The 'regulatory' b-subunit of protein kinase CK2 negatively ... - Nature

Oncogene (2005) 24, 6194–6200

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SHORT REPORT

The ‘regulatory’ b-subunit of protein kinase CK2 negatively influences p53-mediated allosteric effects on Chk2 activation Marina Bjrling-Poulsen1, Simone Siehler2, Lisa Wiesmu¨ller2, David Meek3, Karsten Niefind4 and Olaf-Georg Issinger*,1 1 Institut for Biokemi og Molekylær Biologi, Syddansk Universitet, Odense, Denmark; 2Gyna¨kologische Onkologie, Universita¨tsfrauenklinik, Universita¨t Ulm, Ulm, Germany; 3Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Scotland, UK; 4Institut fu¨r Biochemie, Universita¨t zu Ko¨ln, Ko¨ln, Germany

The ‘regulatory’b-subunit of protein kinase CK2 has previously been shown to interact with protein kinases such as A-Raf, c-Mos, Lyn and Chk1 in addition to the catalytic subunit of CK2. Sequence alignments suggest that these interactions have a structural basis, and hence other protein kinases harboring corresponding sequences may be potential interaction partners for CK2b. We show here that Chk2 specifically interacts with CK2b in vitro and in cultured cells, and that activation of Chk2 leads to a reduction of this interaction. Additionally, we show that the presence of the CK2b-subunit significantly reduces the Chk2-catalysed phosphorylation of p53 in vitro. These findings support the notion that CK2b can act as a general modulator of remote docking sites in protein kinase – substrate interactions. Oncogene (2005) 24, 6194–6200. doi:10.1038/sj.onc.1208762; published online 23 May 2005 Keywords: Chk2; p53; CK2; protein kinase; phosphorylation

Protein kinase CK2 is a pleiotropic serine/threonine kinase consisting of two catalytic a- (and/or a0 -) subunits and two‘regulatory’b-subunits (for reviews see: Guerra and Issinger, 1999; Ahmed et al., 2002; Litchfield, 2003). Evidence is accumulating that CK2 plays a role in cell cycle regulation. For example, it has been shown, in yeast, that CK2b is important for adaptation to G2/M arrest induced by DNA damage (Toczyski et al., 1997). Additionally, studies show that disruption of CK2 in mammalian cells inhibits cell cycle progression at several transitions (Lorenz et al., 1994; Pepperkok et al., 1994; Ford et al., 2000). CK2 phosphorylates topoisomerase II (Cardenas et al., 1992; Daum and Gorbsky, 1998; Escargueil et al., 2000) and transcription factor Six1 during mitosis (Ford et al., 2000), and it has additionally been found to phosphorylate the phosphatases Cdc25B

*Correspondence: O-G Issinger, BMB, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark; E-mail: [email protected] Received 17 December 2004; revised 13 April 2005; accepted 18 April 2005; published online 23 May 2005

(Theis-Febvre et al., 2003) and Cdc25C (Schwindling et al., 2004), thereby increasing the catalytic activity of Cdc25B and inhibiting nuclear uptake of Cdc25C. It is the CK2 holoenzyme that is predominantly found in a cell. However, evidence is accumulating that the individual subunits of CK2 themselves may interact with a number of different proteins in vitro and in vivo (for a review see: Guerra and Issinger, 1999; Korn et al., 1999). An excess of CK2b over CK2a is found in certain tumors (Stalter et al. 1994), and additionally, the crystal structure of the CK2 holoenzyme (Niefind et al., 2001) has revealed that the interface between the a- and b-subunits is relatively small, suggesting that the holoenzyme may be able to dissociate and reassociate. Furthermore, CK2b interacts with a part of the common kinase core in CK2a, suggesting that CK2b may be able to interact with the same domain also in other protein kinases. In fact, interaction of CK2b with other proteins, especially protein kinases, has been shown in several cases, for example A-Raf (Boldyreff and Issinger, 1997), c- Mos (Chen et al., 1997; Chen and Cooper, 1997), p90rsk (Kusk et al., 1999), Lyn (Lehner et al., 2004) and checkpoint kinase 1 (Chk1) (Guerra et al., 2003), the latter finding further supporting a role for CK2b in cell cycle regulation. Based on the evidence for existence of CK2b outside the CK2 holoenzyme in complex with other kinases, and findings that CK2b likely plays a role in cell cycle regulation, we wanted to analyse a possible interaction of CK2b with checkpoint kinase 2, Chk2. Chk2 is a tumor-suppressor protein functioning as a signal transducer in cell cycle checkpoints responding to DNA damage. Established substrates of Chk2 include Cdc25A, Cdc25C, BRCA1 and p53. Phosphorylation of p53 at Thr18 and Ser20 by Chk2 stabilizes the protein, and enhances its potential to positively regulate expression of proteins involved in DNA repair, apoptosis and cell cycle control (for a review see: Bartek et al., 2001; Bartek and Lukas, 2003). However, the established consensus sequence for Chk2 phosphorylation: L-X-RX-X-S/T (O’Neill et al., 2002; Seo et al., 2003), is absent in p53. So how then does p53 become a substrate for Chk2? This question was addressed by Craig et al. (2003) who showed that p53 possesses two docking sites for

