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Molecular and Cellular Biochemistry 191: 229–234, 1999.© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Multiple forms of protein kinase CK2 present inleukemic cells: In vitro study of its origin byproteolysis

Joan Roig,1 Andreas Krehan,2 Dolors Colomer,3 Walter Pyerin,2 EmilioItarte1 and Maria Plana1

1Departament de Bioquímica i Biologia Molecular, Unitat de Ciències, Universitat Autònoma de Barcelona, Bellaterra,Cerdanyola; 2Biochemische Zellphysiologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 3Serveid’Hematologia Biològica, Hospital Clinic, Barcelona, Spain

Abstract

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunitsin vitro. In both cases, CK2α′ was more resistant to these proteases than CK2α. When these proteases were assayed on thereconstituted (α

2β

2 holoenzyme, a 37 kDa α-band, analogous to that observed in AML extracts, was generated which was resistant

to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies withCK2α deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2α. These resultspointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomesevident when CK2 is overexpressed as in AML cells. (Mol Cell Biochem 191: 229–234, 1999)

Key words: protein kinase CK2, leukemia, calcium activated protease, m-calpain, proteasome

Introduction

Protein kinase CK2 is a ubiquitous serine/threonine kinasepresent in all eukaryotic organisms (reviewed in [1–3]). It hasa tetrameric structure with two catalytic (CK2α or CK2α′,of 44 kDa and 38 kDa respectively) and two regulatory(CK2β, of 28 kDa) subunits. CK2β subunit enhances CK2α/α′ activity [4], and also changes substrate specificity andstabilizes the catalytic subunits [5]; it is synthesized in excessof the catalytic subunit α, and the newly synthesized fractionthat is not incorporated to the holoenzyme is degradedthrough a non-lysosomal proteolytic system with a very lowATP requirement [6].

CK2 phosphorylates a broad number of proteins, some ofthem implicated in the control of cell growth and division(reviewed in [7]). Although its function is vital for the cell,

Address for offprints: M. Plana, Departament de Bioquímica i Biologia Molecular, Unitat de Ciències, Universitat Autònoma de Barcelona, Edifici C,Campus de Bellaterra, 08193 -Cerdanyola del Vallès, Spain

the exact role of protein kinase CK2 and its control is not fullyunderstood. CK2 has been implicated in signal transductionpathways, and increased kinase activity has been observedafter serum or growth factor stimulation in different cell types(reconsidered in [8]). Also, mitogenic stimulation can bereduced or eliminated by antisense oligonucleotides com-plementary to CK2 mRNAs or by microinjection of anti-CK2subunit antibodies, indicating that CK2 is necessary for cellcycle progression [9–11]. CK2 activity has been foundelevated in rapidly proliferating tissues and cell lines, in solidtumors, in virus-transformed cells, and in a lympho-proli-ferative disease induced by intracellular parasites (see [12–18]). The α subunit of CK2 has also been shown to act as anoncogene when expressed in lymphocytes of transgenic mice[19]. All this data suggested a relationship between theenzyme and the control of the processes of cell growth and

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division. This would also suggest a role of CK2 in the processesthat leads to transformation and to the apparition of cancer.

High CK2 activity levels have been described in acutemyeloid leukemia (AML) and HL60 cells, an AML derivedcell line when compared to normal myeloid cells. Theincrease in CK2 activity correlates with the amount ofcatalytic protein present, evaluated by western blot. Also,both AML and HL60 cells showed a complex pattern ofCK2α subunits, with two main bands of 44 and 37 kDa. The37 kDa CK2α band was generated in vivo by C-terminalproteolysis of the 44 kDa one [20].

In the present work, we have studied the in vitro proteolysisof recombinant CK2 subunits using the two major non-lysosomal cytoplasmic proteases: calpain and the proteasome.CK2α, CK2α′ and CK2β are substrates of m-calpain (in aCa2+-dependent way) and 20 S proteasome, but not of 26 Sproteasome (in the conditions used). CK2α′ subunit is moreresistant to proteolysis than CK2α, and in the CK2 holoenzymeCK2α subunit is protected against total degradation withgeneration of a 37 kDa band, corresponding to CK2αtruncated by its C-terminus, results analogous to whatobserved in AML cell extracts.

