Casein kinase: the triple meaning of a misnomer.

Biochem. J. (2014) 460, 141–156 (Printed in Great Britain)

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doi:10.1042/BJ20140178

REVIEW ARTICLE

*Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/b, 35131 Padova, Italy †CNR Neuroscience Institute, Via Ugo Bassi 58/b, 35131 Padova, Italy

The term ‘casein kinase’ has been widely used for decades to denote protein kinases sharing the ability to readily phosphorylate casein in vitro. These fall into three main classes: two of them, later renamed as protein kinases CK1 (casein kinase 1, also known as CKI) and CK2 (also known as CKII), are pleiotropic members of the kinome functionally unrelated to casein, whereas G-CK, or genuine casein kinase, responsible for the phosphorylation of casein in the Golgi apparatus of the lactating mammary gland, has only been identified recently with Fam20C [family with sequence similarity 20C; also known as DMP-4 (dentin matrix protein-4)], a member of the four-jointed family of atypical protein kinases, being responsible for the phosphorylation of many secreted proteins. In hindsight, therefore, the term ‘casein kinase’ is misleading in every instance; in the case of CK1 and CK2, it is because casein is not a physiological substrate, and in the case of G-CK/Fam20C/DMP-4, it is because casein is just one

out of a plethora of its targets, and a rather marginal one at that. Strikingly, casein kinases altogether, albeit representing a minimal proportion of the whole kinome, appear to be responsible for the generation of up to 40–50 % of non-redundant phosphosites currently retrieved in human phosphopeptides database. In the present review, a short historical explanation will be provided accounting for the usage of the same misnomer to denote three unrelated classes of protein kinases, together with an update of our current knowledge of these pleiotropic enzymes, sharing the same misnomer while playing very distinct biological roles.

CASEIN: AN ARIADNE’S THREAD IN THE LABYRINTH OF PROTEIN KINASES

substrate, was probably not responsible for the physiological phosphorylation of casein in the tissue where this protein is secreted, i.e. the lactating mammary gland. Later, their prediction proved correct. In hindsight, however, we can say that it was resting on two wrong assumptions, namely that the physiological G-CK (genuine casein kinase) is not present in the liver and that casein itself cannot be expressed in tissues other than the mammary gland. Instead, several decades later, it was shown that G-CK is also present in the liver, as well as in other tissues [4], and that casein fractions are expressed in tissues besides the mammary gland (e.g. [5–7]). As outlined in Figure 1, in the two decades following the discovery of protein kinase(s) being able to phosphorylate casein, this activity was shown to be accounted for by two distinct enzyme activities denoted by a number of different names (Supplementary Online Data and Supplementary Table S1 at http://www.biochemj.org/bj/460/bj4600141add.htm). However, they were finally termed CK1 (casein kinase 1, also known as CKI) and CK2 (also known as CKII) to avoid confusion with the increasing number of physiologically relevant regulatory protein kinases which were discovered in the meantime [e.g. phosphorylase kinase, cAMP- and cGMP-dependent protein kinases, and PKC (protein kinase C)] whose implication in signal transduction was becoming evident and whose physiological targets were already partially known.

In the second half of the 19th Century, the findings of Ulaf Hammarsten [1] that phosphorus was present in the milk protein casein, in addition to carbon, nitrogen, hydrogen, oxygen and sulfur, came as a surprise. Henceforth casein became the prototype of a new class of proteins named ‘phosphoproteins’. These were believed for decades to represent a tiny minority of what is termed the proteome today, before it became clear that at least 50 % of all proteins, and probably even more, undergo phosphorylation during their lifespan. The mode of phosphorus binding to casein remained unclear until Fritz Lipmann [2] in 1933 provided evidence that it is esterified to the side chain of serine residues, to give phosphoserine, a post-translational modification shown to also affect threonine and, later, tyrosine residues. The biochemical mechanism by which casein becomes phosphorylated, however, remained a matter of conjecture until 1954, when Burnett and Kennedy [3] were able to isolate from liver an enzyme preparation that catalyses the phosphate transfer from ATP to casein and coined for this novel activity the name, which is still universally employed, ‘protein (phospho) kinase’. In their paper, Burnett and Kennedy [3] argued that the protein kinase activity, which they discovered using casein as an in vitro

Key words: cancer, casein kinase 1 (CK1), casein kinase 2 (CK2), family with sequence similarity 20C (Fam20C), genuine casein kinase (G-CK), neurodegeneration.

Abbreviations: AI, amelogenesis imperfecta; APC, adenomatous polyposis coli; BMP-15, bone morphogenetic protein-15; CK1, casein kinase 1; CK2, casein kinase 2; DDX3, DEAD-box RNA helicase 3; DMP-4, dentin matrix protein-4; Dvl, Dishevelled; ER, endoplasmic reticulum; Fam20, family with sequence similarity 20; FJ, four-jointed; G-CK, genuine/Golgi casein kinase; GSK3, glycogen synthase kinase 3; IUBMB, International Union of Biochemistry and Molecular Biology; LRP, lipoprotein receptor-related protein; MAP-2, microtubule-associated protein-2; MDM2, mouse double minute 2 homologue; MDR, multidrug-resistant; MRP-1, multidrug-resistance protein-1; PER2, PERIOD circadian protein homologue 2; PRP-1, proline-rich phosphoprotein-1; SCPP, secretory calcium-binding phosphoprotein; SIBLING, small integrin-binding ligand N-linked glycoprotein; SPP1, secreted phosphoprotein 1; Tat, transactivator of transcription; TBB, 4,5,6,7-tetrabromo-1H -benzotriazole; UPR, unfolded protein response. 1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]). c The Authors Journal compilation c 2014 Biochemical Society

Biochemical Journal

Andrea VENERANDO*†, Maria RUZZENE*†1 and Lorenzo A. PINNA*†1

www.biochemj.org

Casein kinase: the triple meaning of a misnomer

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Figure 1

A. Venerando, M. Ruzzene and L. A. Pinna

Casein kinases at a glance

Chronology of casein phosphorylation and related enzymes.

By sharp contrast, the endogenous substrates of CK1 and CK2 remained unknown for more than two decades after their discovery and the possible implication of these two kinases in the phosphorylation of casein was a matter of debate. For a while, CK2 was suspected to be responsible for the endogenous phosphorylation of casein because it was shown to be able to re-phosphorylate the same S-S-S-E-E motifs which are entirely phosphorylated in native casein fractions. It was possible to show, however, that CK2 could only phosphorylate the first and second serine residues of the triplets, but not the third one (consistent with its known consensus S-X-X-E/D/pS), whereas a casein kinase preparation partially purified from the Golgi-enriched fraction of lactating mammary gland readily phosphorylated the second and third serine residues of the triplet. Once the third serine residue was also phosphorylated, the first serine residue underwent phosphorylation, consistent with an S-X-E/pS consensus [8,9]. At that stage, it was clear that the name ‘casein kinase’ was shared by at least three independent enzymes (or families of enzymes) displaying the common ability to readily phosphorylate casein, but neatly differing for their consensus sequences, which are shown in Figure 2. This confusing situation led to the proposal, forwarded to the IUB [International Union of Biochemistry; now known as IUBMB (International Union of Biochemistry and Molecular Biology)] Nomenclature Committee in 1994, to name the two ‘false’ casein kinases just after their acronyms, protein kinases CK1 and CK2, keeping the name ‘casein kinase’ for the ‘genuine casein kinase’ especially expressed in the Golgi apparatus of the lactating mammary gland. This was often denoted by the acronym GEF-CK (Golgi-enriched fraction casein kinase), later shortened to G-CK standing for both ‘genuine’ and ‘Golgi’ casein kinase [4], not to be confused with ‘GCK’ (without hyphen), widely used to denote glucokinase and other enzymes. In the meantime, CK1 and CK2 were turning out to be highly pleiotropic protein kinases, implicated in many cellular functions and their genes were discovered and characterized. Whereas the human CK2 holoenzyme is a heterotetramer composed of two catalytic subunits (α and/or α ) and a dimer of a non-catalytic β c The Authors Journal compilation c 2014 Biochemical Society

Figure 2

Consensus sequence of the casein kinases

The phosphorylatable residue(s) are in red and the specificity determinants are underlined. X denotes any residue, but in the CK2 consensus, these are preferably acidic amino acids.

subunit, CK1 makes up a small family of monomeric isoforms (seven in mammals). In the human kinome, CK1 represents an independent group distantly related to CK2, which instead is phylogenetically close to, but definitely distinct from, the CMGC group (Figure 3). Somewhat paradoxically, G-CK is not there in the kinome. Despite several efforts, it remained an ‘orphan’ enzyme for decades after its biochemical characterization, and only very recently has it been identified as a member of a small family of atypical protein kinases, Fam20C (family with sequence similarity 20C) [10–12] (Figure 3). Previously, however, it had already been shown to be present in tissues other than the mammary gland [4] and to be not a dedicated enzyme committed to the phosphorylation of casein alone, but a kinase as pleiotropic as its false sisters CK1 and CK2, being particularly responsible for the generation of the largest proportion of the phosphosecretome

Casein kinases

Figure 3

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Casein kinases in the ‘kinome’

CK1, CK2 and G-CK/Fam20C are indicated by red circles. Although CK1 and CK2 are members of the kinome, Fam20C belongs to a small family of proteins (FJ) with an atypical putative kinase domain.

