A Molecular and Cellular View of Protein Kinase CK2 (Developments in Molecular and Cellular Biochemi

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Ecto-kinases are powerful regulatory enzymes for protein phosphorylation at the cell surface. They are critical for intercellular communication and transduction of external stimuli. Potential substrates for ecto-kinases are cell adhesion molecules, growth factors and their receptors, coagulation factors and ion channels. In some cases, ecto-kinases are shed from the surface of cells in a substrate-induced manner. Ecto-kinases have been identified as extracellular versions of known intracellular kinases. The pathway involved in their export out of the cell is largely unknown.

The cytosol of mammalian cells contains 5—10 mM ATP. This concentration gradient facilitates an increase in extracellular ATP concentration after cell activation or cell damage. Another important argument for ecto-kinases may be the addition of exogenous substrates, which do not enter the cells, or the use of inhibitors of kinases, which do not penetrate into cells into the labelling medium. Moreover, working on ecto-kinases, it is always an important point to exclude leakage from dead cells. Originally, two ecto-kinases have been described, c-AMP dependent protein kinase 11 and a cyclic nucleotide independent kinase 12 — These ecto-kinases were assumed to bind to lipid-anchored molecules, some of which were additionally shed into the medium of cultured cells.

In addition, there is also evidence for ecto-kinases, which are shed into the culture medium without being previously attached to the cell surface. Notably, there is also evidence for the presence of ecto-phosphatase activity at least on endothelial cells, which was shown by using the membrane impermeable reagent, microcystin LR, which inhibits protein phosphatases PP-1 and PP-2a Moreover, CK2-like kinases have been reported to be secreted from activated platelets, neutrophils and endothelial cells 7 , 16 , Knowledge regarding protein kinases and in particular protein kinase CK2 has increased considerably.

The human kinome consists of protein kinases The common feature of these protein kinases is the transfer of the terminal phosphate group of a nucleotide to a serine, threonine or tyrosine residue of substrate proteins.

Protein kinase CK2: a new view of an old molecular complex

The number of cell proteins, which are phosphorylated by CK2, is increasing rapidly 20 and therefore it is not surprising that CK2 is involved in almost every cell process regulating cell proliferation, cell survival 21 , apoptosis 22 , DNA damage and repair 23 , development and differentiation 24 and the regulation of metabolism CK2 is regarded as a constitutively active enzyme.

With regard to the numerous substrates of CK2 and its implication in numerous basic cell processes it is, however, hard to believe that this kinase is not tightly controlled. Since the tetramers and higher molecular weight complexes differ in their kinase activity, self-aggregation and dissociation seem to be involved in the regulation of CK2.

Thus, there are obvious cell regulators of CK2 47 — 49 and vice versa, as well as other proteins that are regulated by CK2 binding 49 — Phosphorylation and dephosphorylation is often the main mechanism for the regulation of proteins and their activities. This led to the conclusion that phosphorylation and dephosphorylation do not play a major role in the regulation of CK2.

The most important regulatory mechanism involved in CK2 seems to be its subcellular localisation.

Ecto‑protein kinase CK2, the neglected form of CK2 (Review)

Since the beginning of research on CK2, it has become evident that CK2 is located in the nucleus and in the cytoplasm. However, an increasing number of studies reported that CK2 is located in almost every compartment of a eukaryotic cell.

Moreover, the subcellular localisation of CK2 seems to be highly dynamic 55 — This dynamic subcellular localisation enables the CK2 subunits to interact with proteins, which are specific for one particular cell compartment. Besides the nuclear and cytoplasmic localisation, CK2 was found at the plasma membrane 13 , 59 — In recent years, more substrates for CK2 have been identified at the plasma membrane.

Since plasma membrane phosphorylation of proteins by CK2 were neglected thus far, the present review aims to address the role of CK2 as an ecto-kinase. An early observation identified that bone phosphoproteins were phosphorylated by CK2 isolated from detergent extracts of membranous fractions of a day embryonic chick tibia Although there are many early reports on ecto-CK2, little is known regarding the mechanism of how CK2 is exported to the cell surface.

It took approximately 5—7 h after transfection before tagged subunits were detectable on the cell surface When the subunits were expressed individually, they were not detectable externally. De novo protein synthesis is not required for the presence of ecto-CK2 on the cell surface Ecto-CK2 is not released from the cell surface by incubation with phospholipase C, suggesting that ecto-CK2 is not anchored in the plasma membrane via glycosyl-phosphatidyl-inositol-linkage.

