Barbara Guerra

Protein kinase CK2 and its role in cellular proliferation, development and pathology

Protein kinase CK2 is a pleiotropic, ubiquitous and constitutively active protein kinase that can use both ATP and GTP as phosphoryl donors with specificity for serine/threonine residues in the vicinity of acidic amino acids. Recent results show that the enzyme is involved in transcription, signaling, proliferation and in various steps of development. The tetrameric holoenzyme (α2β2) consists of two catalytic α‐subunits and two regulatory β‐subunits. The structure of the catalytic subunit with the fixed positioning of the activation segment in the active conformation through its own aminoterminal region suggests a regulation at the transcriptional level making a regulation by second messengers unlikely. The high conservation of the catalytic subunit from yeast to man and its role in the tetrameric complex supports this notion. The regulatory β‐subunit has been far less conserved throughout evolution. Furthermore the existence of different CK2β‐related proteins together with the observation of deregulated CK2β levels in tumor cells and the reported association of CK2β protein with key proteins in signal transduction, e.g. A‐Raf, Mos, p90rsk etc. are suggestive for an additional physiological role of CK2β protein beside being the regulatory compound in the tetrameric holoenzyme.

Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme.

The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C‐terminally truncated catalytic and two regulatory subunits has been determined at 3.1 Å resolution (Protein Data Bank code: 1JWH). In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C‐terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit as a docking partner for various protein kinases. Furthermore it shows an inter‐domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.

Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 Å resolution

CK2α is the catalytic subunit of protein kinase CK2, an acidophilic and constitutively active eukaryotic Ser/Thr kinase involved in cell proliferation. A crystal structure, at 2.1 Å resolution, of recombinant maize CK2α (rmCK2α) in the presence of ATP and Mg2+, shows the enzyme in an active conformation stabilized by interactions of the N‐terminal region with the activation segment and with a cluster of basic residues known as the substrate recognition site. The close interaction between the N‐terminal region and the activation segment is unique among known protein kinase structures and probably contributes to the constitutively active nature of CK2. The active centre is occupied by a partially disordered ATP molecule with the adenine base attached to a novel binding site of low specificity. This finding explains the observation that CK2, unlike other protein kinases, can use both ATP and GTP as phosphorylating agents.

Protein kinase CK2: evidence for a protein kinase CK2β subunit fraction, devoid of the catalytic CK2α subunit, in mouse brain and testicles

The highest CK2 activity was found in mouse testicles and brain, followed by spleen, liver, lung, kidney and heart. The activity values were directly correlated with the protein expression level of the CK2 subunits α (catalytic) and β (regulatory). The α′ subunit was only detected in brain and testicles. By contrast, Northern blot analyses of the CK2α mRNA revealed a somewhat different picture. Here, the strongest signals were obtained for brain, liver, heart and lung. In kidney, spleen and testicles mRNAs were only weakly detectable. For CK2α′ mRNA distribution strong signals were observed for lung, liver and testicles. In the case of CK2β mRNA the highest signals were found for testicles, kidney, brain and liver. The amount of CK2β mRNA in testicles was estimated to be about 6‐fold higher than in brain. The strongest CK2β signals in the Western blot were found for testicles and brain. The amount of CK2β protein in brain in comparison to the other organs (except testicles) was estimated to be ca. 2–3‐fold higher whereas the ratio of CK2β between testicles and brain was estimated to be 3–4‐fold. Results from the immunoprecipitation experiments support the notion for the existence of free CK2β population and/or CK2β in complex with other protein(s) present in brain and testicles. In all other mouse organs investigated, i.e. heart, lung, liver, kidney and spleen, no comparable amount of free CK2β was observed. This is the first physiological evidence for the existence of a ‘free CK2β’ (or in complex with proteins other than CK2α) in normal animal tissue apart from the hitherto dogmatic association with CK2α in a tetrameric holoenzyme complex.

The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation.

The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro 0.5 – 1 mol of phosphate were incorporated per mol MDM2 protein. The catalytic subunit of protein kinase CK2 (α-subunit) catalyzed the incorporation of twice as much phosphate into the MDM2 protein as it was obtained with the holoenzyme. Polylysine stimulated MDM2 phosphorylation by CK2 holoenzyme threefold in contrast to the α-subunit-catalyzed MDM2 phosphorylation which was reduced by about 66% when polylysine was added. Full length p53, but also a peptide representing a C-terminal fragment of the tumor suppressor gene product p53 (amino acids 264 – 393 which also harbors the CK2β interaction site at amino acids 287 – 340) mimicked the polylysine effect in all respects, ie. stimulation of phosphate incorporation by CK2 holoenzyme and inhibition in the presence of the catalytic CK2 α-subunit. Stimulation by p53264 – 393 was on the average close to twofold and inhibition in the case of the α-subunit-catalyzed MDM2 phosphorylation was about 40%. Phosphorylation of MDM2 by CK2 holoenzyme in the presence of the p21WAF1/CIP1, known to be a potent inhibitor of cyclin-dependent protein kinases, also led to a significant reduction of phosphate incorporation into MDM2 indicating that p21WAF1/CIP1 does not exclusively inhibit cell cycle kinases. Furthermore, these data add new insight into the autoregulatory loop which include p21WAF1/CIP1, MDM2 protein, CK2 and p53.

