Casein Kinase II
Protein kinase CK2 was isolated almost 60 years ago on the basis of its ability to phosphorylate the milk protein casein (Allende and Allende 1995; Venerando et al. 2014). As a result, it was originally designated “casein kinase II,” though this misnomer is less frequently used because of the lack of physiological significance for the phosphorylation of casein by CK2. Ironically, the enzyme/substrate relationship between CK2 and casein illustrates somewhat of a recurring theme. In this respect, over 50 years of research on CK2 has resulted in a compilation of a very large number of putative CK2 substrates, while the functional analysis of these phosphorylation events has lagged comparatively behind. In large part, the identification of many CK2 substrates has been promoted by its relatively simple and somewhat unique preference for acidic specificity determinants (S/T-X-X- E/D/pS/pY) (Meggio and Pinna 2003). Even before the onset of the large-scale identification of phosphorylation sites, there were more than 300 candidate substrates identified with thousands of potential CK2 substrates now predicted on the basis of global phosphoproteomics and computational analyses (Pinna and Allende 2009). The promiscuity of CK2 in both its number of substrates and the cellular functions in which it has been implicated comes as little surprise when considering its lack of strict regulation (Litchfield 2003; Olsten and Litchfield 2004). Work to date has not yielded a unifying mechanism to explain the regulation of CK2 in cells. In this respect, it has been suggested that CK2 is constitutively active, although there is evidence that there are changes in the phosphorylation of some of its substrates in cells suggesting that its activity could in fact be regulated. Despite the uncertainties about its cellular regulation, evidence for pathogenic misregulation of CK2 in various human malignancies was observed over 20 years ago; CK2 activity and expression are elevated in a number of human cancers, while inhibition of CK2 results in cellular death (Duncan and Litchfield 2008; Trembley et al. 2009). This essay will outline structural and enzymatic features of CK2 and will highlight some of the mechanisms that may contribute to its physiological regulation along with an overview of its cellular functions and emergence as a candidate for molecular-targeted therapy.
Structural and Enzymatic Features of CK2
Although CK2β is not strictly required to turn on or turn off the catalytic activity of CK2α or CK2α′, it is apparent that it can affect the activity of the catalytic subunits and modulate substrate selectivity (Meggio and Pinna 2003; Olsten and Litchfield 2004; Bibby and Litchfield 2005). While the majority of CK2 substrates can be effectively phosphorylated by either tetrameric CK2 or free catalytic CK2 subunits, the regulatory CK2β subunit generally enhances thermal stability of the catalytic subunits and confers a modest increase (typically three- to fivefold) in catalytic activity toward most substrates. By comparison, there are other proteins, such as eIF2β, where CK2β is required to enable efficient phosphorylation, or calmodulin, where the presence of CK2β leads to complete inhibition of phosphorylation. The demonstration that calmodulin is phosphorylated at its CK2 sites in cells was among the first evidence provided to suggest that free catalytic subunits exist, and are functionally active, in cells. In a similar vein, CK2-independent functions and regulatory mechanisms of CK2β have been explored (reviewed by Bibby and Litchfield 2005). In this respect, it is interesting to note that CK2β is synthesized in excess of the catalytic subunits. Though it is believed that CK2β is quickly degraded when it is not incorporated into tetrameric CK2 complexes (Zhang et al. 2002), a number of reports highlight potential CK2-independent interactions of CK2β including interactions with a number of other protein kinases (e.g., c-Mos, Chk1, and A-Raf). Collectively, these findings illustrate not only the ability of CK2β to control the substrate specificity of CK2 but also raise the prospect that individual CK2 subunits exist outside the holoenzyme and are governed by unique modes of regulation. At this point, however, the precise mechanisms that regulate tetramer formation and dissociation in cells remain unclear.