[13] (Figure 4). So, 130 years after the discovery that casein is phosphorylated, we can say that this phosphoprotein has played an essential role in tracking out and characterizing three families of kinases, none of which are dedicated enzymes committed to just the phosphorylation of casein, but which, together, are responsible for the generation of up to 50 % of the known phosphoproteome (Figure 4). What do these kinases share, besides their remarkable ability to phosphorylate casein in vitro? Not so much, as we will discuss in the following sections and can be anticipated considering the lack of close homology (Figure 3) and differences in 3D structures (Figure 5). One common feature, besides extreme pleiotropicity, is the acidic nature of the specificity determinants which define their consensus sequences (Figure 2). These include, besides carboxylic side chains, phosphorylated residues, thus making CK1, CK2 and G-CK/Fam20C all susceptible to hierarchical, or ‘primed’, phosphorylation [14] and, at least in principle, to substrate level regulation. This acidophilic character of casein kinases appears to depend on a number of basic residues which are

responsible for substrate recognition (Figure 5). Another common feature seems to be ‘constitutive activity’, a term, however, which has to be used with great caution. All of the three classes of ‘casein kinases’ are catalytically competent in the absence of phosphorylation events which are instead required to ‘turn on’ the majority of other protein kinases. Their catalytic subunits/domains also appear to be active in the absence of other subunits and/or second messengers. Many devices, however, are already known which can modulate the targeting of substrates by CK1 and by CK2, and there are clues that some kind of regulation also applies to G-CK/Fam20C, whose mode of action as a secreted kinase is unique [15].

THE ‘GENUINE’ CASEIN KINASE: FAM20C/DMP-4 (DENTIN MATRIX PROTEIN-4)

Although the kinase responsible for the in vivo phosphorylation of casein remained an orphan enzyme until very recently, its c The Authors Journal compilation c 2014 Biochemical Society

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Figure 4


Contribution of casein kinases to the generation of the whole phosphoproteome (left-hand panel) and of the phosphosecretome (right-hand panel)

The left-hand panel is reproduced from [190] Springer, Mol. Cell. Biochem. 356, 2011, 5–10, Protein kinase CK2 accumulation in oncophilic cells: causes and effects. Ruzzene, M., Tosoni, K., Zanin, c 2011, Springer Science + Business Media, LLC, with kind permission from Springer Science and Business Media. The right-hand panel is reproduced with S., Cesaro, L., Pinna, L.A., Figure 1, permission from [13] Salvi, M., Cesaro, L., Tibaldi, E. and Pinna, L.A. (2010) Motif analysis of phosphosites discloses a potential prominent role of the Golgi casein kinase (GCK) in the generation of human plasma phospho-proteome. J. Proteome Res. 9, 3335–3338. Copyright 2010 American Chemical Society.

biochemical characterization started much earlier, using partially purified preparations of casein kinase from lactating mammary glands [16–18] and from milk [19]. An important achievement of those early studies was the unambiguous demonstration that the consensus sequence of G-CK was different from those of the other two ‘so-called’ casein kinases, CK1 and CK2, being specified by a glutamic acid or a phospho-serine residue at position n + 2 relative to the target serine residue (S-X-E/pS) [8,9]. This outcome on the one hand confirmed the conclusions of a thorough analysis performed previously on the phosphorylated residues found in the various casein fractions, invariably displaying the S-X-E/pS motif [17], but on the other hand, provided the clear-cut demonstration that, albeit similar, the consensus of protein kinase CK2 (S/T-X-X-E/D/pS) is definitely different, thus allowing the development of highly selective peptide substrates suitable for the specific monitoring of each of the three ‘casein kinases’, the ‘genuine’ one (G-CK) [20], CK1 and CK2 [21] (see Supplementary Online Material and Supplementary Table S2 at http://www.biochemj.org/bj/460/bj4600141add.htm). The G-CK-specific peptide, derived from a β-casein phosphosite (β 28–40 ), proved an invaluable tool for the further characterization of this enzyme, whose gene was still unknown and for which antibodies were not available. Given its extreme specificity, it provided the means to demonstrate for the first time that G-CK activity is not restricted to the mammary gland where casein is secreted, but it is also present in the Golgi of several other tissues, notably the liver, brain, spleen and kidneys [4], and it became the gold standard to prove that the enzymes responsible for the phosphorylation of proteins other than casein at S-X-E motifs were indistinguishable from G-CK. Another breakthrough of these early studies was the demonstration that G-CK is not a dedicated casein kinase, but a pleiotropic enzyme, responsible for the phosphorylation of proteins believed previously to be substrates of CK2, like osteopontin [22], and finally suspected to generate up to 70 % of the phosphosecretome found in serum and in cerebrospinal c The Authors Journal compilation c 2014 Biochemical Society

fluid [13]. This kind of phosphoproteomic analysis was facilitated by the notion that the site specificity of G-CK is not only different from, but also more stringent than, those of the other acidophilic ‘casein kinases’. Notably, unlike CK2, G-CK tolerates only glutamic acid and phospho-serine, but neither aspartic acid nor other phospho-residues as specificity determinants, and its ability to phosphorylate the threonine residue instead of serine is negligible. Remarkably, G-CK proved entirely insensitive to any protein kinase inhibitor tested so far on it, including the most promiscuous one, staurosporine, drastically affecting nearly all kinases in the submicromolar range, but displaying no effect on G-CK at concentrations up to 0.5 mM [23]. Although this argument was put forward to speculate that G-CK might not be a member of the kinome, the demonstration that indeed this is the case did not come until 2012 when Tagliabracci et al. [10] provided the incontrovertible evidence that the G-CK responsible for the phosphorylation of serine residues within the S-X-E/pS motif is Fam20C, also termed DMP-4, a member of a small family of atypical kinases [FJ (‘four-jointed’)] unrelated to the main body of the ‘kinome’ (Figure 3). The same conclusion was also reached independently in two other laboratories, through different approaches [11,12]. Although all secreted members of the FJ family share a putative atypical kinase domain, the ability to catalyse the phosphorylation of serine residues in proteins/peptides has been unambiguously demonstrated only in the case of Fam20C. Fam20B phosphorylates xylose within a glycosaminoglycan– protein linkage region [24], but it seems not to be a bona fide protein kinase, whereas in the case of Fam20A (whose mutations cause AI [amelogenesis imperfecta] and gingival hyperplasia in humans [25,26]) and fjx1 (the human FJ) protein kinase activity has been only hypothesized to explain their biological functions. No functional characterization is available for the other two members of the family, Fam198A and Fam198B.

Casein kinases

Figure 5

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3D structures of ‘casein kinases’

In (A), (B) and (C) the crystal structures of rat CK1δ 317 (PDB code 1CKJ), the α catalytic subunit of human CK2 (PDB code 4MD8) and C. elegans Fam20C (PDB code 4KQB) are shown. The glycine-rich loops are coloured deep purple, whereas the activation loops of CK1δ and CK2 α subunits (lacking in Fam20C) are coloured teal. Basic residues believed to be responsible for substrate recognition are coloured blue and denoted by asterisks. The cap-like structure over the N-lobe of Fam20C is indicated by the bracket. The heterotetrameric structure of the human CK2 holoenzyme (composed by two α subunits, in pink and orange, and a β dimer, in teal and cyan) is shown in (D) (PDB code 4MD8), whereas in (E) the structure of a trimeric form of CK2 holoenzyme [75] is reported.

