International Symposium on Neurodegeneration and Neuroprotection
Protein kinase CK2: a newcomer in the ‘druggable kinome’ M.A. Pagano*†, L. Cesaro*, F. Meggio* and L.A. Pinna*†1 *Department of Biological Chemistry and CNR Institute of Neurosciences, University of Padova, viale G. Colombo 3, 35121 Padova, Italy, and †Venetian Institute for Molecular Medicine (VIMM), via Orus 2, 35129 Padova, Italy
Abstract The acronym CK2 (derived from the misnomer ‘casein kinase’ 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n + 3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10–20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.
Introduction: the ‘druggable kinome’ Nearly all aspects of cell life and death are controlled by the phosphorylation of proteins, which is catalysed by protein kinases and reversed by protein phosphatases. The role of protein kinases can be likened to that of interpreters, who translate a variety of signals into biochemical events. To this purpose, protein kinases are often functionally interlinked and highly regulated, forming complex communicative networks. Not surprisingly therefore the deregulation of protein kinases results in cell malfunction, eventually causing neoplastic growth and other diseases. This makes protein kinases attractive targets for drugs to combat cancer as well as several other pathologies, notably diabetes, inflammatory and infectious diseases, stroke, hypertension and neurodegeneration. The attractiveness of protein kinases as targets is enhanced by the fact that they are enzymes, i.e. targetable molecules par excellence, whose biological activity can be turned off easily and precisely by compounds that block the catalytic site. All eukaryotic serine/threonine- and tyrosinespecific protein kinases belong to the largest single family of enzymes (the so-called ‘kinome’) numbering over 500 and accounting for almost 2% of the proteins encoded by the human genome [1]. Although they share similar catalytic domains, these are sufficiently distinctive that it is possible to develop compounds that are selective for a particular or for a few protein kinases. Several protein kinase inhibitors are already in clinical use especially for the treatment of cancers, and many others are undergoing human clinical trials [2,3]. Key words: apoptosis, consensus sequence, druggable kinome, kinase inhibitor, phosphoproteome, protein kinase CK2. 1 To whom correspondence should be addressed (email
[email protected]).
According to a recent analysis [4], the human kinome is the first source of pharmacological targets, possibly accounting alone for >20% of the ‘druggable’ genome. It is generally held that a prerequisite for a kinase being a valuable pharmacological target would be that of being ‘deregulated’ in such a way that, due to mutations or other genetic rearrangements, its activity is constitutively on, even in the absence of specific stimuli. This would exclude from the ‘druggable kinome’ a minority of protein kinases whose constitutive activity is a natural property rather than the outcome of a pathogenic accident. This prediction, however, is likely to be wrong, as exemplified by protein kinase CK2, the prototype of ‘constitutively active’ protein kinases, whose catalytic activity, particularly elevated in a wide variety of tumours and exploited by viruses to phosphorylate proteins that are essential to their life cycle, represents nowadays an enticing target for developing new therapeutic strategies.
The extraordinary pleiotropy of protein kinase CK2 The acronym CK2 is used to indicate a ubiquitous serine/ threonine protein kinase first detected and characterized more than 40 years ago using casein as an artificial in vitro substrate, although it is not committed to casein phosphorylation in vivo. In the human kinome, CK2 occupies a small branch adjacent to, but separate from, the large CMGC subfamily, which includes other cyclin-dependent kinases, MAPKs (mitogen-activated protein kinases) and GSK3 (glycogen synthase kinase 3). Unlike most of the serine/threonine protein kinases which are basophilic or proline-directed enzymes, CK2 recognizes acidic sites generally specified by C 2006
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Figure 1 Substantial contribution of protein kinase CK2 to the generation of eukaryotic phosphoproteomes The phosphopeptides identified in murine embryonic brain (MEB), human colon adenocarcinoma cells (HCA), rat liver (RL) and mouse postsynaptic density preparations (MPDP) were analysed by the presence of phosphoserine/phosphothreonine residues fulfilling the consensus for CK2 (S/T-X1 -X2 -D/E/pS, where X1 is different from proline and X1 /X2 are different from basic residues) (black), other acidophilic/phosphate-directed kinases (grey), proline-directed kinases recognizing the S/T-P consensus (dashed), and other kinases including the basophilic ones (dotted). The number of phosphosites individually found was 470, 215, 296 and 541 in the MEB [8], HCA [9], RL [10] and MPDP [11] databases respectively.