Physiological Regulation of CK2
As mentioned previously, the activity of CK2 in cells is not subject to absolute “on/off” regulation by individual events such as posttranslational modifications, protein/protein interactions, or small molecule secondary messengers that are observed with other major protein kinase families such as MAPKs, CDKs, and PKCs, respectively. Rather, on the basis of structural characteristics (discussed above), the ability to isolate active, recombinant enzyme from bacterial sources (Turowec et al. 2010) and, most importantly, the observation that activity is neither turned on nor turned off in response to a variety cellular stimuli that modulate a large number of other protein kinases (Litchfield et al. 1993), CK2 is considered to be constitutively active. While it is apparent that CK2 lacks strict on/off regulation, it is conceivable that a number of more subtle mechanisms may contribute to its regulation in cells, including protein interactions and temporal or stimulus-specific changes in function that are discussed below (Olsten and Litchfield 2004).
The observation that CK2 phosphorylates proteins in multiple cellular compartments supports the hypothesis that interacting proteins regulate CK2 function by directing it to specific locations within the cell (Olsten and Litchfield 2004; Filhol and Cochet 2009). In this way, specific subpopulations of CK2 could be regulated by interacting proteins that permit spatial access to substrates, much the same way that AKAPs modulate the localization and function of PKA. One protein that may regulate CK2 in this manner is CKIP-1, a PH domain-containing protein that localizes CK2α, but not CK2α′, to the plasma membrane (Canton and Litchfield 2006). Changes in the expression of CKIP-1 induce alterations in cell morphology and the actin cytoskeleton. Since CKIP-1 interacts with CPα, a subunit of the heterodimeric actin-capping protein that can be phosphorylated by CK2, these observations are consistent with a working model suggesting that CKIP-1 could participate in the regulation of the actin cytoskeleton by modulating the CK2-catalyzed phosphorylation of CPα. In addition to CKIP-1, CK2 has a large number of other potential interaction partners (Gyenis and Litchfield 2015; Nunez de Villavicencio-Diaz et al. 2017). Again, given the large number of CK2 substrates located in numerous cellular locations, further elucidation of its interaction partners and investigation of their spatial distribution could reveal important new insights into the regulation and physiological functions of CK2.
Though not necessarily responsible for global changes in CK2 activity, it has been demonstrated that discrete populations of CK2 are modulated in response to specific stimuli (Filhol and Cochet 2009). For example, in response to UV radiation, CK2 interacts with the FACT complex, which consists of SSRP1 and hSPT16 (Keller et al. 2001). Binding of CK2 to the FACT complex results in increased phosphorylation of p53 by CK2 at Ser 392, an event believed to increase the transcription of apoptotic and cell cycle regulatory genes. Likewise, the regulation of specific CK2 protein interactors and substrates has been observed in nocodazole-arrested cells where CK2α has been shown to be phosphorylated at four proline-directed phosphorylation sites that can be phosphorylated by CDK1 in vitro (Fig. 1) (St-Denis et al. 2011). Phosphorylation of these sites within its unique C-terminal domain promotes interactions between CK2α and the peptidyl-prolyl isomerase Pin1 (Messenger et al. 2002). Interactions with Pin1 modulate the ability of CK2 to phosphorylate topoisomerase IIα in vitro at sites that are known to be phosphorylated in mitotic cells. Collectively, the examples of FACT and Pin1 illustrate mechanisms by which the ability of CK2 to phosphorylate specific proteins can be modulated by interactions with other cellular proteins. Given the large number of protein interactions that have been reported for CK2, it is likely that other proteins will exert similar effects to modulate discrete populations of CK2 in cells.
The isoform-specific interactions of proteins such as CKIP-1 and Pin1 also reveal independent forms of regulation for CK2α as compared to CK2α′, an observation consistent with the demonstration of distinct phenotypes for CK2α and CK2α′ knockout mice (Dominguez et al. 2009). CK2α′ knockout mice are viable but exhibit defects in spermatogenesis in males which results in infertility. By comparison, CK2α knockout mice are embryonic lethal with embryos exhibiting significant defects in heart development. Taken together, these contrasting phenotypes suggest unique functions for both catalytic subunits, though CK2α appears more able to compensate for a loss of CK2α′. Furthermore, it is conceivable that unique regulatory mechanisms of CK2α are responsible for its greater importance to murine viability.