The conclusion that Fam20C (DMP-4) accounts for the activity designated previously as ‘genuine’ or ‘Golgi’ casein kinase (i.e. G-CK) is corroborated by the observation that casein kinase activity co-migrates in SDS/PAGE with Fam20C [11] and that all the distinctive features of G-CK could be recapitulated using

recombinant Fam20C as a catalyst [10]. Some of these are summarized in Box 1, including a remarkable preference for Mn2 + over Mg2 + as an activator; a unique consensus, S-X-E/pS, where both the replacement of a serine residue with threonine and glutamic acid with aspartic acid are hardly tolerated (in c The Authors Journal compilation c 2014 Biochemical Society

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Box 1 Some distinctive properties shared by the G-CK isolated from the Golgi apparatus of lactating mammary gland (G-CK) and Fam20C Preference for Mn2+ over Mg2 + Identical consensus (S-X-E/pS) Ability to phosphorylate the specific peptide substrate β 28–40 Insensitivity to staurosporine Ability to phosphorylate many secreted proteins including SPP1, PRP-1 and BMP-15 Ability to recognize the atypical consensus surrounding PRP-1 Ser22 Susceptibility to stimulation by sphingosine

contrast with what generally happens with other serine/threonine kinases); striking insensitivity to staurosporine at concentrations up to 0.5 mM; and the ability to phosphorylate an atypical site on the salivary protein PRP-1 (proline-rich phosphoprotein-1) (Ser22 ).

Structural aspects

Fam20C, as all the other members of the FJ family, displays a weak sequence similarity to PI4KII (phosphoinositide 4-kinase II) [27], but it is not present in the protein kinase complement of the human genome, the so-called ‘kinome’ [28] (Figure 3). Its kcat value is comparable with, albeit somewhat below, that of CK2, if both kinases are assayed with their specific peptide substrates: 0.08 s − 1 [29] compared with 0.8 s − 1 [30]. The structural features underlying the protein kinase activity of Fam20C have been disclosed recently by solving the crystal structure of the Caenorhabditis elegans homologue of human Fam20C, revealing an atypical protein kinase-like fold [29]. Although the catalytic aspartic acid residue present in all protein kinases, as well as the so-called ‘ion pair’ (formed in Fam20C by Lys192 and Glu218 ), another hallmark of protein kinases, are conserved, the glycine-rich loop is altered and makes contact with a unique insertion domain that displays a novel fold and makes a cap-like structure over the N-lobe (Figure 5). The α-helix C, a key element of the upper lobe of protein kinases, is entirely displaced towards the lower lobe. This feature, in conjunction with the lack of the ‘activation loop’ denotes an architecture that is primed for spontaneous catalysis rather than instrumental to dynamic regulation. Also of note is the conservation across Fam20C of different species of three basic residues close to the active site (Arg314 , Lys319 and Lys320 ) which are not present in Fam20A or in Fam20B (both unable to phosphorylate the S-X-E/pS motif) and which might be implicated in the binding of the phosphoacceptor substrate (Figure 5). Indeed, mutation of Arg314 (Arg408 in human Fam20C) decreases activity towards the canonical peptide substrate [29], suggesting that it interacts with the crucial acidic determinant, i.e. the glutamic acid at position n + 2. Lys319 and Lys320 are instead optimally positioned to facilitate the recognition of atypical sites, like the one found in PRP-1 (Ser22 ) which are specified by multiple acidic residues at positions n + 5, n + 6 and n + 7 [31].

Implication in calcification processes

Before being recognized as a protein kinase, Fam20C was already known to be responsible for Raine syndrome, a deadly osteosclerotic bone dysplasia characterized by ectopic calcification [32–34]. In sporadic non-lethal cases of Raine syndrome, dental abnormalities and hypophosphataemia have been reported [35,36]. Severe tooth and bone anomalies and c The Authors Journal compilation c 2014 Biochemical Society

hypophosphataemia are also associated with loss of Fam20C in mice [37–39]. Also of note is that, although the kinase activity of the Fam20C paralogue Fam20A is still a matter of conjecture and its hypothetical substrates are unknown, mutations of Fam20A are causative of AI and ERS (enamel renal syndrome) in humans [25,26,40,41]. It is tempting therefore to correlate the implication of GCK/Fam20C in biomineralization disorders to its ability to phosphorylate at multiple S-X-E/pS motifs the members of the SCPP (secretory calcium-binding phosphoprotein) family. SCPPs fall into two groups, proline/glutamine-rich and acidic SCPPs [or SIBLINGs (small integrin-binding ligand N-linked glycoproteins)] and once phosphorylated, they display high affinity for Ca2 + and regulate biominaralization [42]. Indeed, one SIBLING, osteopontin [SPP1 (secreted phosphoprotein 1)] provided the first example of a protein other than casein that is phosphorylated by G-CK [22]. Osteopontin and other SIBLINGs are also readily phosphorylated by recombinant Fam20C [10]. The observation that Fam20C mutations responsible for Raine syndrome and other biomineralization disorders impair its catalytic activity [10,12] reinforces the view that its kinase activity is critically instrumental to normal calcification. Although the precise mechanism linking the two events is still unclear, a simplistic scheme such as that shown in Supplementary Figure S1 (at http://www.biochemj.org/ bj/460/bj4600141add.htm) could roughly account for the ossification defects observed upon loss-of-function mutations of Fam20C, with special reference to ectopic calcification and hypophosphataemia: both are expected consequences of defective uptake of Ca2 + by hypophosphorylated SCPPs, resulting in an abnormal rise in Ca2 + concentration and precipitation of HA (hydroxyapatite). This causes a decrease in phosphate concentration in the extracellular fluids.

G-CK/Fam20C targets not directly related to biomineralization

Before its identification with Fam20C, G-CK was already suspected to generate alone the largest proportion (70 % or so) of the ‘phosphosecretome’ (Figure 4). Only a relatively minor fraction of these secreted proteins phosphorylated at S-X-E/pS motifs are Ca2 + -binding molecules directly involved in biomineralization. All of the others can be grouped into different classes according to their biological functions, as summarized in Supplementary Table S3 (at http://www.biochemj.org/bj/460/bj4600141add.htm); of note, the numerousness of growth factors and hormones (with special reference to neuropeptide hormones) and of proteolytic enzymes. Also of historical interest may be the observation that some of the phosphosites generated by G-CK/Fam20C belong to the infancy of studies of protein phosphorylation, preceding the elucidation of the sequence of casein. This is the case of two phosphoserine residues in fibrinogen whose surrounding sequence was elucidated in 1962 [42a] (see Supplementary Table S3). Paradoxically, nearly nothing is known about the biological meaning of these phosphorylations and their possible regulatory function. Only in rare cases has phosphorylation of these proteins at S-X-E motifs, presumably by G-CK/Fam20C, been related to physiopathological events. One example is provided by the phosphorylation of aquaporin 2 at Ser256 , whose failure caused by the Glu258 to lysine mutation (preventing Ser256 phosphorylation by G-CK) is responsible for the dominant form of nephrogenic diabetes insipidus [43]; another by the oocytesecreted BMP-15 (bone morphogenetic protein-15) and GDF-9

Casein kinases

(growth differentiation factor-9), whose phosphorylation by GCK [44] has been shown to regulate the bioactivity of these growth factors. On the other hand, the large number of neuropeptide hormones among G-CK/Fam20C targets looks intriguing, and suggests the implication of this kinase in neuroregulation. Pertinent to this may be the observation that defective phosphorylation of salivary proteins at residues displaying the G-CK/Fam20C consensus has been correlated with autism spectrum disorders [45].

Modulation of G-CK/Fam20C: a mediator of sphingosine signalling?

G-CK has proved entirely refractory to all protein kinase inhibitors tested so far, no matter how promiscuous. Especially remarkable is its insensitivity to staurosporine at concentrations up to 0.5 mM [23], a concentration expected to suppress the activity of any member of the kinome, as well as to a large panel of flavonoids, several of which are powerful inhibitors of CK2 and CK1 [11]. This probably reflects the unique features of the active site of Fam20C as compared with canonical protein kinase [29] and will require the design of new scaffolds to produce selective inhibitors. On the other hand, it may be argued that, in contrast with other protein kinases whose pathogenic potential (especially in neoplasia) is due to gain of function, the opposite may apply to Fam20C, whose implication in biomineralization disorders, as far as we can say, correlates with loss of function. This would make desirable the availability of up-regulators rather than downregulators of its activity. The only compound reported so far to display this property is sphingosine, which has been shown to increase severalfold the phosphorylation of PRP-1 by G-CK partially purified from lactating mammary gland [31]. Such an effect of sphingosine has been now confirmed using recombinant Fam20C and shown not to be substrate-directed. It is quite evident also using the canonical peptide, casein and BMP-15 as phosphoacceptor substrates; stimulation is accounted for by both increased V max and decreased K m with respect to ATP (while affinity for the substrate remains unchanged). Quite interestingly, sphingosine efficacy is drastically enhanced if Mn2 + is replaced with Mg2 + in the reaction medium (G. Cozza, E. Tibaldi, V.S. Tagliabracci, J.E. Dixon and L.A. Pinna, unpublished work). It is noteworthy that under these conditions, Fam20C behaves categorically as a ‘sphingosine-dependent kinase’, since in the presence of Mg2 + and absence of sphingosine its activity is negligible, whereas in the presence of 25 μM sphingosine and Mg2+ it equals the one observed with Mn2 + . Future challenges will be to assess whether these in vitro observations reflect a physiological situation, where GCK/Fam20C under basal conditions is quiescent, becoming active in response to stimuli, thus promoting a rise in the level of sphingosine or sphingosine-related second messengers. This in turn will also imply a better understanding of the compartment(s) where G-CK/Fam20C displays its activity and of the devices it exploits to selectively target its numerous substrates.