Since CK2 is known to be elevated in many cancer cells and the observation that an inhibition of CK2 kinase activity leads to apoptosis of at least cancer cells 67 , 68 , there has been a search to identify new efficient and specific inhibitors of CK2 worldwide. Recently, bifunctional inhibitors have been designed, which, on the one hand bind ATP competitively, and on the other hand, mimic phospho-acceptor substrates 69 — On the basis of 4,5,6,7-tetrabromo-1H-benzimidazol TBI , new derivatives were generated, which compete with the phospho-acceptor sites of substrates.

These bifunctional inhibitors of CK2 have impaired cell permeability, which qualifies them for the inhibition of ecto-CK2 Table I lists the substrates, which are shown to be phosphorylated by ecto-CK2. The ecto-kinase activity was inhibited by heparin and by DRB, two compounds that are known to inhibit CK2 kinase activity 73 , Already in , a CK2-like activity was described in thrombin-activated platelets and in the supernatant of these activated platelets. It is known that thrombin triggers at least in endothelial cells, the release of intracellular ATP The presence of elevated extracellular ATP concentration and the presence of ecto-CK2 are an ideal combination for the phosphorylation of cell surface proteins.

Phosphorylated factor Va was more sensitive for active protein C than the non-phosphorylated form, triggering its degradation. This result indicates that ecto-CK2 may play a role in the downregulation of coagulation. Whether this effect is due to an impaired localization of CK2 on the cell surface remains to be elucidated.

Another protein that is phosphorylated by CK2 is C3 5. Phosphorylated fragments derived from C3 cleavage show an increased binding to IgG in serum over that of non-phosphorylated C3. Since CK2 phosphorylation of C3 increases its susceptibility to elastase cleavage, these results suggest an effect of platelet-derived CK2 phosphorylation of C3 to enhance the opsonisation of immune complexes 5. This protein kinase phosphorylates casein and fibrinogen and the enzyme is inhibited by heparin.

These features suggest ecto-CK2 There was a very similar observation, i. The C9 protein, which is a component of the lytic activity of the complement system was found to be phosphorylated by an ecto-kinase 4 , which was subsequently identified as CK2 The ecto-kinase is shed from the plasma membrane and this shed protein kinase also phosphorylates C9 protein C9 phosphorylation was achieved on intact Raji cells and also by shed proteins from the surface of Raji cells.

Ecto-CK2 was identified by immunofluorescence flow cytometry. The three inhibitors did not reduce cell viability. C9 is a blood plasma protein that binds to the C5b-8 complex of the complement system C9 phosphorylation by ecto-CK2 is a protective mechanism against complement-mediated lysis Phosphorylated C9 has a reduced haemolytic activity whereas the inhibition of ecto-CK2 kinase activity enhanced cell killing.

Other examples of substrates for ecto-CK2 are phosvitin and vitronectin. Vitronectin is a glycoprotein, which is present in blood and in the extracelluar matrix. At least for vitronectin, it is known that its phosphorylation is inhibited by DRB. Phosphorylation of vitronectin by ecto-CK2 regulates the adhesion of cells to the extracellular matrix 82 — Both substrates induced the release of ecto-CK2 when vascular smooth muscle cells VSMC were incubated with phosvitin or vitronectin When the cells were adhered to vitronectin, ecto-CK2 was enriched in clusters on the cell surface and then underwent a displacement from the VSMC surface Ecto-CK2 regulates monocyte migration through laminin-1 phosphorylation Ecto-kinase activity was inhibited by heparin and CK2 was identified with CK2-specific antibodies.

An interesting feature of phosphorylated laminin-1 is its interaction with heparin, where phosphorylated laminin-1 binds better to heparin than non-phosphorylated laminin In cell adhesion experiments, a significantly higher amount of cells adhere to phosphorylated laminin-1 Furthermore, phosphorylated laminin-1 promotes cell proliferation as well as monocyte migration.

CK2 was co-immunoprecipitated with an anti Fc-R antibody, indicating that ecto-CK2 is tightly bound to a receptor molecule. Collagen XVII is an example of an integral membrane protein with an extracellular domain, which was phosphorylated by ecto-CK2 at serine and Collagen XVII seems to be an interesting example for hierarchical phosphorylation, because serine is the first phosphorylation event, which generates an acidic environment, which then allows the serine phosphorylation by CK2.

