Cellular Signalling 20 (2008) 19421951

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Cellular Signalling
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells

Greg Vilk a, Jane E. Weber a, Jacob P. Turowec a, James S. Duncan a, Chenggang Wu a, D. Richard Derksen a, Piotr Zien a, Stefania Sarno b, Arianna Donella-Deana b, Gilles Lajoie a, Lorenzo A. Pinna b, Shawn S.C. Li a, David W. Litcheld a,
a b

Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1 Venetian Institute for Molecular Medicine and Department of Biological Chemistry, University of Padova, Padova, Italy

A R T I C L E

I N F O

A B S T R A C T
Protein kinase CK2 exhibits oncogenic activity in mice and is over-expressed in a number of tumors or leukemic cells. On the basis of its amino acid sequence and a wealth of experimental information, CK2 has traditionally been classied as a protein serine/threonine kinase. In contrast to this traditional view of CK2, recent evidence has shown that CK2 can also phosphorylate tyrosine residues under some circumstances in vitro and in yeast. In this study, we provide denitive evidence demonstrating that CK2 also exhibits tyrosine kinase activity in mammalian cells. Tyrosine phosphorylation of CK2 in cells and in CK2 immunoprecipitates is dependent on CK2 activity and is inhibited by the CK2 selective inhibitor 4,5,6,7-tetrabromobenzotriazole. Examination of phosphotyrosine proles in cells reveals a number of proteins, including CK2 itself, which exhibit increased tyrosine phosphorylation when CK2 levels are increased. Peptide arrays to evaluate the specicity determinants for tyrosine phosphorylation by CK2 reveal that its specicity for tyrosine phosphorylation is distinct from its specicity for serine/threonine phosphorylation. Of particular note is the requirement for an aspartic acid immediately C-terminal to the phosphorylatable tyrosine residue. Collectively, these data provide conclusive evidence that CK2 catalyzes the phosphorylation of tyrosine residues in mammalian cells, a nding that adds a new level of complexity to the challenge of elucidating its cellular functions. Furthermore, these results raise the possibility that increased CK2 levels that frequently accompany transformation may contribute to the increased tyrosine phosphorylation that occurs in transformed cells. 2008 Elsevier Inc. All rights reserved.

Article history: Received 24 June 2008 Accepted 3 July 2008 Available online 6 July 2008 Keywords: Protein kinase CK2 Tyrosine phosphorylation Dual-specicity kinase Peptide arrays Protein kinase

1. Introduction Protein kinase CK2 is a ubiquitously expressed protein kinase that is essential for viability and has apparent functions associated with pathways involved in the control of cell proliferation and survival [19]. CK2 has also been shown to exhibit oncogenic activity in transgenic mice and to promote transformation in cultured mammalian cells [1014]. In this regard, it is intriguing that the list of likely physiological targets of CK2 contains a myriad of regulatory proteins including proto-oncogene products such as c-Myc [15], c-Myb [15,16], c-Jun [17], Tal-1 [18] and tumor suppressor gene products such as p53 [19], BRCA-1 [20] and PML [21]. However, a detailed mechanistic understanding of how CK2 participates in transformation and tumorigenesis remains far from complete [22]. Furthermore, since systematic studies to identify its cellular substrates have been hindered by its high constitutive activity and

Corresponding author. Fax: +519 661 3175. E-mail address: litch@uwo.ca (D.W. Litcheld). 0898-6568/$ see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.07.002

enigmatic mode of intracellular modulation, it is certain that many CK2 targets remain to be identied. CK2 has traditionally been viewed as a protein serine/threonine kinase with a preference for substrates with serine/threonine residues embedded within a stretch of acidic amino acids that conform to a rather well dened substrate consensus [1,7]. On the basis of numerous studies primarily performed with synthetic peptides, this consensus has been dened as S/T-X-X-D/E/pS/pY where X can be any amino acid except proline. While the delineation of this consensus has been invaluable in the identication of many potential physiological CK2 substrates, it is evident from the examination of the sites within proteins that are phosphorylated by CK2 that there are CK2 phosphorylation sites that do not conform to this canonical CK2 consensus. For example, one of the DNA damage-inducible phosphorylation sites on p53, S392 in human p53, is efciently phosphorylated by CK2 although it does not match the CK2 recognition consensus [19]. In addition, although CK2 has traditionally been viewed as a serine/ threonine kinase, studies in yeast have raised the specter that CK2 can also phosphorylate tyrosine residues. Notably, in S. cerevisiae, tyrosine phosphorylation of the immunophilin Fpr3 is dependent upon CK2

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activity [23]. Furthermore, CK2 can phosphorylate Y184 on Fpr3 in vitro suggesting that CK2 may directly phosphorylate tyrosine residues on Fpr3 in intact yeast [24]. Although the evidence in yeast, which do not possess any bona de protein tyrosine kinases was indicative of protein tyrosine phosphorylation by CK2, these studies did not provide any insights into the physiological relevance of this observation to mammalian CK2. In particular, although mammalian CK2 was shown to phosphorylate tyrosine-containing peptides in vitro, the kinetic parameters for this phosphorylation were severalfold less favorable than that observed with serine/threonine phosphorylation [24]. Recombinant catalytic subunits of CK2 have also been shown to undergo autophosphorylation on tyrosine residues in vitro [25]. However, these studies did not determine whether CK2 undergoes tyrosine autophosphorylation and/or exhibits protein tyrosine kinase activity in mammalian cells. In the present study, we undertook an investigation of human cell lines with regulated expression of protein kinase CK2 to determine whether altered levels of CK2 resulted in changes in the extent of phosphorylation of its cellular substrates. During the course of this investigation, we made the somewhat unexpected observation that CK2 undergoes tyrosine phosphorylation in mammalian cells. This tyrosine phosphorylation of CK2 is diminished by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and by transfecting cells with mutants of CK2 that are catalytically inactive or that have been shown to be defective in tyrosine autophosphorylation in vitro. Collectively, these results demonstrate that, as appears to be the case in yeast, CK2 also acts as a dual-specicity kinase in mammalian cells. 2. Experimental procedures 2.1. Materials Mouse monoclonal anti-Myc (9E10) antibodies were obtained from the Developmental Studies Hybridoma Bank (Univ. of Iowa). Mouse monoclonal anti-phosphotyrosine (anti-pTyr) antibodies (ptyr-100) and protein phosphatase were purchased from Cell Signaling Technologies (Beverly, MA). Biotin-conjugated mouse antihemaglutinin (3F10) antibodies, mouse monoclonal anti-hemaglutinin (HA) (12CA5) antibodies, and T4 DNA ligase were purchased from Roche Diagnostics. HRP-conjugated goat anti-rabbit and goat antimouse antibodies and the modied-Bradford protein assay reagent were from BioRad. HRP-conjugated streptavidin antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. The Srckinase inhibitor, PP2, was purchased from Calbiochem. TBB, 4,5,6,7tetrabromobenzotriazole, was synthesized as described [2628]. Protein A Sepharose and Protein G Sepharose were obtained from Sigma and Amersham Biosciences, respectively. SuperSignal chemiluminescence and BCA protein assay reagents were obtained from Pierce. The -cyano-4-hydroxy cinnamic acid (4HCCA) matrix and tetracycline were purchased from Sigma. 2.2. Plasmid construction Tyrosine mutants of CK2 (Tyr182Phe, Tyr188Phe, and Tyr182Phe/ Tyr188Phe double mutant) with a C-terminal HA-tag were generated by rst amplifying CK2 (Tyr182Phe) and CK2 (Tyr188Phe) from bacterial expression constructs [25]. Subsequently, C-terminally HAtagged CK2 (Tyr182Phe), CK2 (Tyr188Phe) and CK2 (Tyr182Phe/ Tyr188Phe) constructs were subcloned into the pRc/CMV mammalian expression plasmid. All constructs generated were veried by sequencing. 2.3. Immunoprecipitation of CK2 and immune-complex CK2 assays Tetracycline-inducible human osteosarcoma U2OS cell lines with regulatory expression of CK2 were used in some experiments to