Though some progress has been made regarding the regulation of CK2 via posttranslational modifications and protein/protein interactions, relatively little is known about the transcriptional regulation of CK2. As previously noted, the expression of CK2 is elevated in rapidly dividing cells and a number of human cancers. The precise mechanism by which the expression of CK2, apparent housekeeping genes, is up-regulated in cancers has not been thoroughly characterized, though a number of putative binding sites for various transcription factors have been identified (Pyerin and Ackermann 2003). For example, it is intriguing that all of the human CK2 genes contain response elements for the Ets1 transcription factor that is regulated by mitogen-activated pathways. Other factors that may contribute to the regulation of CK2 levels include ubiquitination and degradation by the proteasome (Zhang et al. 2002).
Cellular Functions of CK2
In consideration of its large number of documented substrates (and the ever-expanding list of prospective substrates that are appearing in rapidly populating phosphoproteomic databases), it is not surprising that CK2 has been implicated in a broad range of cellular processes including cell proliferation and survival, apoptosis, circadian rhythms, viral infection, and transcriptional control (Duncan and Litchfield 2008; St-Denis and Litchfield 2009). Of particular interest to human disease are the observations that CK2 is over-expressed in a number of human cancers, including the kidney, mammary gland, lung, head and neck, and prostate, and that targeted over-expression of CK2 in mice T-cells and mammary glands results in lymphoma and mammary adenocarcinomas (Duncan and Litchfield 2008; Rabalski et al. 2016). Furthermore, synergistic effects in tumor formation are observed in mice when over-expressing CK2 in concert with the Myc or Tal-1 oncogenes or in p53 (−/−) backgrounds (Xu et al. 1999). Conversely, short hairpin RNA (shRNA) knockdown and pharmacological inhibition studies in a variety of model systems result in cell death, reinforcing the notion that targeted inhibition of CK2 represents a promising therapeutic strategy for the treatment of human cancers (Trembley et al. 2009; Ruzzene and Pinna 2010). Though it is clear that global changes in CK2 activity regulate processes pertaining to cellular proliferation and survival, the precise molecular mechanisms by which CK2 exerts these functions are still incompletely understood. In the following discussion, a number of advances regarding the specific role of CK2 in these cellular processes will be highlighted.
CK2 has been implicated in many signaling pathways directly involved in controlling the rate of cellular proliferation (Duncan and Litchfield 2008). As noted above, coordinated over-expression of CK2 and Myc in murine T-cells results in lymphoma development. Supporting the pathogenic synergism between CK2 and Myc is the observation that CK2 phosphorylates Myc and prevents its proteasome-dependent degradation, allowing increased transcriptional activity of proliferative and survival genes. Similarly, CK2 is believed to function at many levels within the Wnt signaling pathway, ultimately promoting β-catenin stability, dissociation from APC, and the transcription of pro-survival and proliferative genes.
It is important to reiterate the vast repertoire of cellular functions involving CK2 and the diversity of its substrates. Even before the inception of predictive analyses of phosphoproteomic databases for putative CK2 substrates, CK2 was believed to have over 300 substrates, including over 60 transcription factors, 80 signaling molecules, and 40 viral proteins (Meggio and Pinna 2003). However, as a result of the constitutive activity of CK2 and its relatively simple consensus motif determinants, some analyses of phosphoproteomic databases have predicted the number of CK2 substrates to number in the thousands (Pinna and Allende 2009). While the systematic identification of all CK2 substrates remains a daunting task, a more complete evaluation of substrates will undoubtedly uncouple the pathogenic and physiological functions of CK2. Along these lines, an important avenue of future research will involve validating putative substrates. One appealing approach will certainly employ bioinformatic consensus motif analyses of phosphoproteomic databases to identify likely physiological targets of CK2, followed by in vitro phosphorylation assays that test the ability of CK2 to directly phosphorylate these sites (Turowec et al. 2010). In this sense, demonstration of direct phosphorylation of putative substrates by CK2 as well as the existence of these phosphorylation sites in cells should be adopted as the gold standard for classifying substrates as bona fide. Furthermore, sorting of biologically relevant CK2 substrates by GO designations will not only act to further clarify the physiological function of CK2 but may streamline the identification of substrates with pathological significance. Likewise, the demonstration that CK2 can participate in complex hierarchical regulatory mechanisms, such as protection of caspase substrates from degradation or acting as a primary or secondary kinase in hierarchical protein phosphorylation, may also be exploited by bioinformatics and/or high-throughput analyses that attempt to expand upon the role of CK2 in signaling pathways pertaining to cellular proliferation (Figs. 3 and 4) (St-Denis and Litchfield 2009; Duncan et al. 2010).