The pharmacological inhibition of CK2 with 4,5,6,7-tetrabromobenzimidazol TBB as well as the use of a non-phosphorylatable alanine mutant revealed that CK2 phosphorylation of collagen XVII regulates ecto-domain shedding.

As a control for the specificity, a dominant negative mutant of CK2 was expressed and shown to be present on the cell surface. The authors of that study suggested that ecto-CK2 phosphorylation is a novel mechanism involved in the regulation of adhesion and motility of epithelial cells Ecto-CK2 seems to be involved in ossification by direct influencing mineral formation through the phosphorylation of osteopontin This observation is in agreement with the detection of stanniocalcin-2 STC-2 , a substrate of ecto-CK2 STC-2 is a proteohormone, which is involved in the regulation of calcium and phosphate homeostasis Using affinity chromatography on urokinase-conjugated Sepharose4B and nano-electrospray mass spectrometry and by immunoblotting, ecto-CK2 was found in a complex with urokinase and with nucleolin 90 , and this complex seems to be highly dynamic on the cell surface.

Ecto-CK2 phosphorylated the cell membrane-associated protein nucleolin. Nucleolin is a phosphoprotein, which shuttles between the nucleus and cytoplasm and which is located on the cell surface Urokinase activates ecto-CK2, leading to the phosphorylation of nucleolin and this phosphorylation is responsible for the translocation of nucleolin into the cell nucleus By contrast, intracellular CK2 is insensitive in the activation by urokinase.

Another example of a complex of ecto-CK2 with cell surface proteins was mentioned earlier, that of the association with Fc-R on monocytes There is a long history of a subclass of CK2 as an ecto-kinase. CK2 was identified by using casein as a substrate and with heparin as an inhibitor. It is now clear that casein is not a natural substrate of CK2.

The kinase committed to the phosphorylation of casein in the Golgi apparatus of the lactating mammary gland is conventionally termed genuine or Golgi casein kinase Heparin, which was used as an inhibitor of CK2, interacts non-specifically with proteins such as cytokines, growth factors, adhesion molecules and proteases Thus, in both cases off-target effects cannot be excluded. The direct identification of CK2 subunits on the cell surface by immunofluorescence or by immunoprecipitation were steps forward in the identification and characterisation of ecto-CK2. Furthermore, the development of new, highly specific, cell impermeable inhibitors of CK2 and phosphorylation experiments with substrates that cannot penetrate into cells have improved the specificity for ecto-CK2.

It remains an open question whether there are also high molecular aggregates of the CK2 holoenzyme on the cell surface. The proteins already identified earlier as substrates for ecto-CK2 show that ecto-CK2 plays a role in blood homeostasis, in thrombosis, in cell adhesion, in Alzheimer disease, calcium homeostasis and in the regulation of the immune system.

Other plasma membrane-associated proteins such as ion channels and receptors for hormones and growth factors, are excellent candidates for ecto-CK2.

This study was supported by the Dr Rolf M. Schwiete Stiftung, Mannheim, Germany project no. Ecto protein kinase activity on the external surface of neural cells. Ecto kinase activities in normal and transformed cells. Eur J Cell Biol. Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation. Paas Y and Fishelson Z: Ekdahl KN and Nilsson B: Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.

Platelet coagulation factor Va: The major secretory platelet phosphoprotein. Two recent papers on protein kinase CK2 formerly known as casein kinase II show how such insights can be gained by the comparison of structural data Niefind et al , with cell-culture studies, in which the dynamics of the protein can be visualized directly using green fluorescent protein GFP -tagged CK2 Filhol et al , CK2 is a multifunctional and almost universal protein kinase that has crucial roles in cell differentiation, proliferation and survival Ahmed et al , ; Litchfield, In addition, recent studies on the Drosophila clock genes provide strong evidence for the involvement of CK2 in the molecular clock machinery Akten et al , ; Blau, Therefore, numerous complementary observations, mostly based on traditional biochemical methods, led to the long-held tenet that CK2 is a strong obligate complex.

Moreover, the identification of several CK2-interacting proteins reinforced the idea that both subunits might have biological functions other than those attributed to the CK2 holoenzyme. However, despite these observations, the reported high-affinity interaction of the CK2 subunits has made hugely controversial the existence of independent subpopulations of these molecules in the cell. To resolve this dispute, the direct visualization of CK2 subunits and an investigation into their interaction behaviour in living cells was required.