monitor tyrosine phosphorylation in cells [29]. Transfections of U2OS cells with the various CK2 constructs were performed using established methods [30]. Transfected parental U2OS cells or osteosarcoma cell lines with tetracycline-regulated expression of CK2 were harvested in TLB buffer (150 mM NaCl, 50 mM TrisCl (pH 7.5), 29.4 g/mL aprotinin, 1 mM PMSF, 20 g/mL leupeptin, and 7 g/ mL pepstatin A) containing 1% NP40 and 0.1% DOC. The cell lysate was sonicated for 3 10 s on ice and then centrifuged in a TLA100.2 rotor at 100 000 rpm at 4 C for 25 min. The supernatant was collected and a BCA protein assay (Pierce) was performed using bovine serum albumin (BSA) as a standard. Equal amounts of protein were incubated with 100 g of anti-HA (12CA5) antibodies for 2 h at 4 C. Thirty L of 1:1 Protein A Sepharose beads in lysis buffer was then added to the immunoprecipitate and incubated with mixing for a further 2 h at 4 C. Following the incubation, the Protein A beads were collected and washed with 430 second washes with TLB buffer containing 1% NP40 and 230 second washes with TLB buffer. The immunoprecipitated CK2 on the beads was then incubated for 30 min at 30 C with 200 200 U of protein phosphatase in a solution composed of 50 mM Tris Cl (pH 7.5), 0.1 mM Na2EDTA, 5 mM DTT, 0.01% Brij 35, and 2 mM MnCl2. To rephosphorylate CK2, the phosphatase cocktail was rst removed by aspiration and the Protein A beads were washed 2 30 s with TLB containing 1 mM sodium vanadate. Re-phosphorylation of the immunoprecipitated CK2 was performed by incubation for 20 min at 30 C in kinase assay buffer (150 mM NaCl, 50 mM TrisCl (pH 7.5), 1 mM DTT, 10 mM MgCl2, and 0.1 mM ATP or GTP). For assays performed with radioactive ATP, each reaction was supplemented with 5 Ci of -32P-ATP. To terminate kinase reactions, kinase assay buffer was removed, 50 L Laemmli buffer added and an aliquot loaded onto a 10% SDS-PAGE gel which was transferred to a PVDF membrane and immunoblotted with anti-biotin-conjugated-HA antibodies (anti-HA-biotin) or anti-phosphotyrosine antibodies (antipTyr). Western blotting was performed using established protocols [31]. Primary antibody concentrations used in these experiments are as follows: 1:2000 dilution anti-pTyr; 1:5000 anti-; 1:5000 anti-; 1:2000 anti-HA; 1:5000 anti-HA-biotin; and 1:2000 anti-Myc. Where indicated, densitometry was performed by scanning immunoblots and performing analysis with ImageQuant v1.2 software (Molecular Dynamics). Levels of pTyr were normalized according to the levels of HA-tagged CK2. Changes in pTyr phosphorylation in response to TBB treatment were normalized according to DMSO treatment. 2.4. Phosphoamino acid analysis Prior to phosphoamino acid analysis, radiolabelled proteins were separated by 10% SDS-PAGE, transferred to Immobilon membranes and detected on membranes by autoradiography. Partial hydrolysis of proteins was performed as previously described [32]. Two-dimensional separations of partial hydrolysis products were carried out according to the method of Cooper et al. [33]. 32P-labelled phosphoamino acids were visualized using a Phosphorimager and quantied using ImageQuant v1.2 software (Molecular Dynamics). 2.5. Large-scale anti-phosphotyrosine immunoprecipitation RS2.31 cells expressing HA-CK2 were seeded into 80150 mm tissue culture dishes in the presence of 1 g/mL tetracycline. On the next day, 40 plates were washed extensively to remove the tetracycline from the medium and allowed to grow another 48 h before harvesting. The RS2.31 cells were then harvested in TLB buffer with 1% NP40/0.1% DOC and cleared of insoluble material. Equal amounts of protein (20 mg) were incubated overnight at 4 C with 20 g of anti-pTyr antibodies. On the next day, 50 l of a 1:1 slurry of Protein A Sepharose was added to the mixture and allowed to tumble at 4 C for an additional 4 h. The beads were then washed 4 times with 1% NP40 in TLB buffer. The Protein A beads were subsequently