CK2 negatively influences p53-mediated allosteric effects on Chk2 activation M Bjrling-Poulsen et al

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Chk2 (Box-II and Box-V, within the DNA-binding domain) which, although located at some distance within the polypeptide chain from the Thr18 and Ser20 phosphorylation sites, influence the interaction such that p53 becomes a substrate for Chk2, despite its lack of the typical consensus sequence. This type of substrate recruitment via remote docking sites resembles the activation of an enzyme by an allosteric effector. It may have widespread significance for Chk2 function (Craig et al., 2003), since similar mechanisms have been established for other protein kinases such as Cdk2 (Luciani et al., 2000), Pdk1, MAPK and GSK3 (Biondi and Nebreda, 2003). In addition to the possibility that CK2b may interact with Chk2, CK2b can interact with p53 (Go¨tz et al., 1999; Schuster et al., 2001). Thus, we wanted to test

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To determine whether the CK2 holoenzyme or the individual CK2 subunits could interact directly with Chk2, we performed GST pull-down experiments (Figure 1). GST-Chk2 did not interact with CK2a or the CK2 holoenzyme (Figure 1a). However, a significant interaction of CK2b with GST-Chk2 was observed, and this interaction proved to be specific for Chk2, since CK2b did not interact with GST alone (Figure 1b). We then analysed whether interaction between Chk2 and CK2b would also take place in mammalian cells. First, interaction was tested in transfected Cos-1 cells, which overexpressed the two proteins (Figure 1c). CK2b was immunoprecipitated with CK2b antiserum, Chk2 with polyclonal Chk2 antibody, and, as negative

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whether CK2b might somehow interfere with p53’s inherent ability to modulate Chk2 activity.