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structures of its catalytic (α) and regulatory (β) subunits were first elucidated in 1987 [46] and their 3D structures, either alone or combined into the holoenzyme, were solved between 1998 and 2001 [30,47,48] (Figure 5). After a pioneering article comparing the properties of ‘multi-potential’ CK1 and CK2 [49], a long series of reviews were published over the last three decades: some of these dealt with CK2 in general (e.g. [50–55]), whereas others addressed specific aspects of CK2, such as its role in cell-cycle regulation [56], the structure of its genes [57], its constitutive activity [58], its extraordinary pleiotropy [59], and its implication in cell survival [60], apoptosis [61], cancer [61–63] and other pathologies [64–66]. Exhaustive repertoires of CK2 inhibitors are also available [67–69]. Recently, a book from the Wiley IUBMB series entirely devoted to CK2 was published [70] in which nearly all structural and functional aspects of this kinase was dealt with. In the present review, we briefly summarize structural and physio/patho-logical aspects with special reference to the most recent breakthroughs and controversial issues. Structural biology of CK2

Native CK2 isolated from animal tissues is a heterotetrameric holoenzyme composed of two catalytic and two regulatory subunits [71]. Two isoforms of the catalytic subunits exist: α and the less represented α . The two isoforms are highly homologous, but coded by two different genes [72], and their relative expression is tissue-specific [73]. The regulatory β subunit plays a role in determining substrate specificity instead of controlling the catalytic activity, i.e. being a constitutively active enzyme. As a result, substrates are classified according to the effect of the β subunit; those that can be not required for phosphorylation (class I substrates), those negatively affecting it (class II substrates) or those that are essential (class III substrate) [54]. This classification is based on in vitro experiments, since the only CK2 form unambiguously identified in vivo so far is the tetrameric holoenzyme. However, some findings suggest the possible occurrence of different aggregation states. An analysis of individual CK2 subunits in living cells revealed dynamic events by which different multimolecular assemblies are possible [74]. Recently, a new crystal structure of CK2 was reported where the β subunit mediates the formation of inactive polymeric structures [75,76] (Figure 5E). If this is confirmed in cells, it would provide an unexpected mechanism of regulation, conferring constitutive activity only to a pool of CK2 molecules which, for some reason, are able to escape the supramolecular organization. An unbalanced expression of regulatory and catalytic subunits has been found to occur in some tumours, and an excess of CK2α over CK2β expression has been correlated to EMT (epithelialto-mesenchymal transition) [77]; moreover, a higher amount of α subunit compared with β subunit has been found in an MDR (multidrug-resistant) leukaemia cell line, where it seems to contribute to the apoptosis-resistant phenotype [78]. It is therefore conceivable that the paradigm of a very stable and constitutively active enzyme, undeniable in vitro, may undergo exceptions in vivo with pathological relevance.

THE TWO ‘PSEUDO’ CASEIN KINASES: CK1 AND CK2 Protein kinase CK2

Physio/patho-logical roles of CK2

In contrast with G-CK (or Fam20C), which belongs to a small family of atypical kinases, both CK1 and CK2 are bona fide members of the ‘kinome’ where they are positioned in distantly related branches (Figure 3). Although CK1 and CK2 were detected very early together, the biochemical and structural characterization of CK2 preceded that of CK1. The primary

As outlined above, CK2 phosphorylates an extraordinarily high number of substrates at sites specified by acidic determinants, mostly fulfilling the S/T-X-X-E/D/pS/pY consensus (Figures 2 and 3). Paradoxically, such pleiotropicity has delayed the discovery of a general physiological function instead of facilitating it, and only recently has a major role been identified c The Authors Journal compilation c 2014 Biochemical Society

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which recognizes this kinase as a global antiapoptotic agent. Indeed, among the many CK2 targets, several proteins have been demonstrated to promote proliferation and/or counteract apoptosis when phosphorylated by CK2. This implies that CK2 is heavily involved in regulating the cell death/survival ratio, whose unbalance is a peculiar feature of cancer cells. Moreover, CK2 affects other hallmarks of cancer, being able to favour angiogenesis and cell migration, inactivating tumour suppressors, and enhancing drug resistance, as reviewed previously [63]. Consequently, CK2 is generally more abundant in tumour cells than in the healthy counterparts [79], and, for all of these reasons, it is considered a promising target for cancer therapy. Several inhibitors have been developed for CK2, with variable potency and selectivity (see the following section), and one compound, CX-4945, has entered clinical trials [80]. Interestingly, tumour cells display a higher sensitivity to CK2 inhibition than do normal cells, and this is interpreted as a consequence of the ‘addiction’ of cancers to CK2 [63]. In other words, normal cells are already used to relatively low levels of CK2 activity, and although during embryogenesis this enzyme is essential for normal cell proliferation/survival [81], in adult cells its reduction is not so critical. On the contrary, the survival of cancer cells relies more on a given CK2 level, not only because this is particularly high, but also because it sustains and potentiates a number of signalling pathways which ensure cancer cell survival, and which are specific and/or hyperactivated in cancer. In this sense, CK2 is considered a lateral player, meaning that it acts in a figurative ‘horizontal’ way on ‘vertical cascades’, since it is constitutively active and ready to co-operate with different signalling often triggered by external stimuli. In tumour cells, where one or a few of these pathways are pathologically activated even in the absence of the signal, a higher CK2 level is an added value for tumour cell survival and propagation. Recently, a new additional site of action has been proposed to explain the antiapoptotic role of CK2, namely its protection from ER (endoplasmic reticulum) stress-induced apoptosis. Consequently, CK2 inhibition is associated with an alteration of the UPR (unfolded protein response) signalling pathway which generates an apoptotic signal [82–84]. This involvement of CK2 in UPR signal may have relevance not only in cancer, but also in neurodegenerative pathologies, where the accumulation of abnormal proteins produces ER stress. The implication of CK2 in regulating the cellular level of many proteins has been known for several years. CK2 controls protein synthesis, with many transcription factors being among its substrates [59]. It can also affect protein degradation, often exerting a protective action, such as in the case of caspase substrates [85], β-catenin [86] or PTEN (phosphatase and tensin homologue deleted on chromosome 10) [87], but in other cases promoting cleavage, such as for the NF-κB (nuclear factor κB) inhibitor IκB [88]. CK2 also has an inhibitory function on proteasome gene expression and activity, exerted by phosphorylating the transcription factor Nrf1 (nuclear factorerythroid 2-related factor 1) [89]. Very recent reports are disclosing a special role for CK2 at the RNA level. For example, it has been found that CK2 increases rRNA synthesis, by phosphorylating the TIF-IA (transcription initiator factor IA) [90]. Interestingly, to do that, CK2 needs to be phosphorylated at Thr13 by Akt (also known as protein kinase B). Since we had demonstrated previously that CK2 phosphorylates and activates Akt [91,92], this new finding adds further complexity to the multifaceted network of connections between these two kinases, suggesting that they mutually potentiate each other for a maximal amplification of their survival signalling. A site of action for CK2 at the RNA level has also been suggested by a recent c The Authors Journal compilation c 2014 Biochemical Society

phosphoproteomic screening of CK2 substrates, among which several proteins belonging to the spliceosomal machinery have been identified, indicating CK2 as a regulator of the spliceosome functions [93]. CK2 as a drug target