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incubated 2 10 min with 100 mM phenyl phosphate in PBS to elute the tyrosine-phosphorylated proteins. Both the beads and the eluate were run on 10% SDS-PAGE and subsequently silver-stained using established procedures [34]. 2.6. In-gel tryptic digestion and mass spectrometric analysis Silver-stained bands were cut out of the SDS-PAGE gel and in-gel digested with trypsin using established protocols with modications with respect to the extraction of peptides [3436]. The extracted peptides were desalted on home-made columns consisting of Eppendorf gel-loader tips packed with a 1 l each of POROS R2 and R3 resins (Applied Biosystems). Matrix-assisted laser desorption/ ionization-time-of-ight (MALDI-TOF) spectra of in-gel digests were examined on a Micromass MALDI-TOF mass spectrometer (Micromass, Manchester, UK) in positive ion reector mode. Spectra were collected between an m/z range of 900 to 4000. The MALDI-TOF analyzer was calibrated using angiotensin I, renin, and adenocorticotrophic hormone clip 1839 in a 1:2:3 ratio. Analysis of the spectra was performed using MassLynx 3.5 software. The list of masses obtained from the mass spectrometric analysis of each trypsindigested band was inputted into the Profound (http://prowl.rockefeller.edu/cgi-bin/ProFound) on-line web site to identify the protein within the silver-stained band. 2.7. Synthesis of peptide arrays The SPOT method was employed in the synthesis of peptide arrays for use in determining the tyrosine kinase specicity of CK2 [37]. Peptides were synthesized on Amino-PEG500-UC540 membranes derivatized with a polyethylene glycol spacer (Intavis AG). Stock solutions of amino acid derivatives (Peptides International) were prepared at a concentration of 0.33 M in a solution of 0.5 M HOBt/ NMP. Immediately prior to each cycle of synthesis, amino acid solutions were activated with the addition of one part DIC solution (1.1 M DIC in NMP) to three parts of the amino acid stock solution. The high-density peptide arrays (1536 peptide spots/8 12 cm membrane) were synthesized using the ASP222 SPOT robot with an extra ne needle tip. The amount of peptide synthesized at each spot on the membrane is estimated at approximately 5 nmol [38]. Each cycle of peptide synthesis consisted of (i) coupled spotting of amino acids on the membrane, (ii) capping of the membrane with a solution of 2% acetic anhydride in DMF (1 30 s, 1 5 min), (iii) wash with DMF (3 2 min), (iv) deprotection with 20% piperidine in DMF (10 min), (v) wash with DMF (4 2 min), (vi) wash with ethanol (2 2 min), and (vii) drying of membranes in a stream of cold air and assembly on the robot for the next cycle of synthesis. After the nal cycle in the peptide synthesis, the membrane was thoroughly dried overnight in a fume hood to prepare the peptides for side chain deprotection. Side chain deprotection was carried out on the membrane in the following steps: (i) deprotection in 95% triuoroacteic acid, 3% triisopropylsilane, 2% water for 2 h, (ii) wash with dichloromethane (4 2 min), (iii) wash with DMF (4 2 min), (iv) wash with ethanol (2 2 min), and (v) dry with a stream of cold air. 2.8. Phosphorylation of peptide arrays Individual peptide arrays were phosphorylated with recombinant CK2 holoenzyme (Calbiochem) or GST-CK2 [39,40] following the protocol of Tegge and Frank [41]. In summary, the peptide array was initially moistened with ethanol to assure solubilization of the peptides in the kinase buffer. The array was then washed with kinase buffer (50 mM TrisCl (pH 7.5), 30 mM MgCl2, 50 mM KCl, 1 mM DTT) and incubated overnight at room temperature in the same buffer. The following day, the incubation buffer was decanted and 6 mL of fresh kinase buffer was added to the array in a reaction trough. The array

and buffer were pre-incubated at 30 C for 1 h with rotation. The kinase reaction was initiated by the addition of 10 M ATP, 10 Ci [-32P]-ATP, and 65 units of either recombinant CK2 holoenzyme or GST-CK2 (1 unit is dened as the amount of enzyme that will catalyze the transfer of 1.0 pmol of phosphate to RRRDDDSDDD/min at 30 C). Reactions were typically performed for 20 min at 30 C, with the exception of assays that were performed for 10 and 40 min to verify the linearity of the enzymatic reaction. To terminate the reaction, the kinase buffer was decanted and the array was washed 15 times with 1 M NaCl and 10 times with water. In order to decrease the background noise on the array, the membrane was incubated in stripping solution (4 M guanidine hydrochloride, 1% SDS, 0.5% -mercaptoethanol) for 1 h in a 40 C water bath with shaking. The membrane was washed several times with water to remove precipitate from the stripping solution, washed with ethanol and dried. The peptide arrays were visualized using a Phosphorimager (Molecular Dynamics) and the amount of 32P incorporated into each spot was determined using ImageQuant TL software (Amersham Biosciences). 2.9. Scoring matrices for prediction of CK2 phosphorylation sites Data obtained from Phosphorimager analysis of peptide arrays were employed to develop scoring matrices for the prediction of candidate CK2 phosphorylation sites. Individual scoring matrices were employed for the prediction of serine phosphorylation sites, threonine phosphorylation sites and tyrosine phosphorylation sites. Furthermore, the prediction of tyrosine phosphorylation sites was performed separately for holoenzyme CK2, for CK2 and for CK2. Scoring matrices were based on the LOGO algorithm [42,43]. The score for each amino acid at a position was determined by comparing the signal

Fig. 1. CK2 is tyrosine phosphorylated in mammalian cells. RS2.31 cells expressing HACK2 (AC), or GV7.21 cells expressing kinase-inactive HA-CK2(K69/M) (B) were grown in the presence (+ TET) or absence ( TET) of tetracycline as indicated. Immunoprecipitations were performed with anti-phosphotyrosine antibodies (A), or with anti-HA antibodies (B,C). In A and B, immunoprecipitates were analysed on immunoblots with anti-CK2 antibodies or with anti-phosphotyrosine antibodies. The Ctrl lane represents an antibody control (i.e. no extract). In B, the upper panels illustrate samples derived from cells expressing active HA-CK2 while samples derived from cells expressing catalytically-inactive HA-CK2(K69/M) are shown in the lower panels. In C, anti-HA immunoprecipitates were treated with or without phosphatase (P'ase) as indicated prior to analysis as in panels A and B.

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for each amino acid to the total signal contributed by all amino acids at that position according to the following formula. Score f a; plog2 f a; p log2
a1 n n f a;p

Where: f (a,p) n log2n

n a1

represents the signal of amino acid a over the total signal at position p represents the number of amino acids examined represents the maximum uncertainty
f a;p

one amino acid has a signicant signal at a particular position, that amino acid will receive a score approaching the highest score. Any amino acid that was not examined was given the average score of those amino acids that were examined. The score of a peptide is derived from the sum of the scores for all of its constituent amino acids. A motifscanning program was developed to retrieve serine, threonine or tyrosine-containing peptides from the Swiss Swiss-Prot database (http://ca.expasy.org/sprot/). Peptides are ranked in descending order. 3. Results 3.1. CK2 is tyrosine phosphorylated in cells and exhibits tyrosine kinase activity in vitro Although earlier studies had dismissed the possibility, recent observations primarily based on usage of bacterially expressed CK2 have raised the prospect that CK2 is tyrosine phosphorylated under some circumstances [25]. However, these studies have yielded limited insights regarding the physiological relevance of the tyrosine phosphorylation of CK2. Consequently, to systematically examine this possibility in mammalian cells, we isolated CK2 from cell lines with tetracycline-regulated expression of either of the closely related

f a; p log2
n n a1

represents the uncertainty at position p

f a;p

log2 f a; p log2

represents the amount of information

presented at position p. Using this formula, two extremes are possible. In one instance, if all amino acids at a position display the same signal strength, which means that there is no preference at that position, this position has maximum uncertainty and every amino acid is scored 0. On the other hand, if only

Fig. 2. CK2 is tyrosine phosphorylated in vitro. (A) CK2-HA (left panels) or catalytically-inactive CK2 (K68/M)-HA (right panels) were isolated by immunoprecipitation performed with anti-HA antibodies. Following treatment of anti-HA immunoprecipitates with phosphatase, the immune-complexes were used for in vitro kinase assays using ATP or GTP as substrate in the presence or absence of a CK2 selective inhibitor (TBB) at a concentration of 60 M or a Src family kinase inhibitor (PP2) as indicated. Following in vitro kinase assays, immune-complexes were analysed on immunoblots using anti-HA or anti-phosphotyrosine antibodies. Three times more immunoprecipitate was analysed on anti-pTyr immunoblots than on anti-HA or anti-CK2 immunoblots. (B) Immune-complex kinase assays of HA-CK2 immunoprecipitates were performed using -32P-ATP in the presence or absence of TBB or PP2. (C) Immune-complex kinase assays using -32P-ATP were performed with CK2-HA (left panel) or catalytically-inactive CK2 (K68/M)-HA (right panel) immunoprecipitates. Phosphoamino acid analysis was performed on the indicated bands. Relative levels of each phosphoamino acid for each of the indicated phosphoproteins are summarized in the tables below each of the panels. Quantitative assessment of phosphoamino acid levels was not possible for weakly phosphorylated bands in C. In these instances, the qualitative presence of each phosphoamino acid is indicated if any of the phosphoamino acids was detectable. Similarly () signies levels of 32P incorporation that were too low to reliably quantify (typically less than 2% of total 32P detected).