Modulation of human checkpoint kinase Chk1 by the regulatory β -subunit of protein kinase CK2

Protein kinase CK2 is a serine/threonine protein kinase involved in various aspects of cellular regulation. The regulatory β-subunit of CK2 exerts a central role not only in mediating formation of tetrameric CK2 complexes but also as a docking partner for several protein kinases. In this study, CK2β is found to interact with the human cell cycle checkpoint kinase Chk1. The Chk1-interacting region of CK2β is localized at the C-terminus and the complex between CK2β and Chk1 is devoid of the catalytic CK2α-subunit. The interaction between CK2β and Chk1 leads to an increase in the Cdc25C phosphorylation activity of Chk1. The screening of several cell lines has revealed that the association between CK2β and Chk1 also occurs in vivo at a different degree. Collectively, these studies confirm the implication of the regulatory β-subunit of protein kinase CK2 in cell cycle regulation and identify a novel mechanism for the activation of Chk1 protein kinase.

The regulatory β-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

Cell-cycle transition from the G2 phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory β-subunit of protein kinase CK2 by RNA interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2β causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1–Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found that CK2β participates in PLK1–Wee1 complex formation whereas its cellular depletion leads to disruption of PLK1–Wee1 interaction and reduced Wee1 phosphorylation at Ser53 and 121. The data reported here reinforce the notion that CK2β has functions that are independent of its role as the CK2 regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.

Regulation of DNA-dependent protein kinase by protein kinase CK2 in human glioblastoma cells

The DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the nonhomologous end-joining pathway of DNA double-strand breaks repair. Although DNA-PK has been biochemically characterized in vitro, relatively little is known about its functions in the context of DNA repair and how its kinase activity is precisely regulated in vivo. Here, we report that cellular depletion of the individual catalytic subunits of protein kinase CK2 by RNA interference leads to significant cell death in M059K human glioblastoma cells expressing DNA-PKcs, but not in their isogenic counterpart, that is M059J cells, devoid of DNA-PKcs. The lack of CK2 results in enhanced DNA-PKcs activity and strongly inhibits DNA damage-induced autophosphorylation of DNA-PKcs at S2056 as well as repair of DNA double-strand breaks. By the application of the in situ proximity ligation assay, we show that CK2 interacts with DNA-PKcs in normal growing cells and that the association increases upon DNA damage. These results indicate that CK2 has an important role in the modulation of DNA-PKcs activity and its phosphorylation status providing important insights into the mechanisms by which DNA-PKcs is regulated in vivo.

Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

BackgroundThe DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks.ResultsIn the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks.ConclusionsTaken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

NFκB signaling is important for growth of antiestrogen resistant breast cancer cells

Resistance to endocrine therapy is a major clinical challenge in current treatment of estrogen receptor-positive breast cancer. The molecular mechanisms underlying resistance are yet not fully clarified. In this study, we investigated whether NFκB signaling is causally involved in antiestrogen resistant cell growth and a potential target for re-sensitizing resistant cells to endocrine therapy. We used an MCF-7-derived cell model for antiestrogen resistant breast cancer to investigate dependence on NFκB signaling for antiestrogen resistant cell growth. We found that targeting NFκB preferentially inhibited resistant cell growth. Antiestrogen resistant cells expressed increased p50 and RelB, and displayed increased phosphorylation of p65 at Ser529 and Ser536. Moreover, transcriptional activity of NFκB after stimulation with tumor necrosis factor α was enhanced in antiestrogen resistant cell lines compared to the parental cell line. Inhibition of NFκB signaling sensitized tamoxifen resistant cells to the growth inhibitory effects of tamoxifen but was not sufficient to fully restore sensitivity of fulvestrant resistant cells to fulvestrant. In support of this, depletion of p65 with siRNA in tamoxifen resistant cells increased sensitivity to tamoxifen treatment. Our data provide evidence that NFκB signaling is enhanced in antiestrogen resistant breast cancer cells and plays an important role for antiestrogen resistant cell growth and for sensitivity to tamoxifen treatment in resistant cells. Our results imply that targeting NFκB might serve as a potential novel treatment strategy for breast cancer patients with resistance toward antiestrogen.