Emergence of CK2 as a Candidate for Molecular-Targeted Therapy
The over-expression of CK2 in a number of human malignancies and its participation in multiple pro-survival signaling pathways have driven the development of pharmacological inhibitors of CK2 for use as candidate lead compounds for molecular-targeted therapy (Sarno and Pinna 2008; Rabalski et al. 2016). In fact, clinical trials have been initiated with two distinct CK2 inhibitors. One of the clinical stage CK2 inhibitors is CX-4945 (silmitasertib), an ATP-competitive inhibitor of CK2 with an IC50 in the nanomolar range (Pierre et al. 2011). The other clinical stage inhibitor is CIGB-300, a cyclic peptide that was isolated from a screen to identify cyclic peptides that bind to the E7 protein of human papillomavirus type 16 (Solares et al. 2009). Although many details of its precise mechanism of action remain to be defined, CIGB-300 appears to inhibit CK2 by interfering with the binding of CK2 to many of its substrates. In addition to these clinical-stage inhibitors, there are a number of other compounds available from commercial sources that have been reported to be potent and/or selective CK2 inhibitors including DRB, emodin, TBB, TBBz, and DMAT (Ruzzene and Pinna 2010). Since the majority of available CK2 inhibitors are ATP-competitive inhibitors, it is important to acknowledge that a main challenge of developing ATP-competitive-specific protein kinase inhibitors relates to the high degree of conservation exhibited among all human protein kinases and to, a lesser extent, other ATP-binding proteins. Indeed, off-target effects of CK2 inhibitors are apparent and need to be considered when exploiting these compounds both as clinical agents and as research tool compounds (Duncan et al. 2008; Gyenis et al. 2013). Another key limitation relating to the utilization of CK2 inhibitors is the lack of bona fide biomarkers (Gyenis et al. 2011). The preclinical utility of CK2 inhibitors to date has generally been measured by gross phenotypic changes, such as apoptosis, as opposed to a decrease in phosphorylation of a particular biomarker (Ruzzene and Pinna 2010). Consequently, by developing tools that directly monitor the phosphorylation status of specific cellular substrates of CK2, the distinction between off-target effects and specific CK2 inhibition would be more easily clarified as biomarker status could be correlated to phenotypic changes. Furthermore, phosphospecific antibodies could also be useful for advancing clinical applications of CK2 inhibitors including the prospect of identifying patients with high levels of CK2 who may be candidates for CK2-targeted therapy and for monitoring clinical response to CK2 inhibition.
Since its discovery in 1954, much has been learned regarding the enzymatic characteristics, physiological regulation, and cellular functions of protein kinase CK2. Structural studies have yielded many insights into the unique features of CK2, such as its constitutive activity and ability to use GTP as a phosphate donor. While CK2 appears to be constitutively active, it is likely that a number of distinct mechanisms such as spatial regulation through protein interactions and possibly the regulated assembly or disassembly of tetrameric CK2 will govern how the phosphorylation of many of its substrates is controlled in cells. However, on the whole, many details regarding the precise manner by which CK2 is regulated in cells remain to be defined. Though the expanding list of CK2 substrates has illuminated many cellular functions, including roles pertaining to cell survival, proliferation, and involvement in diseases such as cancer, it is clear that CK2 has many undefined substrates whose phosphorylation status dictates unknown roles. In this sense, future endeavors should involve the use of phosphoproteomic databases which represent a tremendous resource of physiological protein phosphorylation sites, to guide the identification of bona fide CK2 substrates. In expanding our knowledge of CK2 substrates, it is more likely that the physiological and pathogenic functions of CK2 will be uncoupled and that the generation of convenient biomarkers capable of monitoring CK2 activity in cells will be established. Tools such as these will greatly accelerate the development and benchmarking of current and future generations of inhibitors, which in turn will feedback and drive the discovery of CK2 functions and benefit the treatment of cancers where CK2 hyperactivity contributes to tumorigenesis.
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