Materials and methods

Materials and cells

Human recombinant CK2 subunits were prepared as describedin [22]. m-calpain (from rabbit skeletal muscle, 15–40 U/mgprotein) and the peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin were from Sigma. The 20 Sproteasome (recombinant, from Methanosarcina termophila)was from ICN. Lactacystin was from Biomol Research. Otherchemicals were obtained from Sigma and were of the highestpurity available. HL60 cells were cultured in RPMI 1640media supplemented with 10% (v/v) heat-inactivated fetalcalf serum and 10 µg/ml gentamycin at 37°C under anatmosphere of 5% CO

2.

CK2 holoenzyme reconstitution

Equimolar quantities of human recombinant CK2 subunitswere incubated for 30 min at 30°C in 50 mM Tris/HCl pH 7.5,150 mM NaCl, 0.1 % BSA, for CK2 holoenzyme reconstitution.

26 S proteasome reconstitution

HL60 cells were used as a source of the 26 S proteasome dueto the high content of this proteinase complex in these cells[23]. HL60 cell extracts (obtained by sonication at 4°C, in

50 mM Tris/HCl pH 7.5, 1 mM DTT, and 20% glicerol buffer,and centrifuged at 20,000 g at 40°C for 1 h) were incubatedwith 5 mM ATP at 37°C for 20 min for reconstitution of the26 S proteasome [24]. The reconstitution was monitored bythe increase in proteasome activity, as described below.

Proteolytic assays

Proteolytic activity was assayed at pH 7.5 and 30°C (m-calpain) or 37°C (20 S proteasome). The reaction was stoppedby addition of Laemmli sample buffer [25]. After SDS-PAGE[25], the gels were stained by the Nile Red staining method[26], and the proteins quantified using a Gel Doc system andthe Bio Rad Molecular Analyst 3.0 program. Proteasomeactivity was determined by measuring the fluorescence ofaminomethyl-coumarin liberated from peptides using Sue-LLVYNH-MeC as substrate. The reaction mixture (100 µl)comprised 0.1 M Tris/HCl buffer (pH 9) and 0.1 mM substrate.After incubation at 37°C, the reaction was stopped by additionof 100 µl 10% SDS and 1.3 ml of 0.2 M Tris/HCl pH 9 [27].

Results

Proteolysis of CK2 subunits by m-calpain

Previous studies showed that AML cell extracts which hadhigher levels of protein kinase CK2 (protein and activity) thannormal granulocytes, presented also an heterogeneous patternof CK2α, with two main bands of 42 and 37 kDa, recognizedby antibodies against an N-terminal region of CK2α, but the37 kDa was not recognized by antibodies raised against theC-terminus of CK2α [20].

Incubation of recombinant CK2 subunits with m-calpainat 30°C caused the degradation of all subunits in a calcium-dependent manner (Fig. 1) but CK2α′ was more resistant toproteolysis than CK2α. When m-calpain was incubated withthe reconstituted α

2β

2 holoenzyme a resistance to proteolysis

was observed. No apparent degradation of CK2β was seenand CK2α was partially proteolysed to a 37 kDa form whichwas stabilized against further proteolysis. So the CK2α patternobserved in AML cell extracts can be mimicked by incubationof CK2 holoenzyme with m-calpain in the presence of calcium.

Proteolysis of CK2 subunits by the 20 S proteasome

The recombinant 20 S proteasome from Methanosarcinatermophila was used to study the degradation of the CK2subunits by the multicatalytic proteinase complex. Thisrecombinant protease was able to proteolyse at 65°C (itsoptimal temperature) the free subunits of CK2, having the

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CK2 holoenzyme reconstitution a protective effect against thedegradation process. As at 65°C CK2 subunits could bepartially denatured, we tested the ability of the recombinant20 S proteasome to proteolyse CK2 subunits also at 37°C(Fig. 2). As can be seen the recombinant 20 S proteasomecould proteolyse free CK2 subunits at 37°C, with results verysimilar to which obtained using m-calpain as protease. AgainCK2α′ is more resistant to degradation than CK2α, andholoenzyme reconstitution has a protective effect on bothCK2α and CK2β subunits.

Effect of 20 S proteasome and calpain on several CK2adeletion mutants

To study the structural features of cleavage by both proteasesdeletion mutant of CK2α′ lacking the terminal 41 residues

(CK2α∆350–391) or the C-terminal 36 residues (CK2α∆365–391) were used. Mutants were enzymatically active andcapable of forming CK2 holoenzyme according to catalyticactivity (data not shown).

