Mass spectrometry analysis of a protein kinase CK2beta subunit interactome isolated from mouse brain by affinity chromatography

CK2, an acronym derived from the misnomer "casein kinase 2", denotes a ubiquitous and extremely pleiotropic Ser/Thr protein kinase, the holoenzyme of which is composed of two catalytic (alpha and/or alpha') and two noncatalytic beta subunits acting as a docking platform and the multifarious functions of which are still incompletely understood. By combining affinity chromatography and mass spectrometry, we have identified 144 mouse brain proteins that associate with immobilized CK2beta. A large proportion (60%) of the identified proteins had been previously reported to be functionally related to CK2, and a similar proportion have been classified as phosphoproteins with approximately half of these having the features of CK2 targets. A large number of the identified proteins ( approximately 40%) either are nuclear or shuttle between the nucleus and cytoplasm, and the biggest functional classes of CK2beta interactors are committed to protein synthesis and degradation (32 proteins) and RNA/DNA interaction (20 proteins). Also well represented are the categories of cytoskeletal/structural proteins (19), trafficking proteins (17), and signaling proteins (14). The identified proteins are examined in relation to their functions and potential as targets and/or regulators of CK2, disclosing in some cases unanticipated links between this kinase and a variety of biochemical events.     

Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk1. In this study, we show that Chk1 specifically phosphorylates in vitro the regulatory beta-subunit of CK2. Chymotryptic peptides and mutational analyses have revealed that CK2beta is phosphorylated at Thr213. Formation of a stable complex between CK2beta and Chk1 is not affected by the modification of Thr213 but it does require the presence of an active Chk1 kinase.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

The protein of a new gene,Tctex4, interacts with protein kinase CK2beta subunit and is highly expressed in mouse testis

Casein kinase 2 (CK2) is a ubiquitous, multifunctional eukaryotic serine/threonine kinase that phosphorylates an array of proteins. CK2 is a heterotetramer composed of two catalytic (alpha,alpha(')) and two regulatory (beta) subunits. CK2 plays an essential role in regulatory pathways in cell transformation and proliferation. But the role and function of the individual subunits of CK2, which are not in the holoenzyme, are not yet clear. Northern blot analysis reveals the highest CK2beta activity in mouse testicles and brain. By employing a yeast two-hybrid screen to identify the proteins that interact with CK2beta, we have isolated a cDNA clone encoding a 14-kDa protein with homology to dynein light chains and have designated it as Tctex4. CK2beta interacts specifically with Tctex4 both in a yeast two-hybrid system and in an in vitro interaction assay. Northern blot and in situ hybridization showed that Tctex4 is a novel gene that is expressed in mouse testis.     

Identification of chemical inhibitors of protein-kinase CK2 subunit interaction

Protein kinase CK2 is a multi-subunit complex whose dynamic assembly appears as a crucial point of regulation. The ability to interfere with specific protein-protein interactions has already provided powerful means of influencing the functions of selected proteins within the cell. CK2beta-derived cyclopeptides that target a well-defined hydrophobic pocket on CK2alpha have been previously characterized as potent inhibitors of CK2 subunit assembly [9]. As a first step toward the rational design of low molecular weight CK2 antagonists, we have in the present study screened a collection of podophyllotoxine indolo-analogues to identify chemical inhibitors of the CK2 subunit interaction. We report the identification of a podophyllotoxine indolo-analogue as a chemical ligand that binds to the CK2alpha/CK2beta interface inducing selective disruption of the CK2alpha/CK2beta assembly and concomitant inhibition of CK2alpha activity.     

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.     

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.     

Xenopus paraxial protocadherin inhibits Wnt/β-catenin signalling via casein kinase 2β

Xenopus paraxial protocadherin (PAPC) regulates cadherin-mediated cell adhesion and promotes the planar cell polarity (PCP) pathway. Here we report that PAPC functions in the Xenopus gastrula as an inhibitor of the Wnt/β-catenin pathway. The intracellular domain of PAPC interacts with casein kinase 2 beta (CK2β), which is part of the CK2 holoenzyme. The CK2α/β complex stimulates Wnt/β-catenin signalling, and the physical interaction of CK2β with PAPC antagonizes this activity. By this mechanism, PAPC restricts the expression of Wnt target genes during gastrulation. These experiments identify a novel function of protocadherins as regulators of the Wnt pathway.     