Figure 1 Human CK2b and Chk2 interact in vitro and in transfected cells. (a) Matched amounts of GST and GST-Chk2 were immobilized on glutathione Sepharose 4B beads (Amersham Biosciences) and incubated with 2 mg of CK2a or CK2 holoenzyme. Lanes 1 and 2 contain negative control reactions. In lanes 3 and 4, GST-Chk2 was incubated with CK2a and CK2 holoenzyme, respectively. In lane 5, CK2a was loaded directly on the gel as a positive control. Incubations were performed in binding buffer (20 mM Tris-HCl 7.5, 100 mM NaCl, 2 mM EDTA, 0.1% Igepal, 2 mM DTT, 0.05% BSA, 5% glycerol, protease inhibitors (Complete, Roche)) for 2 h at 41C. Subsequently, the beads were washed and immobilized proteins eluted with SDS-sample buffer. The presence of CK2a was detected by Western blotting, using monoclonal CK2a antibody (1AD9) (Calbiochem). Chk2 was cloned in the pGEX-6P-C vector (Amersham Biosciences) for glutathione S-transferase (GST) tagging and expressed in DH5a cells. In parallel, GST was expressed on its own. Expression of GST and GST-Chk2 in bacterial lysates was compared by SDS– polyacrylamide gel electrophoresis (SDS – PAGE). CK2a and CK2b were cloned in pT7-7 and expressed separately in BL21 (DE3) cells as described by Grankowski et al. (1991). CK2a and CK2 holoenzyme were then purified as described by Guerra et al. (1997). (b) Same analysis as in (a), only CK2a was substituted by CK2b. Immobilized CK2b was detected using monoclonal CK2b antibody (6D5) (Calbiochem). CK2b was purified as described by Guerra et al. (2003). (c) Western blot detection of Chk2 and CK2b expression in Cos-1 cells transfected with empty vector (lane 1) or Chk2-His plus CK2b-MycHis encoding vectors (lane 2). Chk2 was cloned in the pQE-TriSystem vector (QIAGEN), introducing the His-tag. CK2b was cloned into the pcDNA3.1 vector (InVitrogen) as described by Guerra et al. (2003), introducing the MycHis-tag. Cos-1 cells were cultured as described in the legend to Figure 2(a), and transfection was performed using jetPEI transfection reagent (PolyPlus transfection). At 48 h post-transfection, cells were harvested and analysed by Western blotting. Chk2 was detected using monoclonal Chk2-antibody, clone 7 (Upstate) and CK2b as described in (b). (d) Immunoprecipitation of CK2b (lane 1) and Chk2 (lane 3) from co-transfected Cos-1 cells. Lanes 2 and 4 contain negative control reactions. Immunoprecipitations were performed essentially as described previously (Guerra et al., 1999), using polyclonal CK2b antiserum produced by immunization of rabbits with recombinant CK2b (lane 1), polyclonal anti-Chk2 antibody (H-300) (Santa Cruz Biotechnology Inc.) (lane 3) or rabbit preimmune serum (lanes 2 and 4). Precipitates were analysed for Chk2 and CK2b by Western blotting using the antibodies described in (b) and (c) Oncogene


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controls, Cos-1 extracts were precipitated using preimmune serum (Figure 1d). Again, a specific interaction between Chk2 and CK2b was observed. It is noteworthy that the Chk2 antibody precipitates large amounts of exogenous CK2b-MycHis and little endogenous CK2b, suggesting that Chk2 is more likely to interact with free CK2b than with the CK2 holoenzyme. However, some of the co-precipitation of exogenous CK2b with exogenous Chk2 may also be an artefact mediated by the His-tags on both proteins. We then went on to analyse interaction between endogenous Chk2 and CK2b in non-transfected cells. Expression of Chk2 and CK2b was detected in six mammalian cell lines by Western blotting (Figure 2a), and all cells were found to express both proteins, although the expression of Chk2 was very low in Cos-1 cells. We immunoprecipitated CK2b from each cell line and tested for interaction with Chk2 by Western blotting (results not shown). However, a significant and specific interaction between Chk2 and CK2b was only observed in K562 and Jurkat cells (Figure 2b). It is not known, why interaction between Chk2 and CK2b seems to occur solely in suspension cells, but, interestingly, the same phenomenon was observed by Guerra et al. (2003) for the interaction between CK2b and Chk1. In this case, interaction was observed in K562 and Y-79 cells, but not in 3T3, Cos-1, U2OS or HCT116 cells. As a control for the specificity of Chk2 for interaction with free CK2b, we immunoprecipitated CK2a from K562 and Jurkat cells and tested for co-precipitation of Chk2 by Western blotting (results not shown). Surprisingly, small amounts of Chk2 co-precipitated with the CK2a antiserum, although much less than with the CK2b antiserum. This indicates that Chk2 might to some extent interact with the CK2 holoenzyme or the CK2a subunit, in addition to the free CK2b subunit. However, co-precipitation of Chk2 with CK2a from cellular extracts might just reflect the existence of larger cellular complexes involving both proteins rather than a direct interaction. In any case, since no interaction of Chk2 with either the holoenzyme or the CK2a subunit was detected in vitro, we consider the interaction between Chk2 and free CK2b as the most convincing. Activation of Chk2 inhibits its interaction with CK2b To determine the influence of Chk2 activation with respect to its interaction with CK2b, cells were incubated with neocarzinostatin (NCS), which induces DNA double-strand breaks (DSBs) (Povirk, 1996). DSBs lead to activation of ATM, which then phosphorylates Chk2 on Thr68 and activates it (for a review see: Bartek and Lukas, 2003). Cells were incubated with 30 mg/ml NCS and harvested at different time-points. Maximal Thr68-phosphorylation of Chk2 was detected after 1 h of incubation (Figure 2c and d). Thus, for the following immunoprecipitations with activated Chk2, cells were incubated with NCS for 1 h prior to analysis. CK2b was Oncogene