clusters of carboxylic acid and/or pre-phosphorylated side chains downstream from the target residue, with the one at position n + 3 determining the minimum consensus, S/T-XX-E/D/pS. Another peculiar property of CK2 is the usage of GTP as a phosphodonor substrate as good as ATP. However, the most remarkable features of CK2, possibly representing the two sides of the same coin, are on one side constitutive activity and on the other extreme pleiotropy [5–7]. Unlike most protein kinases, which are turned on only in response to specific stimuli, the activity of CK2 catalytic subunits (α and/or α ) is constantly ‘on’ either in the absence or presence of the ‘regulatory’ β-subunits with which they form a heterotetrameric holoenzyme [5,6]. It has been suggested that this may reflect the outstanding pleiotropy of CK2 whose latest repertoire of phosphorylatable targets includes more than 300 proteins implicated in the regulation of many cellular functions, notably gene expression, signal transduction and nucleic acid and protein synthesis [7]. It is likely, however, that this value represents a dramatic underestimate if we scrutinize the sequences of the phosphosites identified in recent phosphoproteome analyses [8–11]. Figure 1 shows that, in four databases collectively including approx. 1522 phosphosites identified in different mammalian tissues, the percentage of sites unambiguously displaying the consensus C 2006
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for CK2 phosphorylation ranges from 18 to 27%, suggesting that CK2 is directly responsible for the generation of a substantial proportion of the eukaryotic phosphoproteome, probably numbering more than 1000 phosphoproteins, assuming that the human phosphoproteome will include 10 000 proteins or so. Note that CK2 alone accounts for this value, whereas the larger proportion (35–58%) of ‘proline-directed’ phosphosites is attributable to dozens of protein kinases sharing the same S/T-P consensus, not to mention the relatively negligible individual contributions of hundreds of basophilic protein kinases that altogether account for the remaining phosphosites (less than 30%). In particular, CK2 accounts for 17.9 and 26.8% of the phosphosites found in the two databases obtained from brain tissue, where CK2 is believed to play particularly important roles [12]. It should be noted that many of these sites belong to proteins that were not previously known to be CK2 substrates, and some play specific functions in neural tissues, such as MAP (microtubuleassociated protein) 1A and 1B, neurofilament medium and light polypeptides, synaptopodin, presenilin 1, survival motor neuron protein and neuron navigator 1. It is expectable that the outstanding pleiotropy of CK2 justifies at least in part its ‘constitutive’ activity. This latter, in turn, is likely to underlie the pathogenic potential of CK2. In fact CK2 has been implicated in several pathologies, including cancer, infectious and cardiovascular diseases and neurodegeneration. In general, the rationale linking CK2 to these pathologies is provided by the nature of some of its natural substrates, which are notoriously implicated in specific diseases. In the case of cancer, however, a striking, albeit coincidental, argument is also the observation that CK2 activity is invariably elevated more in tumour cells than in ‘normal’ cells. Moreover, the oncogenic potential of CK2 catalytic subunits, has been confirmed by transfecting them into cell and animal models where they dramatically enhance the tumour phenotype [13]. An invaluable tool for future studies in this area is provided by a number of recently developed, quite specific, cell-permeant, ATP-site-directed CK2 inhibitors, a few of which are displayed in Table 1. Crystallographic studies in conjunction with mutational analyses have shown that the selectivity of these inhibitors crucially depends on the shape and size of a hydrophobic pocket adjacent to the ATP-binding site, which in CK2 is smaller than in most of the other protein kinases due to a number of unique bulky side chains, generally replaced by smaller ones in the other kinases [19]. Consequently, the double mutant V66A/I174A is much less sensitive than wild-type to a panel of inhibitors [20].
CK2 as a global anti-apoptotic agent A number of structurally unrelated CK2 inhibitors, tested on a variety of cells derived from tumours, including lymphomas, leukaemias, multiple myeloma and prostate carcinoma, display a pro-apoptotic effect which is roughly proportional to their in vitro inhibitory potency [20,21]. The incontrovertible evidence that such a cytotoxic effect is really due to CK2
International Symposium on Neurodegeneration and Neuroprotection
Table 1 Cell-permeant CK2 inhibitors Compound
Structure
IC50 in vitro (µM)
Commercial availability
Reference
TBB
0.50
Yes
[14]
DMAT
0.14
Yes
[15]
IQA
0.39
No
[16]
NBC
0.30
No
[17]
DBC
0.10
No
[17]
Ellagic acid
0.04
Yes
[18]
inhibition has been provided with HEK-293 cells (human embryonic kidney cells) by showing that apoptosis induced by the CK2 inhibitor K27 is abrogated if the cells are transfected with the CK2 V66A/I174A mutant [21], which is 11-fold less sensitive to K27 than wild-type CK2. Several mechanisms are already known by which CK2 can counteract programmed cell death, including acceleration of IκB (inhibitory κB) degradation by calpain, generation of cleavage-resistant sites in a variety of caspase protein substrates, activation of the caspase inhibitor protein ARC (apoptosis repressor with caspase recruitment domain), potentiation of the Akt/PKB (protein kinase B) pathway (see [22] and references therein) and facilitation of DNA repair [23]. The emerging view therefore is that CK2 is essential to survival because it counteracts ‘premature’ apoptosis whenever this can be avoided without compromising ‘healthy’ viability; however, abnormally high CK2 activity may also prevent programmed cell death where this is timely and appropriate, thus enhancing, for example, the tumour phenotype of neoplasia, whose key feature is deregulation of apoptosis.
