Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2alpha or GFP-CK2beta revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2beta, CK2alpha can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2alpha is dramatically changed by its association with CK2beta, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.     

Highlighting protein kinase CK2 movement in living cells

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (α < eqid1 > β₂) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol {23}: 975–987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio–temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

CK2β interacts with and regulates p21-activated kinases in Drosophila

The role of CK2β has been defined as the regulatory subunit of protein kinase CK2, which is a heterotetrameric complex composed of two CK2β and two catalytic active CK2α subunits. The identification of other serine/threonine kinases such as A-Raf, Chk1, and c-Mos that interact with and are regulated by CK2β has challenged this view and provided evidence for functions of CK2β outside the CK2 holoenzyme. In this report we describe the first interaction of Drosophila CK2β outside the CK2 holoenzyme with p21-activated kinase (PAK) proteins. This interaction is seen for distinct PAK and CK2β isoforms. In contrast to the CK2α–CK2β interaction, dimer formation of the CK2β subunits is not a prerequisite for binding of PAK proteins. Our results support the idea that CK2β can bind to PAK proteins in a CK2α independent manner and negatively regulates PAK kinase activity.     

Subcellular localization of protein kinase CK2

More than 46 years ago, Burnett and Kennedy first described protein kinase CK2 (formerly known as casein kinase 2) in liver extracts. Since then, protein kinase CK2 has been investigated in many organisms from yeast to man. It is now well established that protein kinase CK2 is a pleiotropic and ubiquitous serine or threonine kinase, which is highly conserved during evolution. A great number of studies deal with substrates of CK2, but the fact that over 160 substrates exist is more confusing than elucidatory. The holoenzyme is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. There is now increasing evidence for individual functions of the subunits that are different from their functions in the holoenzyme. Furthermore, more and more studies describe interacting partners of the kinase that may be decisive in the regulation of this enzyme. A big step forward has been the determination of the crystal structure of the two subunits of protein kinase CK2. Now the interactions of the catalytic subunit of CK2 with ATP as well as GTP and the interaction between the regulatory subunits can be explained. However, cellular functions of protein kinase CK2 still remain unclear. In the present review we will focus our interest on the subcellular localization of protein kinase CK2. Protein kinase CK2 is found in many organisms and tissues and nearly every subcellular compartment. There is ample evidence that protein kinase CK2 has different functions in these compartments and that the subcellular localization of protein kinase CK2 is tightly regulated. Therefore studying the subcellular localization of protein kinase CK2 may be a key to its function.     

CK2 and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles

Several peripheral membrane proteins associated with clathrin-coated vesicles (CCVs) are reversibly phosphorylated, but it is not clear precisely which protein kinases are involved. In order to address this question directly, we have isolated highly purified CCVs from porcine brain. The peripheral membrane proteins have been removed and assayed for kinase activity using the CCV peripheral membrane proteins as substrate. The major kinase activity identified has a molecular mass of 40 kDa, is inhibited by known specific inhibitors of the protein kinase CK2 and is recognised by an antibody specific to CK2. We show that CK2 is responsible for the phosphorylation of the majority of CCV-associated proteins that are subject to phosphorylation. Intriguingly, CK2 is inactive when associated with CCVs but becomes active once the clathrin coat has been removed. The medium subunit of the AP2 adaptor complex (μ2) is not a substrate for CK2, but is phosphorylated by a second kinase that we show to be cyclin G-associated kinase (GAK/auxilin2). Unlike the situation for the CK2 substrates, μ2 is a substrate for GAK/auxilin2, both in intact CCVs and in solution. In addition, we show that the 'stripped' CCV membranes that remain once the peripheral membrane proteins have been removed from CCVs inhibit CK2 but not GAK/auxilin2 activity.     

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.     

Fluxes through cytosolic and mitochondrial creatine kinase, measured by P-31 NMR

The kinetic properties of the cytoplasmic and the mitochondrial iso-enzymes of creatine kinase from striated muscle were studied in vitro and in vivo. The creatine kinase (CK) iso-enzyme family has a multi-faceted role in cellular energy metabolism and is characterized by a complex pattern of tissue-specific expression and subcellular distribution. In mammalian tissues, there is always co-expression of at least two different CK isoforms. As a result, previous studies into the role of CK in energy metabolism have not been able to directly differentiate between the individual CK species. Here, we describe experiments which were directed at achieving this goal. First, we studied the kinetic properties of the muscle-specific cytoplasmic and mitochondrial CK isoforms in purified form under in vitro conditions, using a combination of P-31 NMR and spectrophotometry. Secondly, P-31 NMR measurements of the flux through the CK reaction were carried out on intact skeletal and heart muscle from wild-type mice and from transgenic mice, homozygous for a complete deficiency of the muscle-type cytoplasmic CK isoform. Skeletal muscle and heart were compared because they differ strongly in the relative abundance of the CK isoforms. The present data indicate that the kinetic properties of cytoplasmic and mitochondrial CK are substantially different, both in vitro and in vivo. This finding particularly has implications for the interpretation of in vivo studies with P-31 NMR. (Mol Cell Biochem 174: 33–42, 1997)     