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isoforms of CK2. It is evident from Fig. 1 that HA-tagged CK2 is present in anti-phosphotyrosine immunoprecipitates (panel A) and that a tyrosine-phosphorylated protein which appears to co-migrate with HA-CK2 is present when CK2 is immunoprecipitated (panel B). Notably, these bands are absent when these cells are grown in the presence of tetracycline which suppresses the expression of HACK2. Moreover, tyrosine phosphorylation of CK2 is not observed when catalytically-inactive CK2 is isolated from cells (lower panel in Fig. 1B) and is eliminated upon phosphatase treatment of immunoprecipitated HA-CK2 validating the specicity of the immunoreactivity (Fig. 1C). Similar ndings were obtained with a cell line that exhibits tetracycline-regulated expression of CK2-HA, the other major catalytic subunit of CK2 (data not shown), indicating that both isozymic forms of CK2 undergo tyrosine phosphorylation in cells. To further characterize the tyrosine phosphorylation of CK2, immunecomplexes containing CK2 were dephosphorylated as in Fig. 1C and then subjected to in vitro re-phosphorylation assays. As illustrated in Fig. 2A, tyrosine phosphorylation of CK2-HA was readily detected by antiphosphotyrosine antibodies following the re-phosphorylation assays. To determine whether the tyrosine re-phosphorylation of CK2 was the result of co-precipitating protein tyrosine kinases, re-phosphorylation assays were performed in the presence of a selective inhibitor of CK2 (TBB) and PP2, a selective inhibitor of Src family protein tyrosine kinases which have been shown to phosphorylate CK2 in vitro [44]. In these assays, tyrosine phosphorylation is dramatically inhibited by TBB, but not by PP2, consistent with the view that tyrosine phosphorylation occurs through an autocatalytic event. The ability of GTP to substitute for ATP as a phosphate donor in the re-phosphorylation assay is also consistent with the conclusion that CK2 is responsible for tyrosine phosphorylation since CK2 is one of the few protein kinases able to utilize GTP as a substrate. Finally, tyrosine phosphorylation is not observed with catalyticallyinactive CK2 in re-phosphorylation assays (Fig. 2A, right panels). Collectively, these observations indicate that tyrosine re-phosphorylation of CK2 in these assays is dependent on CK2 activity, a somewhat unexpected observation in light of the traditional classication of CK2 as a protein serine/threonine kinase. Similar ndings were obtained with HACK2 (data not shown) indicating that both CK2 isoforms display tyrosine autophosphorylation activity. To conrm that CK2 is tyrosine phosphorylated and to examine the relative levels of phosphotyrosine in relation to phosphoserine and

Fig. 4. Tyrosine phosphorylation of Y182F and Y188F mutants of CK2. U2OS osteosarcoma cells were transfected with pRc/CMV constructs encoding wild-type CK2-HA (WT) or mutants of CK2-HA with tyrosine to phenylalanine mutations at positions 182 and 188. At 48 h following transfection, anti-HA immunoprecipitates were performed and analysed by immunoblots using biotin-conjugated anti-HA antibodies (A) or anti-phosphotyrosine antibodies (B). Control lanes include an antibody without extract (Ab) and a transfection performed with an empty vector (Vector). Five times more immunoprecipitate was analysed on anti-pTyr immunoblots than on the anti-HA immunoblot.

Fig. 3. Tyrosine phosphorylation of CK2 is inhibited by TBB in cells. RS2.31 cells were treated with the CK2 selective inhibitor (TBB) at a concentration of 60 M, with a Src family kinase inhibitor (PP2) at a concentration of 2 M or with carrier (DMSO) for 2 h prior to the induction of HA-CK2 by the removal of tetracycline from the growth medium. Following 12 h of induction in the continued presence of these agents, HACK2 was isolated by immunoprecipitation with anti-HA antibodies from 2.5 mg of whole cell lysate and analysed on immunoblots using anti-HA antibodies (A) or antiphosphotyrosine antibodies (B). Controls include a lane containing anti-HA antibodies without extract (Ab) or an immunoprecipitation performed from cells grown in the presence of tetracycline (+ TET) which prevents the induction of HA-CK2. Three times more immunoprecipitate was analysed on anti-pTyr immunoblots than on the anti-HA immunoblots.

phosphothreonine, re-phosphorylation assays were repeated in the presence of -32P-ATP (Fig. 2B, C). While a number of phosphoproteins are evident, with either HA-CK2 (Fig. 2B) or CK2-HA (Fig. 2C), two of the most prominent phosphoproteins co-migrate with the catalytic (HA-CK2 or CK2-HA) and regulatory subunits (Myc-CK2) of CK2. Notably, in accordance with immunoblot analysis performed with anti-phosphotyrosine antibodies, phosphoamino acid analysis clearly reveals that either of the catalytic CK2 subunits contains substantial levels of phosphotyrosine. Furthermore, phosphorylation of the catalytic subunits was appreciably decreased with kinase-inactive CK2-HA (Fig. 2C) or in the presence of the CK2 inhibitor TBB but not in the presence of PP2, a tyrosine kinase inhibitor (Fig. 2B). As expected the regulatory CK2 subunit is almost exclusively phosphorylated on serine with no evidence of phosphotyrosine. Collectively, these observations conrm that the catalytic subunits of CK2 do indeed undergo tyrosine phosphorylation and indicate that tyrosine re-phosphorylation of CK2 in these assays is dependent on CK2 activity. Experiments illustrated in Figs. 1 and 2 suggest that tyrosine phosphorylation of CK2 is in large part dependent on the activity of CK2 itself. To extend this observation, we examined the tyrosine phosphorylation state of CK2 in cells that were treated with the selective CK2 inhibitor TBB or with the Src family kinase inhibitor PP2. As was the case with in vitro re-phosphorylation assays (Fig. 2), TBB evokes a dramatic decrease in the tyrosine phosphorylation of CK2 suggesting that CK2 activity is required for a large part of its tyrosine phosphorylation in mammalian cells (Fig. 3). Densitometric analysis of Fig. 3 revealed that TBB decreased tyrosine phosphorylation of HACK2 by approximately 75% whereas the tyrosine phosphorylation of HA-CK2 was unaffected by PP2. We do not know whether the incomplete inhibition of tyrosine phosphorylation of CK2 by TBB is the result of incomplete inhibition of CK2 or whether it indicates that other protein tyrosine kinases contribute to the tyrosine phosphorylation of CK2 in cells. However, decreased tyrosine phosphorylation of CK2 is observed upon examination of catalytically-inactive CK2 (Fig. 1B). 3.2. Tyrosine phosphorylation of CK2 is diminished by mutation of Tyr182 and Tyr188 In vitro studies with puried recombinant CK2 had shown that mutation of Tyr182, and to a lesser extent Tyr188, to phenylalanine drastically reduces tyrosine autophosphorylation [25]. Accordingly,