As a consequence of the pro-survival action ubiquitously played by CK2 and its alleged essential role, for a long time its targeting was not considered a feasible therapeutic strategy. However, its emerging involvement in supporting different pathologies, first of all cancer, has prompted studies of cellular treatment with specific inhibitors, leading to the observation that its requirement for cell viability is less general than predicted. Several papers have been published reporting that a reduction in CK2 activity induces apoptosis in tumour cells, but is almost ineffective on healthy cells (reviewed in [63]), and this is now explicable by the phenomenon of cancel cell addiction to CK2, as described above. Although TBB (4,5,6,7-tetrabromo-1H-benzotriazole), one of the first CK2 inhibitors used in cell experiments [94], is still largely used, a wide repertoire of compounds is now available, with some of them being much more effective and selective than TBB; they belong to different structural classes, most are ATP-competitive inhibitors, and many have shown efficacy in cells. Their biochemical properties have been already described in recent reviews [67–69]; in the present article, we focus on specific examples of their usage for in vivo studies, with the caveat that offtarget effects of CK2 inhibitors have been amply documented by functional proteomics analyses [95,96], and represent a limitation for the use of these compounds to dissect the physio/pathological implications of this kinase. In these cases, conclusions grounded on usage of individual inhibitors are not reliable, unless corroborated by complementary approaches (e.g. different structurally unrelated inhibitors and/or kinase silencing). From a more empirical standpoint, however, the in vivo efficacy of CK2 inhibitors is worthy to note in any case, regardless of availability of the proof of concept that the observed phenotype alterations are directly mediated by CK2. The first employment of CK2 inhibitors in an animal model was reported by Ljubimov et al. [97] showing that these compounds prevented retinal neovascularization in a mouse model of oxygeninduced retinopathy. Another pioneering study of CK2 inhibitor administration in animals employed TBB and DRB (5,6-dichloro1-β-D-ribofuranosylbenzimidazole) in several pain models [98], whereas in the study by Kim et al. [99], brain injections of TBCA [(E)-3-(2,3,4,5-tetrabromophenyl) acrylic acid] revealed a relationship between CK2 inhibition and superoxide anion production. However, the majority of the studies have been performed to demonstrate the efficacy of CK2 inhibition in murine xenograft cancer models. They were initially performed with TBB or its derivatives, such as in [82,100] or with different classes of compounds such as ellipticine derivatives [101], or haematein [102], but a great impulse to the field came from the discovery, and later the commercialization, of a new compound, CX-4945, launched by Cylene Pharmaceutical as the first CK2 inhibitor to enter clinical trials in humans [80,103]. Since then, many studies have shown the efficacy of this compound in reducing tumour size, mostly in murine xenograft models (e.g. [84,104,106,107]). As far as human experimentation is concerned, at present, CX-4945 has been reported to be safe, bioavailable, possess a good pharmacological profile without significant toxicity [108] and successfully pass Phase I clinical trials (http://www.senhwabiosciences.com). A very intriguing case is provided by CIGB-300, the only other CK2-related compound undergoing investigation in clinical

Casein kinases

trials. This is a cyclic peptide (formerly known as P15), originally identified from the screening of a random cyclic peptide phage display library for its ability to prevent the phosphorylation of papillomavirus E7 protein by CK2 [109]. This peptide, once fused to the cell-penetrating HIV-derived Tat (transactivator of transcription) peptide, gave rise to the compound termed CIGB300, which is able to induce apoptosis in different tumour cell lines and to reduce solid tumour growth in mice [110]. The human trials of CIGB-300 have started for the treatment of patients with cervical malignancies, and it has been reported as safe, well tolerated and able to produce signs of clinical benefit [111,112]. Moreover, it has been found to inhibit cell migration and angiogenesis [113]. Despite all of these encouraging results, the mechanism of action of CIGB-300 is still poorly understood. Although initially defined as a CK2 inhibitor, preliminary data [114– 116] indicate that in vitro CK2 activity (assayed with a variety of substrates) is not inhibited by CIGB-300, the only known exception being provided by an eIF2β (eukaryotic initiation factor 2β)-derived peptide which requires the presence of CK2β to be phosphorylated by CK2α [117]. Consequently, the CIGB-300 targets identified in cells are only a few specific CK2 substrates, such as the nucleolar protein B23/nucleophosmin [118], which is also responsible for its nucleolar localization [119]. It should be noted, moreover, that both in vitro and in cell effects of CIGB-300 are strictly dependent on the fusion between the P15 and Tat sequences, being the two individual segments of CIGB-300 almost ineffective alone [120]. Taken together, these observations indicate that the mode of action of CIGB-300 may be more complicated than expected assuming just a substratedirected CK2 inhibition, and that, in view of its promising clinical effects, a detailed investigation is still required in order to rationalize and optimize its therapeutic employment. Targeting CK2 may also represent a valuable strategy to overcome drug resistance since CK2 inhibitors are exploitable in order to induce death in apoptosis-resistant cells [78,121,122], where they also facilitate drug uptake and accumulation inside the cell. This is expected, at least in MDR cells overexpressing efflux pumps such as P-gp (permeability glycoprotein) and MRP-1 (multidrug-resistance protein-1), since these proteins are CK2 substrates [123,124] whose direct regulation by CK2 has been reported [124]. Moreover, several examples of combined treatments of CK2 inhibitors and conventional anti-tumour drugs have been reported, where the drug doses required to induce cell death are significantly lowered by CK2 inhibition (e.g. [78,82,103,104,122,125–130]). Interestingly, the sensitivity to interferon-β as an antiviral factor was also found to be increased by CK2 inhibitors [131]. Besides chemical inhibitors, other strategies have been proposed and experimented to block CK2 activity, such as the administration of antisense oligonucleotides towards CK2α and CK2α , which proved effective in reducing tumour size in mice xenograft models [132]. As far as the delivery strategy is concerned, in the case of oligonucleotides, but also for inhibitors, the usage of nanocapsules has been exploited to specifically target the CK2-blocking agents in cancer cells [133,134]. Protein kinase CK1

In contrast with CK2, which for a while was suspected to coincide with the ‘genuine’ casein kinase because it is able to phosphorylate serine residues that are also phosphorylated in native casein, CK1, whose phosphorylation of casein is primed by phosphoresidues, affects serine residues which are not phosphorylated in the native protein substrate and fulfil the pS-X-X-S primed

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canonical consensus shown in Figure 2. Therefore the ‘casein kinase’ activity of CK1 is a serendipitous event, entirely relying on pre-existing phospho-serine residues and it disappears if casein is dephosphorylated. The genetic and molecular characterization of CK1 was somewhat slower than that of CK2, the first primary structures of CK1 isoforms having been elucidated in 1991 [135], revealing a number of peculiarities with respect to protein kinases whose sequences were already known, CK2 included. Together with TTBK (tau tubulin kinase), and VRK (vacciniarelated kinase) families, CK1 forms a small distinct group within the kinome (Figure 3). CK1 can be shortly defined as a monomeric, constitutively active, ubiquitously expressed and second-messenger-independent serine/threonine protein kinase that is evolutionarily conserved within eukaryotes. In mammals, CK1 is present with seven isoforms (α, β, γ 1 , γ 2 , γ 3 , δ and ε), and their related alternative splicing variants, encoded by distinct genes. All CK1 isoforms share a highly conserved kinase domain of approximately 300 amino acids that differs from most other protein kinases for the presence of the sequence S-I-N instead of A-P-E in the kinase subdomain VIII [136]. Outside the kinase domain, CK1 family members display little homology with each other and differ in length and amino acid composition of their Nand C-termini. The highly variable C-terminal extensions have been implicated in both subcellular targeting and regulation of the activity [137,138]. Although the crystal structure of enzymes belonging to the δ (PDB codes 1CKI and 1CSN) and γ (PDB codes 2CMW, 2C47 and 2CHL) isoforms have been solved [139,140], revealing the common 3D bilobal structure of protein kinases (Figure 5A), structural information for CK1α is not yet available. CK1δ and CK1ε give rise to the so-called δ/ε subfamily characterized by a long C-terminal tail responsible for regulating its own enzyme activity. CK1α is closely related to the δ/ε subfamily with a shorter C-terminal domain that has been demonstrated to undergo autophosphorylation [138]. The autophosphorylation of the C-terminal region of CK1α, CK1δ and CK1ε inhibits the activity of the kinase domain, although, in vivo, phosphatases keep it constitutively active in many cases [137,138]. The third and most remote subfamily is formed by the CK1γ subfamily characterized by the ability of its members to anchor to the plasma membrane through their unique C-terminal palmitoylation sites [141]. The CK1 family falls in the category of ‘acidophilic’ protein kinases because substrate recognition is often specified by negatively charged side chains, with special reference to phosphoserine, abundant in casein fractions. Indeed, early studies mostly performed with casein and synthetic substrates revealed that CK1 behaves as a phosphate-directed protein kinase able to phosphorylate with high efficiency serine/threonine residues displaying the consensus sequence pS/pT-X-X-S/T, also referred to as the ‘primed’ canonical consensus of CK1 (Figure 2), which would imply a mechanism of ‘hierarchical phosphorylation’ [14]. However, it soon became apparent that CK1 does not phosphorylate only primed substrates, rather it often acts as a priming kinase itself, its intervention being required to generate the consensus sequence for other phosphate-directed kinases [e.g. GSK3 (glycogen synthase kinase 3)]. Although in some instances, clusters of acidic residues can replace phosphoresidues as CK1 specificity determinants [21,142], scrutiny of sites targeted by CK1 discloses a complex situation where the CK1 family members are also able to phosphorylate ‘noncanonical’ sites specified by different and sometimes elusive local determinants generally in combination with remote recognition elements. c The Authors Journal compilation c 2014 Biochemical Society

