of Pdx-1. We used fragments of Pdx-1 as well as phosphorylation mutants for experiments with protein kinase CK2. Transactivation was measured by reporter assays using the insulin promoter. Our data showed that Pdx-1 is phosphorylated by protein kinase CK2 at amino acids thr231 and ser232, and this phosphorylation was implicated in the regulation of the transcription factor activity of Pdx-1. Furthermore, inhibition of protein kinase CK2 by specific inhibitors led to an elevated release of insulin from pancreatic β-cells. Thus, these findings identify CK2 as a novel mediator of the insulin metabolism.

The role of protein kinase CK2 in the regulation of the insulin production of pancreatic islets

An appropriate regulation of the insulin production and secretion in pancreatic β-cells is necessary for the control of blood glucose homeostasis. The pancreatic duodenal homeobox factor-1 (Pdx-1) is among the various factors and signals which are implicated in the regulation of the insulin synthesis and secretion in the pancreatic β-cells. Recently, we identified Pdx-1 as a substrate for protein kinase CK2. Since CK2 is implicated in the regulation of many different cellular signaling pathways we now asked whether it might also be involved in the regulation of the insulin regulation in β-cells. Here, we show that insulin treatment of β-cells resulted in an elevated CK2 kinase activity. On the other hand down-regulation of CK2 activity by quinalizarin led to an elevated level of insulin. These results demonstrate that CK2 is implicated in the insulin regulation on pancreatic β-cells.