Maize protein kinase CK2: regulation and functionality of three beta regulatory subunits

Biochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2alpha), thus lacking CK2beta regulatory subunits. To investigate the existence and functionality of CK2beta regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full-length cDNAs encoding CK2beta regulatory subunits (CK2beta-1, CK2beta-2 and CK2beta-3) and a cDNA coding for a novel CK2alpha catalytic subunit, CK2alpha-3. The pattern of expression of all these alpha/beta subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2alpha subunits are constitutive, CK2beta subunits are expressed differentially during embryo development. The yeast two-hybrid system and pull-down assays have been used to study specific interactions between the different subunits. While CK2alpha subunits are unable to self-associate, preferential interactions between alpha/beta isoforms and beta/beta isoforms can be predicted. Furthermore, we show that maize CK2alpha/beta subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2beta subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2beta regulatory subunits.     

Chemical proteomics and functional proteomics strategies for protein kinase inhibitor validation and protein kinase substrate identification: Applications to protein kinase CK2

Since protein kinases have been implicated in numerous human diseases, kinase inhibitors have emerged as promising therapeutic agents. Despite this promise, there has been a relative lag in the development of unbiased strategies to validate both inhibitor specificity and the ability to inhibit target activity within living cells. To overcome these limitations, our efforts have been focused on the development of systematic strategies that employ chemical and functional proteomics. We utilized these strategies to evaluate small molecule inhibitors of protein kinase CK2, a constitutively active kinase that has recently emerged as target for anti-cancer therapy in clinical trials. Our chemical proteomics strategies used ATP or CK2 inhibitors immobilized on sepharose beads together with mass spectrometry to capture and identify binding partners from cell extracts. These studies have verified that interactions between CK2 and its inhibitors occur in complex mixtures. However, in the case of CK2 inhibitors related to 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), our work has also revealed off-targets for the inhibitors. To complement these studies, we devised functional proteomics approaches to identify proteins that exhibit decreases in phosphorylation when cells are treated with CK2 inhibitors. To identify and validate those proteins that are direct substrates for CK2, we have also employed mutants of CK2 with decreased inhibitor sensitivity. Overall, our studies have yielded systematic platforms for studying CK2 inhibitors which we believe will foster efforts to define the biological functions of CK2 and to rigorously investigate its potential as a candidate for molecular-targeted therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

The emerging CK2 interactome: insights into the regulation and functions of CK2

Protein kinase CK2 represents a small family of protein serine/threonine kinases implicated in a variety of biological processes including events relating to cell proliferation and survival. Notably, CK2 displays oncogenic activity in mice and exhibits altered expression in several types of cancer. Accordingly, a detailed understanding of the cellular functions of CK2 and elucidation of the mechanisms by which CK2 is regulated in cells is expected to contribute to understanding its role in tumorigenesis with the prospect of novel approaches to therapy. While CK2 has traditionally been viewed as a tetrameric complex composed of two catalytic and two regulatory subunits, mounting evidence suggests that its subunits may have functions independent of tetrameric CK2 complexes. In mammals, as is the case in the budding yeast Saccharomyces cerevisiae, there are two isozymic forms of CK2, adding additional heterogeneity to the CK2 family. Studies in yeast and in human cells demonstrate that the different forms of CK2 interact with a large number of cellular proteins. To reveal new insights regarding the regulation and functions of different forms of CK2, we have examined the emerging interactomes for each of the CK2 subunits. Analysis of these interactomes for both yeast and human CK2 reinforces the view that this family of enzymes participates in a broad spectrum of cellular events. Furthermore, while there is considerable overlap between the interactomes of the individual CK2 subunits, notable differences in each of the individual interactomes provides additional evidence for functional specialization for the individual forms of CK2.     

Crystal structure of brain-type creatine kinase at 1.41 A resolution

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue--as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 A resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2-12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure. The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 A from the structure without substrates.     

Mapping of the human casein kinase II catalytic subunit genes: two loci carrying the homologous sequences for the alpha subunit.