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Especially telling is, in this respect, the multi-phosphorylation by CK1 of the transcription factors family NF-AT (nuclear factor of activated T-cells) [143], β-catenin [144,145], APC (adenomatous polyposis coli) [146] and p53 [147] providing examples of proteins devoid of canonical consensus sequences (either primed by previous phosphorylation or specified by acidic clusters upstream) and nevertheless readily phosphorylated by CK1 through a recognition mechanism relying on both remote docking sites and local atypical specificity determinants. It should be noted, however, that if the pLogos [148] drawn from CK1α and CK1δ phosphosites available in the database are considered (Supplementary Figure S2 at http://www. biochemj.org/bj/460/bj4600141add.htm), the contribution of canonical consensuses becomes quite evident; these are revealed by the positive selection of glutamic and aspartic acid residues at upstream positions (especially evident at positions n−1, n−2, n−4, n−5 and n−6 in the CK1δ pLogo) and of the serine residue at position n−3, and more in general recurrent within S-X-X-S motifs. This latter feature, especially remarkable in the CK1α pLogo, is probably reflecting the canonical primed consensus of CK1 (pS-X-X-S), where the priming phospho-residue (at position n−3) has lost its phosphate due to protein phosphatases acting before MS analysis of the sample. A more comprehensive and detailed phosphoproteomic analysis will allow the assessment of the validity of this interpretation. If it proves correct, we must assume that up until now the relevance and the frequency of primed phosphorylation events catalysed by CK1 have been underestimated. A kinase in need of control

CK1 enzymes have been implicated in a variety of cellular events, including chromosome segregation, spindle formation, circadian rhythm, nuclear import, Wnt pathway and apoptosis [149,150]. Deregulation of CK1 isoforms has been described in neurodegenerative and sleeping disorders [151], and in cancer [152]. Although in vitro CK1 behaves as a constitutively active enzyme, its activity in cells is susceptible to several mechanisms of regulation such as inhibitory autophosphorylation (see above), proteolytic cleavage of the C-terminal tail, subcellular localization and compartimentalization. Stimulation of cells by insulin or viral transformation, as well as treatment with topoisomerase inhibitors or γ -irradiation, leads to elevated CK1 activity and/or protein levels [153–155]. On the contrary, increased membrane concentration of phosphatidylinositol 4,5-bisphosphate reduces CK1α activity in erythrocytes and neuronal cells [156]. These studies have prompted the development of molecules capable of modulating the activity of this family of kinases. To date, different chemical families of ATP-competitive smallmolecule inhibitors of CK1 are available [151] whose caveats and limits for in vivo studies are similar to those outlined for CK2 (see above). The first CK1-specific inhibitor characterized was the quinoline CKI-7, an ATP-competitive molecule unable to discriminate between CK1 isoforms [157]. Later, the oxoindole IC261 displayed a remarkable isoform specificity against α compared with δ/ε isoforms and good cell permeability. This compound induces conformational changes thus stabilizing the kinase– inhibitor complex [158]. Another tool was provided by the imidazole scaffold. PF670462 and PF-4800567 from Pfizer, two potent CK1ε inhibitors with IC50 values in the nanomolar range and quite selective kinase profiles have been shown to alter circadian rhythm in c The Authors Journal compilation c 2014 Biochemical Society

rodent models [159]. D4476, another imidazole derivative, is a highly specific ATP-competitive CK1δ inhibitor recommended for cell-based assay [160]. In a previous study, other imidazole derivatives have been found to be potent CK1 inhibitors [161]. By using a virtual screening approach, the aminoanthraquinone scaffold was also identified as a versatile structure for the design of new and specific CK1 inhibitors [162]; in particular, the ATP-competitive 1,4-diaminoanthraquinone displayed a marked selectivity against CK1δ with a potency 5-fold higher with respect to IC261. If inhibition of CK1 isoforms represented a useful strategy for treating pathologies caused by CK1 overexpression, the possibility of up-regulating CK1 was also explored as a pharmacological tool. Pertinent to this is the controversial case of pyrvinium pamoate, a well-known FDA (U.S. Food and Drug Administration)-approved anthelmintic drug which, during a high-throughput screening of small molecules, was reported to be a potent inhibitor of the Wnt pathway due to its ability to act as an allosteric activator of CK1α [163]. Consequently, pyrvinium pamoate has been proposed and used as a tool for assessing the implication of CK1 in a number of cell functions [150,164,165]. However, any direct effect of this molecule on the catalytic activity of all CK1 isoforms has been subsequently ruled out, its inhibition of Wnt signalling probably being accounted for by an indirect effect on Akt activation [166]. Subcellular localization and compartimentalization appear to be critical for CK1 functionality. As a result of its sequestration to specific intracellular structures or protein complexes, the kinase can join specific pools of substrates and/or modulators [167– 169]. A very telling example is provided by CK1 participation in the Wnt pathway. In the absence of Wnt ligands, βcatenin is progressively phosphorylated by CK1 and GSK3β that take part with APC, Dvl (Dishevelled) and PP2A (protein phosphatase 2A) in a multiprotein complex (the ‘destruction complex’) promoting ubiquitination and proteasome degradation of β-catenin. Whereas, Wnt agonists bind to Frizzled–LRP5/6 (lipoprotein receptor-related protein 5/6), the LRP co-receptor is phosphorylated by CK1, thus recruiting axin and β-catenin to the membrane, resulting in the dissociation of the various components of the destruction complex [149,150,170]. In the same way, CK1, as well as GSK3, may play opposite roles: at the membrane level (LRP phosphorylation) it acts as an agonist, whereas in the destruction complex it is an antagonist of Wnt signals [170]. This highlights the crucial relevance of micro-compartmentalization in determining CK1 functions. Although it is generally held that CK1 family members are constitutively active enzymes, recently the DDX3 (DEAD-box RNA helicase 3) has been identified as a regulatory subunit of CK1 in the Wnt/β-catenin network [171]. DDX3 acts at the level of Dvl that is required for LRP6 activation. DDX3 promotes Dvl2 phosphorylation not only by recruiting CK1ε to Dvl2 cytoplasmic oligomerization sites (punctae), but also by increasing CK1ε activity. In connection with that, signalosome formation is enhanced thereby protecting β-catenin from degradation. In vitro DDX3 stimulates the activity of different CK1 isoforms by decreasing the K m with respect to ATP, independently of the CK1 C-terminal inhibitory autophosphorylation. Pathological implications of CK1

Implication of CK1 in neurodegenerative disorders dates back to 1984 when a CK1 preparation was reported to be responsible for the incorporation of up to 40 phosphate groups into MAP-2 (microtubule-associated protein-2) [172].