immunoprecipitated from both untreated and NCStreated K562 and Jurkat cells, and co-precipitation of Chk2 was analysed by Western blotting (Figure 2e). For both cell lines, we found that interaction of Chk2 with CK2b decreased after incubation with NCS, indicating that activated Thr68-phosphorylated Chk2 does not interact as well with CK2b as quiescent Chk2. Activated Chk2 oligomerizes (for a review see: Ahn et al., 2004), and possibly the monomeric, nonphosphorylated form of Chk2 is more capable of interacting with CK2b. Thus, CK2b may not have a large direct effect on Chk2 activity once it is activated, but it may, by its interaction, inhibit activation of quiescent Chk2. In any case, our finding that CK2b interacts with Chk2 represents further evidence that CK2b may be involved in cell cycle regulation. Although the interaction clearly involves only a small fraction of the two proteins in the cell, it may still be of significance for the function of Chk2. Effects of CK2b with respect to Chk2 activity In vitro Chk2 kinase assays were performed, using the synthetic substrate peptide, Chktide, and adding increasing amounts of CK2b to the reaction (results not shown). No significant effect of CK2b was observed on the Chk2-mediated phosphorylation of Chktide, indicating that CK2b does not directly affect the active site in Chk2. Additionally, since Chktide is a small peptide substrate, it may interact relatively easily with the active site in Chk2 even in the presence of an interaction partner. Next, we tested the effect of CK2b on the Chk2catalysed phosphorylation of native, full-length p53. p53 was chosen as a model full-length substrate, based on the finding by Craig et al. (2003) that p53 Box-II and Box-V domains can allosterically activate Chk2 toward p53 Box-I (Thr18 and Ser20), and the fact that CK2b interacts stably with p53 (Appel et al., 1995; Go¨tz et al., 1999). We wanted to analyse whether CK2b interacting with Chk2, p53 or both might influence the ability of Chk2 to phosphorylate p53. Since Chk2 and CK2b interact only weakly, especially when Chk2 is active, the interaction between CK2b and p53 is likely more important for the Chk2-mediated phosphorylation of p53 than the direct interaction between Chk2 and CK2b. Thus, p53 was preincubated with increasing amounts of CK2b, before Chk2 was added and the phosphorylation reactions initiated. Representative results are shown in Figure 3a and b. The histogram in Figure 3c shows a quantitative determination of the p53 phosphorylation in the presence of increasing amounts of CK2b. When adding the double molar amount of CK2b dimer compared to p53 tetramer, p53 phosphorylation decreased by ca. 40%, and adding four times CK2b led to a loss of ca. 60% of the original p53 phosphorylation. Importantly, addition of four times BSA compared to p53 on a molar basis did not significantly affect the Chk2-mediated phosphorylation of p53, confirming that the interaction between CK2b and p53/Chk2 is specific.


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Figure 2 Interaction between endogenous Chk2 and CK2b. (a) Western blot detection of Chk2 and CK2b expression in selected cell lines, using the monoclonal antibodies described in Figure 1b and c. The housekeeping protein, b-actin, was detected as a control for equal loading, using monoclonal anti-b-actin antibody (A5441) (Sigma). Cos-1, F9 and HeLa cells were maintained in Dulbecco’s MEM supplemented with 10% fetal bovine serum (FBS) and nonessential amino acids. MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1 mM Na-pyruvate, and nonessential amino acids. Jurkat and K562 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 2 mM L-glutamine. FBS was from Bio Whittaker Europe, while all other components of the growth media were from Gibco BRL. (b) CK2b was immunoprecipitated from K562 and Jurkat cells (lanes 1 and 3) as described in Figure 1(d). In control reactions, rabbit preimmune serum was used for immunoprecipitation (lanes 2 and 4). Precipitates were analysed for Chk2 and CK2b by Western blotting, using the antibodies described in Figure 1b and c. (c and d) Analysis of Chk2 Thr68phosphorylation in K562 and Jurkat cells, respectively. Cells were incubated with 30 mg/ml neocarzinostatin (NCS) for the time-periods indicated, then lysed in 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM EDTA, 1 mM Na3VO4, 30 mM NaPPi, 10 mM NaF, 100 nM okadaic acid and, protease inhibitors (Complete, Roche) for 30 min on ice. Thr68-phosphorylated Chk2 was detected by Western blotting, using polyclonal Phospho-Chk2 (Thr68) antibody (Cell Signaling Technologies). b-Actin was detected as a control for equal loading. NCS was a gift from Dr Hiroshi Maeda (Kumamoto University, Japan). (e) CK2b was immunoprecipitated from control (lanes 2 and 4), and NCS-treated (lanes 3 and 5) K562 and Jurkat cells, and precipitates were analysed for Chk2 and CK2b as described in (b). Lane 1: negative control, using preimmune serum for immunoprecipitation from control K562 cells