Incubation of full length CK2α with calpain or 20 Sproteasome generated a digestion product with electrophoreticmobility identical to CK2α∆350–391 (Fig. 3). Even CK2α∆365–391 was degraded to the 37 kDa band but no degradationwas observed of CK2α∆350. These results pointed that bothproteases were acting on the C-terminal region of CK2α.

Effect of the reconstituted 26 S proteasome onrecombinant CK2 subunits

In order to determine if the 26 S proteasome was able toproteolyse CK2 subunits, an extract of HL60 cells (which

Fig. 1. Proteolysis of hrCK2 subunits by calpain. 1 µg of each recombinantCK2 subunits alone or reconstituted as holoenzyme (indigated α, α′, β and(α2β2) were incubated for 30 min at 37°C either alone or with of 0.1 U ofm-calpain, in the absence or in the presence of 2 mM CaC12–. Incubationwas stopped by adding Laemmli sample buffer plus boiling. The sampleswere applied to an SDS-PAGE and stained with Coomassie blue.

Fig. 2. Effect of recombinant 20 S proteasome on CK2 subunits. 1 µg of each recombinant CK2 subunits alone (α, α′, β) or reconstituted as holoenzyme(α2β2) were incubated at 37°C with 0.2 µg of recombinant 20 S proteasome. At the indicated times the reaction was stopped by adding Laemmli samplebuffer and the samples applied to 12% SDS-PAGE. Gels were stained with Nile Red and densitometric data were represented in A. In B the Coomassie bluestained gel is shown.

Fig. 3. Effect of 20 S proteasome and calpain on several CK2α deletionmutants. 1 µg of CK2α and the different CK2α deletion mutants (CK2αwild type, wt; CK2α∆365–391, 365; CK2α∆350–391, 350) reconstitutedas holoenzyme with equimolar quantities of CK2β were incubated at 37°Cwith 0.2 µg of recombinant 20 S proteasome for 6 h (Prot2OS), or for 30min at 37°C with of 0.1 U of m-calpain in the presence of 2 MM CaC12

(Calp.). The reaction was stopped by adding Laemmli sample buffer plusboiling and the samples applied to 12% SDSPAGE. Coomassie blue stainedgel is shown.

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have a high content of proteasome, [23]) was incubated with5 mM ATP in order to reconstitute the 26 S proteasome asdescribed in Materials and methods. ATP promotes 26 Sproteasome reconstitution from the 20 S proteasome andother protein complexes as the 19 S caps, and is necessaryfor proteasome 26 S activity. The process can be monitoredby the increase in proteasome activity against peptides as Suc-LLVYNH-MeC.

HL60 cell extracts were able to degrade CK2 subunits inthe absence of calcium, and this was due to the proteasomeactivity as confirmed by using lactacystin (a specificproteasome inhibitor). Reconstitution of the 26 S proteasomewhile increasing the proteolytic activity on the peptide Suc-LLVYNH-MeC, decreased the capacity of the extracts todegrade CK2α, CK2α′ and CK2β (Fig. 4)

Discussion

Complex patterns of CK2α bands can usually be observedin different cells and tissues which presented levels abovenormal of this protein kinase. This is highly evident in AMLand HL-60 cells where two major CK2α bands were found.A complex pattern of catalytic subunits, considering CK2αand CK2α′ together, has also been reported for Krebs IImouse ascites tumor cells [13] and kidney carcinoma [14].Also, in regenerating rat liver a 36 Mr isoform of the

α-subunit has been described [28]. The presence of this CK2α3

subunit increases after partial hepatectomy or laparotomy, andcorrelates with the decrease in the 42,000 Mr α-subunit.Proteolytic degradation was regarded as responsible for theoccurrence of these bands. However, the proteases responsiblefor this degradation have not been identified yet.

Previous work done in lymphoid cell lines by Lüscher andLitchfield [6], demonstrate that CK2β was synthesized as asurplus of CK2α and the fraction of CK2β that is notincorporated to the holoenzyme is degraded through anon-lysosomal proteolytic system with a very low ATPrequirement. Our work [20] has also shown that in transformedcells that have high amounts of CK2α, this catalytic subunitcould be also degraded in an ATP-independent way.