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we examined the phosphorylation of Tyr182 and/or Tyr188 by expressing mutant forms of CK2 with Tyr to Phe mutations at each of these sites in mammalian cells. As illustrated in Fig. 4, mutation of Tyr182 or Tyr188 leads to a dramatic decrease in the tyrosine phosphorylation of CK2 while mutation of both residues results in what appears to be complete loss of tyrosine phosphorylation. These results are in good agreement with what was seen in vitro and suggest that Tyr182 and Tyr188 are major sites of tyrosine phosphorylation. We cannot however exclude the possibility that the catalytic subunits of CK2 may also be tyrosine phosphorylated at other sites that may be dependent on the integrity of Tyr182 and/or Tyr188. 3.3. Tetrameric CK2 holoenzyme contains tyrosine-phosphorylated catalytic subunits Although the nding that CK2 displays tyrosine kinase activity challenges the traditional view of CK2 as a protein serine/threonine kinase, it is notable that a recent report had demonstrated tyrosine autophosphorylation of either bacterially expressed and puried CK2 or CK2 [25]. The regulatory CK2 subunit inhibited this autophosphorylation event suggesting that tyrosine phosphorylation may be restricted to the isolated catalytic CK2 subunits [25], although the physiological relevance of these observations within mammalian cells was not examined. Consequently, to evaluate the tyrosine phosphorylation of CK2 in the context of tetrameric CK2 complexes that were formed within mammalian cells, CK2 was isolated by reciprocal immunoprecipitations of either the CK2 or CK2 subunits. As illustrated in Fig. 5, tyrosine-phosphorylated CK2-HA is detected in complexes that have been isolated using either anti-HA antibodies to isolate CK2-HA or with anti-Myc antibodies to isolate Myc-CK2. Although these results do not provide any information as to whether CK2-HA was phosphorylated in the presence or absence of CK2, these results clearly illustrate that tetrameric CK2 complexes do indeed contain tyrosine-phosphorylated CK2-HA.
Fig. 6. Tyrosine phosphorylation proles in cells with elevated CK2 expression. Tetracycline was removed from growth medium of RS2.31 cells to induce the expression of HA-CK2 for 48 h. As a control, identical cultures of RS2.31 cells were maintained in the presence of tetracycline to prevent expression of HA-CK2. Extracts (20 mg of total protein) from each of these cell cultures were subjected to immunoprecipitations using 20 g of anti-phosphotyrosine antibodies. Proteins were eluted from the immunoprecipitates using 100 M phenyl phosphate. (A) Eluted proteins were visualized by silver stain following SDS-PAGE. (B) Eluted proteins were analysed on immunoblots using anti-phosphotyrosine antibodies. Bands that are elevated in samples derived from cells with elevated expression of HA-CK2 are marked with arrowheads. An asterisk denotes a band that was identied by mass spectrometry as CK2. The positions of the heavy and light chains of IgG and the molecular weight markers are indicated.

3.4. Elevated expression of CK2 correlates with increased tyrosine phosphorylation proles On the basis that CK2 displays protein tyrosine kinase in mammalian cells, it may be predicted that increased expression of CK2 would yield increased tyrosine phosphorylation of itself and potentially other targets. To test this prediction, we examined the tyrosine phosphorylation proles in cells with tetracycline-regulated expression of CK2 by performing anti-phosphotyrosine immunoprecipitations from cells grown in the presence or absence of tetracycline. In these cells, elevated expression of CK2 yields increases in a number of proteins as observed by silver staining of proteins isolated in these immunoprecipitates and by immunoblot analysis of these immunoprecipitates using anti-phosphotyrosine antibodies (Fig. 6). Detection of proteins in the former case does not necessarily indicate direct tyrosine phosphorylation of the observed protein since some of these proteins could be isolated through complex formation with other tyrosine-phosphorylated protein. In this respect, phosphorylation by CK2 could result in indirect changes in tyrosine phosphorylation through alterations in tyrosine kinase or phosphatase activity. One of the major phosphotyrosine-containing proteins (denoted by on Fig. 6) was identied by peptide mass ngerprinting and mass spectrometry as CK2 itself, a result that is consistent with the characterization of this protein as a tyrosinephosphorylated protein (Figs. 1 and 2). Attempts to identify other bands by mass spectrometry did not yield denitive identication. Nevertheless, the banding pattern observed in Fig. 6 suggests that a number of proteins in addition to CK2 exhibit elevated tyrosine phosphorylation in cells with increased CK2 levels. Coupled with the observation that CK2 exhibits protein tyrosine kinase activity in these cells, it is tantalizing to speculate that some of these proteins may in fact be direct targets for CK2. 3.5. Evaluation of the specicity determinants for CK2 phosphorylation using peptide arrays

Fig. 5. Tyrosine-phosphorylated CK2 exists in tetrameric CK2 holoenzyme complexes in mammalian cells. Extracts prepared from RS3.22 cells expressing CK2-HA together with Myc-CK2 were subjected to immunoprecipitation with anti-HA or anti-Myc antibodies. Immunoprecipitates were analysed on immunoblots using biotin-conjugated anti-HA antibodies (A), anti-phosphotyrosine antibodies (B), or anti-Myc antibodies (C). Anti-HA and anti-Myc antibodies were loaded as controls without cell extracts in adjacent lanes. The positions of CK2-HA, Myc-CK2 and the heavy and light chains of immunoglobulin G are noted. Five times more immunoprecipitate was analysed on anti-pTyr immunoblots than on the anti-HA or anti-Myc immunoblots.