The human serine/threonine protein casein kinase II (CK II) contains two distinct catalytic subunits, alpha and alpha 1, which are encoded by different genes. A combination of segregation analysis of rodent-human hybrid cells and chromosomal in situ hybridization have localized the human CK II-alpha DNA sequence to two loci: 11p15.5-p15.4 and 20p13. In contrast, the CK II-alpha' gene has been mapped to chromosome 16 by somatic cell hybrid analysis. Taken together with our previous assignment of the CK II regulatory beta-subunit gene to 6p12-p21, these results indicate that although the products of these genes form a single biological complex, they are encoded on different human chromosomes. Further analysis should determine whether both loci of CK II-alpha are functional, or perhaps one of the two constitutes a pseudogene.     

Cytokinin conjugation: recent advances and patterns in plant evolution

Cytokinin (CK) conjugates are important in plant development because they regulate active CK concentrations, CK transport, storage, and irreversible inactivation. While numerous CK conjugates have been identified in higher plants, the biological functions of these compounds, their location within cells and tissues, and the enzymes and genes involved in their regulation are not clearly understood. In this paper, recent advances are reported which have occurred through the study of transgenic plants containing the ipt or rolC genes, the identification of new regulatory enzymes affecting CKs, and the characterization of new CK conjugates. In addition, a survey of the literature is presented which examines the pattern of CK conjugates found in different plant taxa. Based on current knowledge, it appears that green algae, mosses, and ferns contain relatively few CK conjugates of isopentenyl adenine (iP) and zeatin (Z). In contrast, higher land plants, such as gymnosperms and angiosperms, contain a more complex set of CKs, primarily conjugates of Z and dihydrozeatin (DHZ). This suggests that the pattern of CK conjugation has become more complex in parallel with the increasing complexity of higher plants.     

Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells

Protein kinase CK2 exhibits oncogenic activity in mice and is over-expressed in a number of tumors or leukemic cells. On the basis of its amino acid sequence and a wealth of experimental information, CK2 has traditionally been classified as a protein serine/threonine kinase. In contrast to this traditional view of CK2, recent evidence has shown that CK2 can also phosphorylate tyrosine residues under some circumstances in vitro and in yeast. In this study, we provide definitive evidence demonstrating that CK2 also exhibits tyrosine kinase activity in mammalian cells. Tyrosine phosphorylation of CK2 in cells and in CK2 immunoprecipitates is dependent on CK2 activity and is inhibited by the CK2 selective inhibitor 4,5,6,7-tetrabromobenzotriazole. Examination of phosphotyrosine profiles in cells reveals a number of proteins, including CK2 itself, which exhibit increased tyrosine phosphorylation when CK2 levels are increased. Peptide arrays to evaluate the specificity determinants for tyrosine phosphorylation by CK2 reveal that its specificity for tyrosine phosphorylation is distinct from its specificity for serine/threonine phosphorylation. Of particular note is the requirement for an aspartic acid immediately C-terminal to the phosphorylatable tyrosine residue. Collectively, these data provide conclusive evidence that CK2 catalyzes the phosphorylation of tyrosine residues in mammalian cells, a finding that adds a new level of complexity to the challenge of elucidating its cellular functions. Furthermore, these results raise the possibility that increased CK2 levels that frequently accompany transformation may contribute to the increased tyrosine phosphorylation that occurs in transformed cells.     

Serum-stimulated cell cycle entry of fibroblasts requires undisturbed phosphorylation and non-phosphorylation interactions of the catalytic subunits of protein kinase CK2

Protein kinase CK2 is a pleiotropic Ser/Thr kinase occurring as alpha2beta2, alpha'2beta2, or alphaalpha'beta2 tetramers. A requirement in serum-stimulated cell cycle entry in both the cytoplasm and the nucleus of human fibroblasts for phosphorylation(s) by CK2 has been concluded from stimulation inhibition by microinjected antibodies against the regulatory subunit (beta). We have now examined this idea more directly by microinjection-mediated perturbation of phosphorylation and non-phosphorylation interactions of the catalytic subunits (alpha and alpha'), and by verifying the supposed matching of the cellular partition of CK2 subunits in the fibroblasts employed. While immunostaining and cell fractionation indicate that the partitions of subunits indeed match each other (with their predominant location in the nucleus in both quiescent and serum-stimulated cells), microinjection of substrate or pseudosubstrate peptides competing for the CK2-mediated phosphorylation in vitro resulted in significant inhibition of serum stimulation when placed into the nucleus but not when placed into the cytoplasm. Also inhibitory were nuclear but not cytoplasmic injections of antibodies against alpha and alpha' that affect neither their kinase activity in vitro nor their complexing to beta. The data indicate that the role played by CK2 in serum-stimulated cell cycle entry is predominantly nuclear and more complex than previously assumed, involving not only phosphorylation but also experimentally separable non-phosphorylation interactions by the catalytic subunits.