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In a recent paper, Filhol and colleagues observed the individual CK2 subunits on a short timescale in living cells using live-cell fluorescent imaging Filhol et al , Overall, the recovery kinetics showed that the majority of the two subunits are not present in a common holoenzyme. This apparent difference in mobility was also evident at the level of their nuclear translocation: Qualitative analysis of kinetic parameters indicates that the nuclear accumulation of the two CK2 subunits might proceed in a sequential manner through different mechanisms.

This observation indicates that the residence time of the catalytic subunit in the nucleus might be shorter than that of the regulatory subunit. These observations also reveal that the molecular interaction between the CK2 subunits, which was once thought to be stable, is in fact highly dynamic. The assembly process of such macromolecular complexes is reminiscent of the dynamic and transient assembly of RNA polymerase I pol I. This possible intersubunit flexibility led the authors to propose that the CK2 holoenzyme is a transient heterocomplex Niefind et al , , which is consistent with the live-cell imaging data.

Intersubunit flexibility indicated by the crystal structure of the CK2 holoenzyme. The subcellular dynamics of the CK2 subunits and the transient nature of their interaction, as revealed by imaging and X-ray crystallography studies, are hallmarks of intracellular signalling molecules.

Introduction

Most non-obligate interactions have a regulatory role, which, in the case of CK2, is illustrated by the notable changes in substrate specificity of its complexed and non-complexed catalytic subunit Martel et al , ; Pinna, Instead, binding of this regulatory subunit might result in the phosphorylation of a range of substrates that are not, or are only weakly, phosphorylated in its absence. As CK2 substrates localize to many different subcellular compartments, a dynamic rather than a static interaction of the CK2 subunits should increase the kinase specificity and ensure that the relevant form of the catalytic subunit is present at each of these locations.

Indeed, fluorescence-correlation spectroscopy FCS analysis has provided evidence for the existence of fast- and slow-moving populations of both CK2 subunits Filhol et al , These considerations lead to a central question: Intuitively, to become non-obligate, a strong transient complex such as the CK2 holoenzyme needs to physicochemically regulate the transient interaction of its subunits through changing their binding affinity by orders of magnitude. Accordingly, it is important to consider that each CK2 subunit resides in a crowded environment with many potential binding partners with different surface properties.

Ecto‑protein kinase CK2, the neglected form of CK2 (Review)

This gives rise to the speculation that, at any one time in any given cell, CK2 is regulated by several protein—protein interactions. If the binding of the CK2 subunits is assumed to be essentially stochastic in nature, this interaction might be controlled by: Therefore, conformational changes could shift the balance in favour of, or against, binding between the two subunits. This hypothesis is supported by FCS analysis in mammalian cells, which showed that a fraction of each individual CK2 subunit is engaged in different high-molecular-weight complexes Filhol et al , These observations are consistent with the data from a proteome-wide analysis of native protein complexes in yeast, which showed the differential integration of the individual CK2 subunits in many functional multiprotein complexes Gavin et al , ; Ho et al , There are several ways to address the biological relevance of this dynamic behaviour of CK2.

First, each subunit has many potential phosphorylation sites; therefore, some insight could be gained through a systematic study of the behaviour of mutant CK2 subunits under various phosphorylation conditions. This should also encourage the development of specific peptide or non-peptide inhibitors of the interaction between the CK2 subunits. Third, higher order CK2 structures between CK2 tetramers that show differential catalytic activity have been characterized in vitro Glover, ; Valero et al , The reversible association of different CK2 conformers in vivo might account for regulated changes in their activity.

Clearly, this idea would add another level of complexity in interpreting the in vivo behaviour of CK2 Pinna, CK2 subunits might not act solely as a single holoenzyme entity; they might also be organized into discrete subcomplexes, each containing a different set of partners or substrates. Additionally, specific phosphorylation events could control the integration of the appropriate combinations of each individual CK2 subunit into these assemblies, so that they interact correctly with substrates Fig 2.

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This would artificially favour the high-affinity interaction of the CK2 subunits Fig 3 ; see supplementary information online for calculations. A schematic view of several dynamic pools of CK2 subunits. In a living cell, high local concentrations of interacting partners, substrates or accessory proteins that are present in specific cellular locations might stabilize weakly associating interactions of each CK2 subunit, leading to the transient formation of macromolecular complexes C I and C II.

The segregation of individual CK2 subunits might therefore contribute to signal specificity by sequestering the CK2 subunits in different pathways. Alternatively, CK2 subunits can bind to each other with high affinity.

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