The recombinant p53 was tested for DNA-binding activity by EMSA analysis (Figure 3d), and since it did bind DNA, we concluded that it was folded correctly and existed as a tetrameric protein.

It is unclear exactly how CK2b inhibits Chk2catalysed phosphorylation of p53. Inhibition is not due to competition between CK2b and p53 for Chk2 phosphorylation, since a C-terminally Oncogene


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Figure 3 CK2b inhibits Chk2-catalysed p53 phosphorylation. (a) Coomassie stained SDS-gel containing proteins from Chk2/p53 phosphorylation assays, with addition of increasing amounts of CK2b (1/2–4 times the molar amount of p53 tetramer). For phosphorylation assays, we used 250 ng Chk2 (1.9 pmol Chk2 dimer), 3 mg p53 (16 pmol p53 tetramer) and 0.4 – 3.2 mg CK2b (8–64 pmol CK2b dimer). 4.3 mg BSA (64 pmol monomer) was used as a negative control for the effect of CK2b (gel not shown). Reactions were mixed in kinase buffer (10 mM MOPS pH 7.0, 0.5 mM EDTA, 50 mM ATP, 7.5 mM MgCl2), and CK2b/BSA was incubated with p53 for 20 min on ice before addition of Chk2 and 5 mCi [g-32P] ATP (Hartmann Analytic, 3000 Ci/mmol). Phosphorylation was carried out at 301C for 30 min and terminated by addition of SDS-sample buffer. Human p53 was expressed from the pET-19b vector (Novagen) in BL21 (DE3) cells. It was insoluble and was subsequently solubilized and refolded as described by Bell et al. (2002). Refolded p53 was purified by Ni-NTA (QIAGEN) affinity chromatography and dialysed against 30 mM Napyrophosphate pH 7.5, 50 mM KCl, 5% (v/v) glycerol and, 2 mM DTT. Human Chk2 was expressed in Sf9 cells, using the BacVector3000 Transfection Kit (Novagen). Recombinant baculoviruses were produced by co-transfection of Sf9 cells with the chk2/pQETriSystem vector and BacVector-3000 DNA. Sf9 cells were cultured at 281C in BacVector Insect Cell Medium (Novagen) supplemented with 5% FBS (Bio Whittaker Europe). At 72 h after infection with recombinant baculovirus, cells were harvested and Chk2 was purified by Ni-NTA affinity chromatography then dialysed against 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM DTT, 270 mM sucrose, 0.1 mM EGTA and, protease inhibitors (Complete, Roche). Recombinant human CK2b was produced as described in Figure 1(b). (b) Autoradiograph of the gel in (a). (c) Quantitative analysis of Chk2-catalysed p53 phosphorylation. Each bar in the histogram represents the mean phosphorylation of p53 (from six independent assays) given as a ratio to the autophosphorylation of Chk2 (p53-P /Chk2-P). The phosphorylation of p53 in the absence of CK2b/BSA was set to 100%. Standard errors are shown by error bars. (d) Recombinant p53 was analysed for DNA-binding activity by electrophoretic mobility shift assays (EMSA). Reactions contained 50 pM radio-labeled DNA and 10 nM competitor tRNA and either 100 ng PAb421 p53 antibody (Calbiochem) (lane 1), 500 ng negative control protein, p38 (lane 2), no protein (lane 3), 500 ng p53 (lane 4), 250 ng p53 (lane 5) or 250 ng p53 with 100 ng PAb421 (lane 6). The image displays the upper half of a representative gel. Arrows indicate DNA-bound p53 and DNA-bound p53 in complex with antibody. The TGCCT repeat from the ribosomal gene cluster, a p53-binding site (Kern et al., 1991; Farmer et al., 1992), was [32P]-labeled by PCR, purified and quantified as described previously (Boehden et al., 2004). Additionally, reactions were electrophoresed and autoradiographed as described previously (Boehden et al., 2004)