The demonstration above that mammalian CK2 can phosphorylate tyrosine residues in vitro and that CK2 itself is tyrosine phosphorylated in mammalian cells raises the prospect that other proteins are tyrosine phosphorylated by CK2 in cells. To facilitate efforts to address this possibility, studies were performed using peptide arrays comprising sequences derived from CK2 or CK2 as well as peptides modeled after Fpr3-derived tyrosine phosphorylation sites which had been

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previously shown to be phosphorylated by CK2 [23,24]. Arrays with CK2-derived peptides did not reveal any sequences that were signicantly phosphorylated by CK2 (data not shown) suggesting that linear peptide sequences based on CK2 do not effectively recapitulate the site(s) that are phosphorylated in the intact protein. By comparison, as previously shown, sequences based on Fpr3 were signi cantly phosphorylated by CK2 [23,24]. Accordingly, we employed oriented peptide arrays based on Fpr3-derived sequences [38] to perform a comprehensive positional scan of residues that inuence phosphorylation by CK2. Examination of Fig. 7 reveals that, as expected, acidic residues in the +1 and the +3 position are selective determinants for phosphorylation of serine (panel A) or threonine (panel B) residues. Furthermore, although not strictly required, acidic residues in other positions are favorable determinants for serine/ threonine phosphorylation. By comparison, tyrosine phosphorylation by tetrameric CK2 reveals that an aspartic acid in the +1 position is a requirement for phosphorylation (Fig. 7C). Similarly, recombinant CK2 (Fig. 7D) or CK2 (Fig. 7E) also exhibit a requirement for aspartic acid in the +1 position. Furthermore, with tetrameric CK2 (Fig. 7C), recombinant CK2 (Fig. 7D) or recombinant CK2 (Fig. 7E) other positions exhibit less selectivity than the +1 position although it is intriguing that there may be some preference for aspartic acid at the 2 position and that aspartic acid may actually be a negative determinant at the 1 position. Collectively, these data corroborate the concept that the substrate specicity of CK2 for tyrosine phosphorylation is signicantly distinct from its serine/threonine phosphorylation [24] and provide a framework for further investigation directed towards the search for proteins that are tyrosine phosphorylated by CK2 in mammalian cells. As a logical extension of the identication of specicity determinants for tyrosine phosphorylation by CK2, we were interested in the identication of candidate proteins containing these specicity determinants. Accordingly, scoring matrices using the data obtained from peptide arrays illustrated in Fig. 7 were employed to predict candidate CK2 substrates phosphorylated at serine (Table 1A), threonine (Table 1B) or tyrosine residues (Table 2). In the interests of

brevity, only the top scoring peptides from each of the scoring matrices are illustrated with comprehensive lists of the top 1536 scoring peptides available as Supplementary data. Examination of Table 1 demonstrates that serine or threonine phosphorylation sites predicted on the basis of peptide array data conform to the previously dened CK2 consensus (i.e. S/T-X-X-E/D) validating the utility of these arrays for delineation of the specicity determinants for CK2. Further reinforcing the utility of these predictions is the observation that several of the predicted serine or threonine phosphorylation sites are indeed phosphorylated in cells as indicated by their presence in Phosida (http://www.phosida.com) and PhosphoELM (http://phospho. elm.eu.org) databases. It is also notable that at least 2 of the highest scoring predicted CK2 substrates, namely elongation factor 1-beta [45] and VAMP4 [46] have previously been identied as CK2 substrates. Collectively, these observations indicate that the scoring matrices based on delineation of specicity determinants using peptide arrays can be employed to make valid predictions regarding CK2 substrates. Examination of Table 2 reveals predicted tyrosine-phosphorylated substrates for CK2. With CK2 holoenzyme and with CK2 or CK2, the highest scoring peptides all contained an aspartic acid in the +1 position. However, whereas the highest scoring peptides predicted for the CK2 holoenzyme contained a number of additional acidic residues, it is notable that the highest scoring peptides for CK2 or CK2 contain hydrophobic residues, particularly on the N-terminal side of the predicted phosphorylation site. These predictions also reveal that while there may be differences between the CK2 holoenzyme and isolated catalytic subunits, the predicted substrates for CK2 and CK2 are very similar. In fact, there are a number of overlapping candidate phosphorylation sites evident in the top 10 highest scoring peptides for each of these two CK2 isoforms (Table 2B vs. Table 2C). Since the prospect that CK2 exhibits tyrosine kinase activity in mammalian cells has not been previously considered, it is not as yet possible to determine whether or not any of these predicted tyrosinephosphorylated CK2 substrates are indeed phosphorylated by CK2 in cells. Nevertheless, it is anticipated that prediction of candidate tyrosine-phosphorylated CK2 substrates could represent an important

Fig. 7. Peptide arrays to evaluate the specicity determinants for phosphorylation by CK2. Peptides containing serine (A), threonine (B) or tyrosine (C) as phospho-acceptor were synthesized on separate peptide arrays using the Fpr3-derived sequence DEDDDIXDEEDFDL with X designating the position of the respective phospho-acceptor. Substitutions were made into these sequences at each position with the amino acids indicated across the bottom of each array. (D, E) Similar peptide arrays with tyrosine as phospho-acceptor were separately synthesized and kinase assays performed as described in the Experimental section using recombinant CK2 holoenzyme (AC), recombinant GST-CK2 (D) or GST-CK2 (E). Following phosphorylation and extensive washing as described in the Experimental section, 32P incorporation into peptides was assessed with a Phosphorimager.

G. Vilk et al. / Cellular Signalling 20 (2008) 19421951 Table 1 Predicted CK2 substrates based on scoring matrix from data in Fig. 7 Sequence Protein name Phosidaa PhosphoELMa

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A. Substrates with serine phosphorylation sites (predicted from Fig. 7A) FLFEGLSDDEDDF Ubiquitin-like protein 7 X NLLEDDSDEEEDF Vesicle-associated membrane protein 4 X DDIFDGSDDEEES Charged multivesicular body protein 2b DIDLFGSDDEEES Elongation factor 1-beta X DGGWEWSDDEFDE Serine/threonine-protein kinase OSR1 X DEDDEESDEEEEE Death domain-associated protein 6 (Daxx) DSETSDSDDEWTF RNA polymerase-associated protein RTF1 homolog DIDLFGSDNEEED Elongation factor 1-delta (EF-1-delta) X DVIVIDSDDEEED Nucleoprotein TPR SLLFDTSDDEELR Fasciculation and elongation protein zeta 2 EEGDVDSDEEEEE Major centromere autoantigen B DEFDLDSDDELQI PHD nger protein 8