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More recently, alterations of CK1 homoeostasis have been related to several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseaeses (CK1δ isoform) [151], the familial advanced sleep phase syndrome (CK1δ and CK1ε isoforms) [173], but also to infectious diseases [174], such as hepatitis C (CK1α isoform) [175] and leishmaniasis [176,177], and to cancer (CK1α, CK1δ and CK1ε isoforms) [150,152]. In the brain, different CK1 isoforms are present and regulate synaptic transmission mediated by NMDA (N-methylD-aspartate)-type glutamate receptors. CK1δ and CK1ε are key components of the circadian rhythm, the innate clock that grants adaptation of the organism to the daily cycle, since they phosphorylate PERIOD proteins on multiple sites promoting their degradation [178]. Therefore mutations of CK1 or of its targets are related to altered rhythm. For instance, mutation of Ser622 , the phosphosite that primes multiple phosphorylation of human PER2 (PERIOD circadian protein homologue 2) protein by CK1, is causative of familial advanced sleep phase syndrome [173]. In patients with Alzheimer’s disease, the CK1δ mRNA level is >30-fold higher than normal [179] and β-amyloid protein, the major component of cerebrovascular and plaque amyloid in Alzheimer’s disease, stimulates CK1 activity, contributing to the overall abnormal protein phosphorylation observed in this disorder [180]. Pertinent to this is the ability of CK1 (in particular, its δ isoform) to hyperphosphorylate tau protein causing a conformational change that alters the binding of tau to microtubule destabilizing microtubule network [181]. Lewy bodies, typical of Parkinson’s disease, are mainly composed of α-synuclein, whose phosphorylation by CK1 has been reported to regulate the propensity to form fibrils, although the mechanism remains unclear [182]. In addition to the crucial implication of CK1 in neurodegenerative diseases, a link between CK1 and tumorigenesis has been also outlined. Altered expression or activity of CK1 family members have been described in several different tumours: in renal cell carcinoma, the expression of CK1γ 3 is increased [183], whereas CK1δ/ε have been implicated in colon and pancreatic cancer progression [184]; moreover, CK1ε decreased expression and high frequency mutations appear to be involved in mammary carcinogenesis [149]. Inactivation of p53 tumour suppressor functions is a common feature of many human cancer cells. CK1α and CK1δ have been reported to phosphorylate p53 at sites involved in the interaction with its negative regulator MDM2 (mouse double minute 2 homologue) [147,185,186]. Of note is that MDM2 itself is phosphorylated by CK1δ/ε within its acidic domain. Therefore CK1 contributes directly and indirectly to the down-regulation of p53. Also, the implication of CK1 in the Wnt/β-catenin pathway, where it participates both as a positive and negative regulator (see above), highlights the relevance that dysregulation of this kinase may have in tumorigenesis. Furthermore, CK1 has been reported to have the potential to increase metastatic processes as it promotes the interaction of nm23-H1 (non-metastatic clone 23-H1; or nucleoside diphosphate kinase A) with h-prune in breast cancer models [187], whereas silencing of CK1 δ/ε prevents TGFβ (transforming growth factor β)-induced metastases [188].

in the detection and characterization of three classes of protein kinases responsible altogether for the generation of the largest proportion of the phosphoproteome. Only one of these recently identified with an atypical protein kinase of the FJ family, Fam20C, already known to be responsible for Raine syndrome, is the genuine casein kinase committed with the phosphorylation of casein, as well as of a plethora of other secreted proteins. These include many calcium-binding proteins implicated in biomineralization, but also proteolytic enzymes, neuropeptides, growth factors and hormones, suggesting that abnormal expression and/or functionality of G-CK/Fam20C may be implicated in an ample spectrum of pathologies, besides calcification disorders. It is expected that investigation in the field will undergo a burst similar to, but more abrupt than, that occurred with CK2 and CK1 in the two decades just gone. Most of the current interest in CK1 and CK2 is fostered by their implication in global diseases, with special reference to neoplasia and neurodegeneration. Considered for a long time as ‘undruggable targets’ due to their pleiotropicity and purported essential role, it later became clear that, at least in the case of CK2, pharmacological down-regulation with beneficial effects in a wide range of tumours is a realistic goal. In the case of the implication of CK1 in neoplasia, it is not so clear-cut due to the existence of various isoforms with possibly different, if not opposite, roles in malignancy. Rather, it is expected that isoformspecific targeting of CK1 will become an appealing strategy for the treatment of neurodegenerative disorders. At variance with CK1 and CK2, which are both typical members of the kinome, the discovery of a member of the FJ family as the biochemical entity responsible for the genuine casein kinase activity discloses entirely new prospects in the field of protein phosphorylation, providing the unprecedented paradigm of a secreted kinase. In fact, it is a matter of debate whether Fam20C phosphorylates its numerous substrates concomitantly with their common secretion in the lumen of the Golgi apparatus or whether it may also operate once entirely released into the extracellular fluids. It is hard to figure out how its active form, generated in the Golgi lumen after cleavage of the signal peptide, could traffic into other cellular compartments. This deprives Fam20C of one of the most powerful devices governing kinase specificity, i.e. variable subcellular localization [189]. Consequently, a number of questions arise, concerning the mode of regulation of this pleiotropic secreted protein kinase, how it can discriminate among its huge repertoire of targets (all specified at local level by the same S-X-E/pS motif) and whether these phosphorylation events are irreversible or not, implying, if they are not, the concerted intervention of, to date, unidentified protein phosphatase(s). In addition to these basic questions, many other less theoretical and more practical issues need to be addressed concerning the functional consequences of the myriad individual phosphorylation events promoted by Fam20C, the pathologies which could be related to its altered expression/functionality and the development of reagents that are able to affect its activity. On top of this, the history of casein kinases provides a memorable lesson about the importance of curiosity-driven research not only for the advancement of knowledge, but also for addressing practical problems related to human health and wellness.

Conclusion and perspectives

FUNDING

More than 130 years after the discovery that casein is a phosphoprotein, we can say that this protein has been instrumental

We received support from Associazione Italiana per la Ricerca sul Cancro (AIRC) [grant number IG-10312]. c The Authors Journal compilation c 2014 Biochemical Society

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Biochem. J. (2014) 460, 141–156 (Printed in Great Britain)

doi:10.1042/BJ20140178

SUPPLEMENTARY ONLINE DATA REVIEW ARTICLE

Casein kinase: the triple meaning of a misnomer Andrea VENERANDO*†, Maria RUZZENE*†1 and Lorenzo A. PINNA*†1 *Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/b, 35131 Padova, Italy †CNR Neuroscience Institute, Via Ugo Bassi 58/b, 35131 Padova, Italy

NOMENCLATURE OF CASEIN KINASES

The term ‘protein phospho kinase’, later shortened to ‘protein kinase’, was first coined in 1954 to denote the liver enzyme(s) capable of phosphorylating casein in vitro [1]. Later, however, the same name was used to indicate the growing list of proteinphosphorylating enzymes undergoing discovery in the meantime and whose biological functions and endogenous targets were, at least partially, already known, thus rendering the nomenclature of protein kinases an increasingly complicated issue. In this context, those kinases, first detected for their ability to phosphorylate casein, but whose endogenous substrates still were entirely unknown, started being denoted after the artificial substrate used for their in vitro assay; ‘casein’ kinases and, less frequently, ‘phosvitin’ kinases. To discriminate between the two ‘so-called’ casein/phosvitin kinases, different conventions were adopted in the individual laboratories working in the field, referring to, e.g. the ability to use GTP or only ATP as a phosphate donor (casein kinase G compared with A), the generation of either both phospho-threonine and phospho-serine or only phospho-serine in αS1 casein (casein kinase TS compared with S), not to say their ‘rediscovery’ as kinases responsible for the phosphorylation of glycogen synthase (glycogen synthase kinase 5, glycogen synthase kinase 0.7) and troponin-T (troponin-T kinase). This led to a very redundant and confusing nomenclature whose most frequently used synonyms and acronyms are summarized in Table S1. At the end, the most conventional among these criteria, i.e. elution order from DEAE-cellulose, prevailed, leading to the acronyms CK1 (or CKI) and CK2 (or CKII), which are now universally adopted and recommended instead of the full names casein kinases 1 and 2, because neither CK1 nor CK2 is the bona fide casein kinase committed to endogenous casein phosphorylation. This latter was first isolated from a ‘Golgi-enriched fraction’ of lactating mammary gland and thereafter denoted by the acronym GEF-CK, later shortened to G-CK, and only identified recently

1

with the secreted protein Fam20C, also known as DMP-4 (Table S1). A pertinent question would be whether CK1, CK2 and GCK/Fam20C are the only protein kinases able to phosphorylate casein in vitro. The answer is no, since many protein kinases, not only serine/threonine-, but also tyrosine-specific kinase can phosphorylate in vitro casein to some extent, albeit much less efficiently than the three ‘casein kinases’. Although these kinases are nearly inactive on a number of basic proteins often used as artificial substrates of many basophilic and prolinedirected kinases, like MBP (myelin basic protein), histones and protamines, the opposite applies to the other kinases, eventually far preferring basic proteins over casein. A notable exception, however, is provided by some members of the Polo-like kinase subfamily, notably PLK2 and PLK3, which are turning out to be acidophilic in nature and able to readily phosphorylate casein in vitro ([2] and references therein). Nevertheless, for historical reasons, PLKs were never included in the heterogeneous group of ‘casein kinases’. DISTINCTIVE PROPERTIES OF INDIVIDUAL CASEIN KINASES

Whenever a crude biological preparation readily phosphorylates casein, it may be desirable to establish which kind of casein kinase is responsible for such an activity. This can be easily accomplished taking advantage of a number of unique properties, as summarized in Table S2. In particular, CK2 is the only casein kinase able to use GTP instead of ATP as a phosphate donor. In addition, GCK/Fam20C displays a unique preference for Mn2 + over Mg2 + and is insensitive to 100 μM staurosporine, which is sufficient to suppress the activity of CK1 and CK2, although both are more refractory to staurosporine than the majority of other kinases. Furthermore, for each casein kinase, specific peptide substrates have been developed which are unaffected by the other two as well as, to the best of our knowledge, any other protein kinase.