The results presented here demonstrate that the two mainnon-lysosomal proteolytic systems of the cell (namely,calpain and the 20 S proteasome) are able to degrade in vitroprotein kinase CK2 subunits. This proteolytic degradation isprevented when CK2 subunits were associated within theholoenzyme α

2β

2, and is calcium-dependent in the case of

calpain. In both cases degradation occurs by the C-terminalend of the protein, as revealed for the further resistance toproteolysis shown by the C-terminal deletion mutants.

Our results also show that CK2α′ is more resistant thanCK2α to proteolytic degradation by both calpain and the 20S proteasome. To our knowledge, this is one of the few knowndifferences between CK2α and CK2α′ and could be relatedto the data presented by Antonelli et al. [29], where it isshown that CK2α′ is more resistant to thermal inactivation.

The multicatalytic proteinase complex also known as 20S proteasome is the main responsible protease for non-lysosomal pathways of intracellular protein turnover, beingthe active core of the 26 S proteasome complex that functionsin the ubiquitin pathway. 20 S proteasome activity is ATPindependent, very abundant (up to 0.5–1% of the total proteinin tissue homogenates)[30], and highly expressed in humanleukaemic cell lines as in HL-60 cells [23].

While the 20 S proteasome is acting in vitro on CK2subunits, no degradation was observed due to the 26 Sproteasome, at least without any additional modification asubiquitination. Our results do not exclude that the ubiquitin/26 S proteasome system could also control the amount ofCK2 subunits or CK2 holoenzyme as a whole present in thecell. The presence of a destruction box motif in CK2β [31]point in this way.

Since protein kinase CK2 is -as far as it is known- notregulated in its catalytic activity by second messengers orcomponents of known signaling cascades, alternative waysof regulation also have to be considered. One such regulatorymechanism may be proteolytic degradation.

Considering that catalytic subunits display differentsubstrate specificity while free (calmodulin-like substrates)or associated with CK2β in the holoenzyme (casein-like

Fig. 4. Comparison of CK2 subunits degradation by 20 S proteasomepresent in HL-60 cell extracts and by the reconstituted 26 S proteasomefrom the same extracts. Total HL-60 extracts were assayed on theSue-LLVYNH-MeC peptide as described in Materials and methods (40 µgof extracts), or incubated with 1 µg of each hrCK2 subunit for 15 min at37°C (2 µg of extracts). These incubations were stopped by adding Laemmlisample buffer and the amount of each subunit was monitored by densitometricanalysis of Nile red stained gels. The same experiment was done using thesame quantities of HL60 extracts pre-incubated at 37°C for 20 min in thepresence of 5 mM ATP (conditions used to reconstitute 26 S proteasome).Both experiments were compared by plotting the results obtained with the26 S proteasome in respect to the 20 S form.

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substrates) the importance of controlling the amount of freeCK2 subunits should be a need for the cell. Moreover, recentworks [32–34] show that CK2 subunits can be found in thecells individually and even have a defined physiological roleinteracting with other partner’s and changing their properties,so control of individual CK2 subunits levels could havedifferent effects on the physiology of the cell.

The data shown in this work are an indication of the in vivoprocesses that may control the amount of CK2 subunitswithin the cell. The hypothesis is that the 20 S proteasome(and perhaps calpain in a calcium-controlled way) is theproteolytic system that degrades the CK2 subunits which arenot incorporated to the holoenzyme. As in other cases ofmonomer degradation [35], the association within theoligomer could be a way to prevent this degradation. Insome cases the protease could also partially degrade CK2αassociated to CK2β cutting off its C-terminal tail althoughwe do not know the physiological significance of this limitedproteolysis. Obviously the deleted region is not only freelyaccessible to proteolytic cleavage but also not required forthe subsistence of the quaternary structure.

Acknowledgements

This work was supported by grants PB95-0610 from DGYCIT,HA993-96 from MEC Spain, from CT96-0047 (BIOMED 2)E.C. and DY2-2/2 from the Deutsche Forschungsgemein-schaft. J. Roig is recipient of a fellowship from DGR(Generalitat de Catalunya, Spain). The authors want to givethanks to the ‘Laboratori d’anàlisi i fotodocumentació’ UAB(Bellaterra).

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