X X X

B. Substrates with threonine phosphorylation sites (predicted from Fig. 7B) EESDDETDGEDGE Zinc nger and BTB domain-containing protein 45 EEDEEETDDSDTW Zinc ngers and homeoboxes protein 1 GLDEEDTDFEEED Serine/threonine-protein kinase Nek3 X GYDDWETDEDEES Tripartite motif-containing protein 26 PGEDGDTDEEFWI Cat eye syndrome critical region protein 2 EEEEEDTDEDDHL RNA polymerase II subunit A C-terminal domain phosphatase EDDDFETDSDDLI Chromodomain-helicase-DNA-binding protein 2 SEEESETEEESED Tripartite motif-containing protein 44 DDWNEDTDGEWEA Calmegin precursor EEMESETEEETIF G-protein coupled receptor-associated sorting protein 1 QWSEEETEDEEEE Proliferating-cell nucleolar antigen p120 X X EDEDEDTEEDEEE PR domain zinc nger protein 10 The presence of individual phosphopeptides in Phosida (http://www.phosida.com/) [63] or PhosphoELM (http://phospho.elm.eu.org/) [64] databases is indicated (X). Only the 12 highest scoring peptides are illustrated. Presentation of the 1536 top scoring predicted CK2 substrates is provided as Supplementary data.
a

are related in sequence to protein serine/threonine kinase but exhibit the ability to autophosphorylate and/or phosphorylate exogenous substrates, on serine/threonine and tyrosine residues. This now appears to be the case for CK2 that turns out to also exhibit the ability to autophosphorylate on tyrosine residues and to phosphorylate tyrosine residues within exogenous substrates both in vitro and in vivo although it had been consistently classied as a protein serine/threonine kinase on the basis of its sequence and a plethora of in vitro and in vivo evidence. In this study, mutation of tyrosine residues, Tyr182 and Tyr188, that had previously been shown to be important for the in vitro tyrosine autophosphorylation of isolated recombinant CK2 or CK2 [25] resulted in a dramatic decrease in the extent of tyrosine phosphorylation. These results suggest that the previously observed in vitro autophosphorylation of CK2 does indeed occur within mammalian cells. As previously noted, Tyr182 and Tyr188 are of interest because of their proximity to the activation loop which is involved in the regulation of many protein kinases. In this respect, Tyr182 is actually included within the activation loop where it makes important contacts with the N-terminal segment which in CK2 keeps the catalytic site in its active conformation [50,51]. For the most part, the decreased tyrosine phosphorylation that we observed with the Tyr182/Phe and Tyr188/Phe mutants corresponded with the results obtained in vitro with the isolated recombinant CK2 subunits. However, it is noteworthy that we observe tyrosine phosphorylation of catalytic CK2 subunits isolated from mammalian cells whereas tyrosine phosphorylation was

Table 2 Prediction of candidate tyrosine-phosphorylated CK2 substrates using scoring matrix based on data presented in Fig. 7 Sequence Protein name

resource for consideration of the role of CK2 in catalyzing tyrosine phosphorylation in mammalian cells. 4. Discussion Although protein kinase CK2 has traditionally been classied as a protein serine/threonine kinase, recent studies have raised the prospect that CK2 may phosphorylate tyrosine residues in yeast and under some circumstances in vitro [2325,47,48]. However, analysis of the kinetic parameters for phosphorylation of tyrosyl peptides by CK2 in vitro suggested that serine/threonine phosphorylation is signicantly more favorable than tyrosine phosphorylation within similar structural contexts [24]. Furthermore, it might have been envisaged that protein serine/threonine kinases such as CK2 that exhibit a low level of protein tyrosine kinase activity would have to compensate for the absence of dedicated protein tyrosine kinases in organisms such as yeast where these latter enzymes are absent. Consequently, the physiological relevance of the protein tyrosine kinase activity of CK2 in mammalian cells that possess a myriad of dedicated protein tyrosine kinase was questionable. The data presented in this paper denitively demonstrates that CK2 can indeed phosphorylate tyrosine residues also in mammalian cells. Furthermore, increased tyrosine phosphorylation of a number of proteins, including CK2 itself, accompanies elevated expression of CK2 in human osteosarcoma cells. Despite the fact that earlier classications had suggested that there was a clear distinction between protein tyrosine kinases and protein serine/threonine kinases, the identication of protein kinases such as MKK1/2 that efciently phosphorylate threonine and tyrosine residues within substrate protein leads to the classication of dual-specicity kinases [49]. In general, dual-specicity kinases are those kinases that

A. Substrates predicted on the basis of phosphorylation with CK2 holoenzyme (Fig. 7C) DEEEEGYDDDDDD Serine/threonine-protein kinase RIO1 EDEEEDYDDDEEE Transcription initiation factor IIA subunit 1 EEDEDLYDCVENE Proto-oncogene vav. WEDEDFYDSDDDT Kanadaptin (kidney anion exchanger adapter protein) EEEELAYDWSDNN Microtubule-actin cross-linking factor 1 DNWDDTYDYWCDP Lethal(3)malignant brain tumor-like protein DGWDSEYDQWVDC Lethal(3)malignant brain tumor-like 2 protein ESSEDQYDDLYVF B-cell scaffold protein with ankyrin repeats EDEEDEYDYDYES Phospholipase C-epsilon-1 EEEEEEYDEEEEE Transcription elongation factor SPT5 B. Substrates predicted on the basis of phosphorylation with CK2 (Fig. 7D) LDLYDNQ Leucine-rich repeat-containing protein 49 Protein phosphatase 1 regulatory subunit 7 QDLYDNP Claudin-18 LLLYDNA Ubiquitin ligase protein MIB2 FDLYDDW SH3 domain-binding protein 1 (3BP-1) LFLYDDD DNA (cytosine-5)-methyltransferase 3-like LRLYDNP Leucine-rich repeat-containing protein 15 precursor (hLib) Leucine-rich repeat-containing protein 17 precursor (p37NB) FDLYDVD Copine-5 (Copine V) Copine-8 (Copine VIII) LDLYDDF Chondroitin sulfate proteoglycan 5 precursor FNLYDDW Pre-mRNA-processing-splicing factor 8 DDLYDQD Lipolysis-stimulated lipoprotein receptor C. Substrates predicted on the basis of phosphorylation with CK2 (Fig. 7E) FDLYDDW SH3 domain-binding protein 1 (3BP-1) VELYDHD Dysferlin (Dystrophy-associated fer-1-like protein) PDLYDDD Septin-4 FDLYDVD Copine-5 (Copine V) Copine-8 (Copine VIII) LFLYDDD DNA (cytosine-5)-methyltransferase 3-like PSPYDHD SH3 protein expressed in lymphocytes homolog FNLYDDW Pre-mRNA-processing-splicing factor 8 WALYDHL Complement component 1 Q subcomponent-binding protein, LELYDRD U3 small nucleolar RNA-associated protein 15 homolog LDPYDRH RNA-binding protein 4 (RNA-binding motif protein 4) For brevity, only the 10 highest scoring peptides are illustrated. Presentation of the 1536 top scoring predicted CK2 substrates in each category is provided as Supplementary data.