Pharmacological perspectives In perspective, the usage of cell-permeant CK2 inhibitors devoid of undesirable side effects may provide a new strategy to combat several kinds of tumours as well as infections by viruses, which are known to exploit the CK2 activity of the host cell to phosphorylate proteins essential to their life cycle. In particular, whenever a therapeutic strategy is based on
induction of apoptosis (e.g. chemotherapy and radiotherapy), it is expectedly hampered by elevated CK2 activity and therefore CK2 inhibitors may display an adjuvant effect. Advantage can be also taken of structural differences between human CK2 and its counterparts in parasitic protozoa, such as Leishmania and Plasmodium species, to design selective inhibitors toxic to the parasite yet not to the host.
This work was supported by grants from the Italian MIUR (Ministero dell’Istruzione, dell’Universita` e della Ricerca Scientifica) (PRIN 2004), AIRC (Associazione Italiana Ricerca sul Cancro) and EC (PROKINASERESEARCH 503467).
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13 Tawfic, S., Yu, S., Wang, H., Faust, R., Davis, A. and Ahmed, K. (2001) Histol. Histopathol. 16, 573–582 14 Sarno, S., Reddy, H., Meggio, F., Ruzzene, M., Davies, S.P., Donella-Deana, A., Shugar, D. and Pinna, L.A. (2001) FEBS Lett. 496, 44–48 15 Pagano, M.A., Andrzejewska, M., Ruzzene, M., Sarno, S., Cesaro, L., Bain, J., Elliott, M., Meggio, F., Kazimierczuk, Z. and Pinna, L.A. (2004) J. Med. Chem. 47, 6239–6247 16 Sarno, S., De Moliner, E., Ruzzene, M., Pagano, M.A., Battistutta, R., Bain, J., Fabbro, D., Schoepfer, J., Elliott, M., Furet, P. et al. (2003) Biochem. J. 374, 639–646 17 Meggio, F., Pagano, M.A., Moro, S., Zagotto, G., Ruzzene, M., Sarno, S., Cozza, G., Bain, J., Elliott, M., Donella Deana, A. et al. (2004) Biochemistry 43, 12931–12936 18 Cozza, G., Bonvini, P., Zorzi, E., Poletto, G., Pagano, M.A., Sarno, S., Donella-Deana, A., Zagotto, G., Rosolen, A., Pinna, L.A. et al. (2006) J. Med. Chem. 49, 2363–2366
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19 Battistutta, R., Sarno, S. and Zanotti, G. (2005) in Inhibitors of Protein Kinases and Protein Phosphatases (Pinna, L.A. and Cohen, P.T.W., eds), pp. 125–156, Springer-Verlag, Berlin, Heidelberg 20 Sarno, S., Salvi, M., Battistutta, R., Zanotti, G. and Pinna, L.A. (2005) Biochim. Biophys. Acta 1754, 263–270 21 Piazza, F.A., Ruzzene, M., Gurrieri, C., Montini, B., Bonanni, L., Chioetto, G., Di Maira, G., Barbon, F., Cabrelle, A., Zambello, R. et al. (2006) Blood 108, 1618–1707 22 Di Maira, G., Salvi, M., Arrigoni, G., Marin, O., Sarno, S., Brustolon, F., Pinna, L.A. and Ruzzene, M. (2005) Cell Death Differ. 12, 668–677 23 Loizou, J.I., El-Khamisy, S.F., Zlatanou, A., Moore, D.J., Chan, D.W., Qin, J., Sarno, S., Meggio, F., Pinna, L.A. and Caldecott, K.W. (2004) Cell 117, 17–28
Received 23 June 2006