truncated CK2b1165 protein, which was not phosphorylated by Chk2, inhibited p53 phosphorylation as much as full-length CK2b (results not shown). However, some binding of CK2b to Chk2 may in part reduce Chk2 activity towards p53, and additionally, the interaction of CK2b with p53 very likely inhibits the allosteric activation of Chk2. Oncogene

CK2b has been shown to interact with the p53 sequence 325GEYFTLQIRGRERFEMFREL344 by peptide screening assays (Go¨tz et al., 1999). Interestingly, the amino-acid stretch from 323 to 355 has additionally been found to be essential for p53 tetramerization. The tetramerization domain is very robust and it is unlikely that just one mutation could disturb the oligomerization


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function (Soussi and May, 1996). However, interaction with a larger molecule, such as CK2b, could induce structural constraints influencing the interaction with other proteins such as Chk2. Thus, CK2b may have a dual function in inhibition of p53 activity. First, CK2b interacts with and directly inhibits binding of p53 to DNA (Prowald et al., 1997), and second, interaction of CK2b with p53 (and possibly also Chk2) inhibits Chk2mediated phosphorylation and activation of p53. CK2b as a general provider/modulator of remote docking sites in protein kinase – substrate interactions For decades, the specificity of protein phosphorylation was considered to be governed exclusively by a specific ‘consensus sequence’ around the phospho-acceptor site, which is recognized by the active site region of the corresponding protein kinase. In recent years, however, the fact that specific interactions by docking sites remote from the active site can contribute significantly to the enzyme – substrate recognition has attracted more and more attention in protein kinase research. These interactions resemble the allosteric activation or inhibition of an enzyme by a small molecule, with the only difference that the allosteric effector is part of the macromolecular substrate itself. The existence of allosteric docking sites on protein kinases and their substrates allow more complex networks of control and signaling in the cell plus the development of more sophisticated drugs. Therefore, knowledge about them is essential both for a cell biological understanding and for pharmaceutical applications.

Within the CK2 holoenzyme (Niefind et al., 2001), the CK2b dimer has no direct contact to the active sites of the two CK2a subunits or to the established catalytic key elements of protein kinases (Huse and Kuriyan, 2002). This observation is consistent with a ‘background’ role of CK2b: its effect on the catalytic activity of an associated kinase is strongly dependent on the context, that is, on the respective substrate and the presence of further effector molecules. With small peptide substrates, CK2b is in general activating – as has been shown for CK2a (Boldyreff et al. 1993) – or neutral as found here for Chk2. Yet, where macromolecular substrates are concerned, remote interactions come into play. These can enhance the catalytic activity or suppress it as in the case of CK2a and the substrate calmodulin (Meggio et al., 1992) or in the case of Chk2 and p53, which we have presented here. Of course, the emerging role of CK2b as a general provider/modulator of docking sites requires a cellular pool of CK2b not irreversibly associated with CK2a. The existence of such a pool has become likely with the elucidation of the structural features of the CK2 holoenzyme and the already described findings of free CK2 subunits (Stigare et al., 1993; Goldberg, 1999; Niefind et al., 2001). Acknowledgements We thank Hans H Jensen for technical support and Dr B Boldyreff (KinaseDetect) for purification of p53 and Chk2. This work was supported by the Danish Cancer Society, Grant No. 002521109210 and the Danish Research Council, Grant No. 21-01-0511 to O-G Issinger, plus the Deutsche Forschungsgemeinschaft, Grants Wi 1376/3-1 to L Wiesmu¨ller and NI 643/1-2 to K Niefind.

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