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not detectable when recombinant CK2 was isolated from bacteria [44]. We do not know the precise reason for this apparent discrepancy but speculate that factors present in mammalian cells may modulate its tyrosine phosphorylation in a manner distinct from that observed with recombinant CK2 in bacteria. It is also notable that, since the regulatory CK2 subunit was previously shown to inhibit the in vitro autophosphorylation of catalytic CK2 subunits, it may have been somewhat unexpected to detect tyrosine-phosphorylated CK2 within tetrameric CK2 complexes. However, it is important to emphasize that our studies do not determine whether tyrosine phosphorylation occurred prior to, or after, formation of tetrameric CK2 complexes. Although our results suggest that tyrosine phosphorylation of Tyr182 and Tyr188 make a major contribution to the tyrosine phosphorylation of CK2, our results do not exclude the possibility that these residues indirectly inuence the tyrosine phosphorylation of CK2, through autophosphorylation or through phosphorylation by other protein tyrosine kinases. In this regard, CK2 has recently been shown to be tyrosine phosphorylated in Jurkat cells, in cells expressing activated forms of Abl and by Src family protein tyrosine kinases in vitro [44,52]. Although the ability of CK2 to phosphorylate tyrosine residues on exogenous substrates had been previously recognized, the functional relevance of this observation was overlooked. In particular, the physiological importance of these ndings had been questioned because of the unfavorable kinetic parameters that had been observed for tyrosine phosphorylation in vitro [24] and because of the overwhelming body of research that has been focused on serine/ threonine phosphorylation by CK2. The former observation was suggestive of an in vitro artifact hardly reecting a biologically relevant property. However, more recent studies in yeast have reinforced the physiological relevance of tyrosine phosphorylation by CK2 since the inhibition of CK2 by TBB together with the over-expression of low molecular weight protein tyrosine phosphatases resulted in severely impaired growth [53]. The present study also clearly demonstrates that CK2 does indeed phosphorylate tyrosine residues within mammalian cells. This is an important nding because it expands the possibilities for the identication of its cellular targets. It may be tempting for example to speculate that the increased levels of CK2 that are invariably observed in tumors and transformed cells [1114] may contribute to aberrant proliferation by catalysing the mis-phosphorylation or hyper-phosphorylation of tyrosine residues that play a central role in these processes. In this regard, it is intriguing that emodin, an anti-cancer agent that was originally reported to be a protein tyrosine kinase inhibitor [54], inhibits CK2 more potently than it does protein tyrosine kinases [55]. In light of the ability of CK2 to phosphorylate tyrosine residues in mammalian cells, it may be of interest to re-address the mechanisms by which emodin and related agents affect tumor cell growth and survival. In addition to CK2 itself, the only protein that has been shown thus far to be tyrosine phosphorylated by CK2 in cells is the immunophilin Fpr3 in yeast [23]. Given that CK2 has distinguished itself as an enzyme with a very large number of substrates [19], it is almost certain that other physiological targets for the tyrosine kinase activity of CK2 will be identied in the future. In this regard, it is noteworthy that the expression of a dominant negative form of small tyrosine phosphatase 1 (Stp1) in yeast led to the identication of 12 tyrosine-phosphorylated targets [56]. Remarkably, all twelve contained tyrosyl sequences with the features of CK2 phospho-acceptor sites suggesting that these proteins may be candidate CK2 substrates. It may also be signicant that, while the tyrosine phosphorylation site of Fpr3 bears a resemblance to the canonical consensus for serine/threonine phosphorylation by CK2 [24], the sequence of the autophosphorylation sites of the catalytic subunits of CK2 is distinct from this consensus. It is conceivable that local events that occur during autophosphorylation permit the phosphorylation of sub-optimal sites. Furthermore, as illustrated by the peptide arrays (Fig. 7) the determinants for tyrosine phosphorylation by CK2 do not rigorously conform to its canonical consensus for serine/

threonine phosphorylation. The substrate specicity of CK2 as deduced from peptide arrays in the present study was consistent with results obtained by kinetic analysis of soluble tyrosine peptide substrates [24], highlighting the requirement for aspartic acid at Tyr + 1. Furthermore, the proposed consensus sequence conforms to a molecular model of interactions between the CK2 active site and a tyrosine peptide as established by Marin et al. [24]. In this model, Lysine 195 and Lysine 198 in the activation loop of CK2 interact with the carboxylate side chain of aspartic acid at Tyr+ 1. The aspartic acid at Tyr 2 protrudes into the active site of the kinase, whereas the Tyr 1 and Tyr + 3 are less likely to interact with the catalytic region of CK2. With respect to possible targets for the tyrosine kinase activity of CK2 in mammalian cells, it is interesting to note that the target specicities of other kinases including the serine/threonine kinase PKA and the tyrosine kinase Abl have been successfully determined using peptide array analysis [38,57]. In both cases, the substrate specicity was already established through synthetic peptides and display libraries. Peptide array libraries that were phosphorylated with PKA and Abl generated results that were consistent with the accepted consensus sequences. These results were further assessed with bioinformatics approaches to predict in vivo target sites. The Scansite program (http://scansite.mit.edu) successfully identied known substrates of PKA and Abl in the Swiss-Prot database using the specicity motif derived from each peptide array. An analogous strategy was employed in the present study to generate a list of potential substrates of CK2 (Table 2). Given the complex roles of CK2 in various aspects of cell regulation [58], it is not inconceivable that the proteins identied by this bioinformatics analysis will include proteins that turn out to be bona de CK2 substrates. It is also noteworthy that potential tyrosine phosphorylation sites (i.e. Y preceded by a hydrophobic residue and followed by D) reside within CK2 itself. In this respect, the LGTEDLY255DYIDKY site in CK2 was recently shown to be tyrosine phosphorylated by Src-related kinases Lyn and c-Fgr in an in vitro assay [44]. Although PP2 inhibited this tyrosine phosphorylation of CK2, it is noteworthy that the associated tyrosine phosphorylation was also prevented by the CK2 specic inhibitor TBB that is entirely ineffective on Src kinase activity. Moreover, there is a degree of sequence conservation surrounding tyrosine 255 in CK2 and CK2 among animals, and the side chain of this residue is surface exposed. With this information it is tempting to speculate that CK2 catalyzes an intermolecular phosphorylation of tyrosine 255 on the CK2 subunit. It is also conceivable that the phosphorylation of Ser/Thr residues by other kinases might generate the consensus for subsequent tyrosine phosphorylation by CK2. In a way this is also the case for the phosphorylation of Tyr184 of the yeast immunophilin Fpr3 whose phosphorylation by CK2 is potentiated by the previous phosphorylation of Ser186 [23]. Another tantalizing linkage between CK2 and the regulation of protein tyrosine phosphorylation in mammalian cells is the association between CK2 and protein tyrosine phosphatases such as CD45 [59,60], PTP-S2 [61] and possibly PTEN [62]. These and possibly other tyrosine phosphatases associated with CK2 could closely regulate tyrosine phosphorylation on CK2 itself as well as the targets of CK2. Overall, the demonstration that CK2 displays dual-specicity kinase activity in mammalian cells adds another dramatic layer of complexity to a family of enzymes that has already been implicated in a broad array of regulatory events. For the future, it is clearly imperative that the ability of CK2 to phosphorylate tyrosine residues be taken into consideration when deciphering its complex role(s) in biological processes such as transformation and tumorigenesis. Acknowledgements This work was supported by grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada with funds from the Canadian Cancer Society to DWL, from the Natural

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Sciences Engineering Research Council and the Ontario Research and Development Challenge Fund to GL, from AIRC and the European Commission, PROKINASERESEARCH 503467 (to LAP) and Genome Canada (to SSCL and DWL). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellsig.2008.07.002. References
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