Correspondence may be addressed to either of these authors (email [email protected] or [email protected]). c The Authors Journal compilation c 2014 Biochemical Society


Figure S1

Hypothetical mechanism accounting for ossification disorders caused by defective activity of Fam20C

The potential of SCPPs to bind large amounts of Ca2 + destined to biomineralization processes is dependent on their phosphorylation at multiple sites (several dozen or even more) by Fam20C. The consequences of suppressing Fam20C kinase activity are shown in red. SCPPs stop sequestering Ca2 + whose concentration rises, causing the precipitation of HA and a decrease in Pi . This in turn promotes ectopic calcification and hypophosphataemia, both often observed in biomineralization disorders.


Casein kinases

Figure S2

pLogos generated from the sites phosphorylated by (A) CK1α (103 sites) and (B) CK1δ (52 sites)

Constructed by Luca Cesaro (University of Padova) with the data available in PhosphoSitePlus® (http://www.phosphosite.org) [3] by using the pLogo program (http://plogo.uconn.edu) [4].


A. Venerando, M. Ruzzene and L. A. Pinna Table S1 kinases’

Synonyms/acronyms which have been used to denote ‘casein

For details see the Nomenclature of casein kinases section and [5,6]. NI, nuclear I; NII, nuclear II. Casein kinase

Synonyms/acronyms

Protein kinase CK1

Casein/phosvitin kinase-A, casein/phosvitin kinase-S, glycogen synthase (casein) kinase-1, cyclic nucleotide and Ca2 + -independent protein kinase NI Casein/phosvitin kinase-G, casein/phosvitin kinase-TS, glycogen synthase kinase 5, glycogen synthase kinase 0.7, troponin-T, Cyclic nucleotide and Ca2 + -independent protein kinase NII GEF-CK, G-CK, Fam20C and DMP-4

Protein kinase CK2

Genuine casein kinase

Table S2

Biochemical criteria useful for the fast identification of individual casein kinases

The specific peptide substrates were designed to be specifically phosphorylated only by one class of ‘casein kinase’ and for being assayed by the phosphocellulose paper procedure [7]. Underlined residues indicates residues that are undergoing phosphorylation.

CK1 CK2 Fam20C/G-CK

Co-substrate(s)

Preferred activator

Staurosporine (100 μM)

Specific peptide substrate

ATP ATP and GTP ATP

Mg2 + Mg2 + Mn2 +

Inhibited Inhibited Unaffected

RRKDLHDDEEDEAMSITA RRRADDSDDDDD KKIEKFQSEEQQQ

Table S3 A selection of secreted proteins not related to biomineralization which are phosphorylated at the S-X-E/pS motifs, grouped according to their function The data are taken from [8] unless indicated otherwise. ACTH, adrenocorticotropic hormone; GDF-9, growth differentiation factor-9; PCSK9, proprotoin convertase subtilisin/kexin type 9. Function

Protein(s) phosphorylated

Storage proteins Proteases

Casein and ovoalbumin [9] Fibrinogen [10], PCSK9, coagulation Factor IX and pepsin BMP-15, GDF-9 [11] and gastrin Secretogranins 1 and 2, proenkephalin A, ACTH, 7B2, chromogranin A, corticotropin [12] and melanotropin [12] Amyloid precursor protein

Growth factors/hormones Neuropeptide hormones

Precursor protein


Casein kinases REFERENCES 1 Burnett, G. and Kennedy, E. P. (1954) The enzymatic phosphorylation of proteins. J. Biol. Chem. 211, 969–980 PubMed 2 Salvi, M., Trashi, E., Marin, O., Negro, A., Sarno, S. and Pinna, L. A. (2012) Superiority of PLK-2 as α-synuclein phosphorylating agent relies on unique specificity determinants. Biochem. Biophys. Res. Commun. 418, 156–160 CrossRef PubMed 3 Hornbeck, P. V., Kornhauser, J. M., Tkachev, S., Zhang, B., Skrzypek, E., Murray, B., Latham, V. and Sullivan, M. (2011) PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 40, D261–D270 CrossRef PubMed 4 O’Shea, J. P., Chou, M. F., Quader, S. A., Ryan, J. K., Church, G. M. and Schwartz, D. (2013) pLogo: a probabilistic approach to visualizing sequence motifs. Nat. Methods 10, 1211–1212 CrossRef PubMed 5 Pinna, L. A. (1994) A historical view of protein kinase CK2. Cell. Mol. Biol. Res. 40, 383–390 PubMed 6 Pinna, L. A., Meggio, F. and Marchiori, F. (1990) Type-2 casein kinases: general properties and substrate specifity. In Peptides and Protein Phosphorylation (Kemp, B. E., ed.), pp. 145–169, CRC Press, Boca Raton

7 Glass, D. B., Masaracchia, R. A., Feramisco, J. R. and Kemp, B. E. (1978) Isolation of phosphorylated peptides and proteins on ion exchange papers. Anal. Biochem. 87, 566–575 CrossRef PubMed 8 Tagliabracci, V. S., Pinna, L. A. and Dixon, J. E. (2013) Secreted protein kinases. Trends Biochem. Sci. 38, 121–130 CrossRef PubMed 9 Henderson, J. Y., Moir, A. J., Fothergill, L. A. and Fothergill, J. E. (1981) Sequences of sixteen phosphoserine peptides from ovalbumins of eight species. Eur. J. Biochem. 114, 439–450 CrossRef PubMed 10 Blombäck, B., Blombäck, M., Edman, P. and Hessel, B. (1962) Amino acid sequence and the occurrence of phosphorus in human fibrinopeptides. Nature 193, 883–884 CrossRef 11 Tibaldi, E., Arrigoni, G., Martinez, H. M., Inagaki, K., Shimasaki, S. and Pinna, L. A. (2010) Golgi apparatus casein kinase phosphorylates bioactive Ser-6 of bone morphogenetic protein 15 and growth and differentiation factor 9. FEBS Lett. 584, 801–805 CrossRef PubMed 12 Browne, C. A., Bennett, H. P. J. and Solomon, S. (1981) Isolation and characterization of corticotropin- and melanotropin-related peptides from the neurointermediary lobe of the rat pituitary by reversed-phase liquid chromatography. Biochemistry 20, 4538–4546 CrossRef PubMed

Received 5 February 2014/14 March 2014; accepted 18 March 2014 Published on the Internet 13 May 2014, doi:10.1042/BJ20140178


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Endometrioid carcinoma of the prostate: a misnomer?

Casein Kinase 2--A Kinase that Inhibits Brown Fat Formation.

Purification of casein kinase I and isolation of cDNAs encoding multiple casein kinase I-like enzymes.

"Mental Deficiency": A Misnomer?

The consensus sequences for cdc2 kinase and for casein kinase-2 are mutually incompatible. A study with peptides derived from the beta-subunit of casein kinase-2.

Separation of glycogen synthase kinase 2 from casein kinase.

A synthetic beta-casein phosphopeptide and analogues as model substrates for casein kinase-1, a ubiquitous, phosphate directed protein kinase.

"Side" effects: a misnomer.

Is "long face" a misnomer?

Postpartum psychosis: a valuable misnomer.

A plea to change the misnomer ECT.

Subcellular localization of casein kinase I.

Polyamine binding activity of casein kinase II.

DNA binding activity of casein kinase II.

Canine hemifacial spasm: a misnomer?

Localized mediastinal amyloidosis: A misnomer?

Sporadic cretinism: a dangerous misnomer.

Casein kinase 1 promotes synchrony of the circadian clock network.

Synthetic peptide substrates for casein kinase II.

Aberrant casein kinase II in Alzheimer's disease.

Casein kinase 1δ-dependent Wee1 protein degradation.

Casein kinase II activity in psoriatic epidermis.

Casein kinase II is a predominantly nuclear enzyme.