Protein p21(WAF1/CIP1) is phosphorylated by protein kinase CK2 in vitro and interacts with the amino terminal end of the CK2 beta subunit

Protein kinase CK2 is a ubiquitous protein that phosphorylates multiple substrates and is composed of catalytic (alpha, alpha') and regulatory (beta) subunits. Abundant evidence relates CK2 to the regulation of cell division. p21(WAF1/CIP1) is a potent inhibitor of cyclin-dependent kinases and of DNA replication and acts as a key inhibitor of cell cycle progression. In this work we examine the relation between these two important proteins. The interaction between the CK2 beta regulatory subunit of CK2 and p21(WAF1/CIP1) has been confirmed. Using a pull-down assay and fusion constructs of glutathione transferase with fragments of CK2 beta and other mutants, it was possible to define that the N-terminal (1-44) portion of CK2 beta contains a p21(WAF1/CIP1) binding site. CK2 reconstituted from recombinant alpha and beta subunits can phosphorylate p21(WAF1/CIP1) in vitro. This phosphorylation is greatly enhanced by histone H1. p21(WAF1/CIP1) can inhibit the phosphorylation of substrate casein by CK2. This inhibition, however, seems to be due to competition by p21(WAF1/CIP1) as an alternate substrate since in order to observe inhibition it is necessary that the concentration of p21 be of the same order of magnitude as the casein substrate concentration. This competition is not related to the binding of p21(WAF1/CIP1) to CK2 beta because it can also be observed when, in the absence of CK beta, CK alpha is used to phosphorylate casein in the presence of the p21.     

Characterisation of the biochemical and cellular roles of native and pathogenic amelogenesis imperfecta mutants of FAM83H

The majority of mutations identified in patients with amelogenesis imperfecta have been mapped to FAM83H. As FAM83H expression is not limited to the enamel, how FAM83H contributes to amelogenesis is still largely unknown. We previously reported that members of the FAM83 family of proteins interact with and regulate the subcellular distribution of the promiscuous serine-threonine protein kinase CK1 family, through their shared N-terminal DUF1669 domains. FAM83H co-localises with CK1 isoforms to speckle-like structures in both the cytoplasm and nucleus. In this report, we show FAM83H, unlike other FAM83 proteins, interacts and colocalises with NCK1/2 tyrosine kinase adaptor proteins. This interaction is mediated by proline-rich motifs within the C-terminus of FAM83H, specifically interacting with the second and third SH3 domains of NCK1/2. Moreover, FAM83H pathogenic AI mutant proteins, which trigger C-terminal truncations of FAM83H, retain their interactions with CK1 isoforms but lose interaction with NCK1/2. These AI mutant FAM83H proteins acquire a nuclear localisation, and recruit CK1 isoforms to the nucleus where CK1 retains its kinase activity. As understanding the constituents of the FAM83H-localised speckles may hold the key to unravelling potential substrates of FAM83H-associated CK1 substrates, we employed a TurboID-based proximity labelling approach and uncovered several proteins including Iporin and BAG3 as potential constituents of the speckles.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2

The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity.     

Regulation of caspase pathways by protein kinase CK2: identification of proteins with overlapping CK2 and caspase consensus motifs

Apoptosis, or programmed cell death, is a vital cellular process often impaired in diseases such as cancer. Aspartic acid-directed proteases known as caspases cleave a broad spectrum of cellular proteins and are central constituents of the apoptotic machinery. Caspases are regulated by a variety of mechanisms including protein phosphorylation. One intriguing mechanism by which protein kinases can modulate caspase pathways is by blocking substrate cleavage through phosphorylation of residues adjacent to caspase cleavage sites. To explore this mechanism in detail, we recently undertook a systematic investigation using a combination of bioinformatics, peptide arrays, and peptide cleavage assays to identify proteins with overlapping protein kinase and caspase recognition motifs (Duncan et al., Sci Signal 4:ra30, 2011). These studies implicated protein kinase CK2 as a global regulator of apoptotic pathways. In this article, we extend the analysis of proteins with overlapping CK2 and caspase consensus motifs to examine the convergence of CK2 with specific caspases and to identify CK2/caspase substrates known to be phosphorylated or cleaved in cells. Given its constitutive activity and elevated expression in cancer, these observations suggest that the ability of CK2 to modulate caspase pathways may contribute to a role in promoting cancer cell survival and raise interesting prospects for therapeutic targeting of CK2.     

CK2 beta, which inhibits Mos function, binds to a discrete domain in the N-terminus of Mos

Progesterone stimulates G2-arrested Xenopus oocytes to synthesize Mos, a MAPK kinase kinase required for the coordinated activation of cdc2 and the G2/Meiosis I (MI) transition. Mos leads to activation of MAPK, Rsk, and the inhibition of the cdc2 inhibitor Myt1. Previous work identified CK2 beta as a Mos-interacting protein, and suggested that CK2 beta acts as a negative regulator by setting a threshold above which newly made Mos must accumulate to activate MAPK. However, it had not been demonstrated that CK2 beta directly inhibits Mos. We report here that Mos (52-115) is required for CK2 beta binding and can serve as a portable binding domain. To test whether CK2 beta acts at the level of Mos or on a downstream component, we took advantage of previous work that showed injection of Mos arrests rapidly dividing embryonic cells. We find that coinjection of CK2 beta and Mos into embryonic cells inhibits the ability of Mos to arrest cell division. In contrast, CK2 beta does not inhibit the mitotic arrest induced by injection of active Rsk. These results argue that CK2 beta directly binds and inhibits Mos rather than a downstream component, and support that CK2 beta functions as a molecular buffer that prevents premature MAPK activation and oocyte maturation.     

Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta. Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.     

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.