Table Of ContentA MOLECULAR AND CELLULAR VIEW OF PROTEIN KINASE CK2
A Molecular. and Cellular View of
Protein Kinase CK2
Edited by
KHALIL AHMED E.CHAMBAZ
Cellular and Molecular Biochemistry Laboratoire de Biochemie des Regulations Cellulaires Endocrines
Research Laboratory (151) INSERM U244
V.A. Medical Centre DBMS, CEA Grenoble, 17 rue des Martyrs
One Veterans Drive F-38054
Minneapolis, MN 55417 Grenoble Cedex 9, France
O.G.ISSINGER
Biokemisk Institut
Odense Universitet
Campusvej 55, DK-5230
Odense M, Denmark
Reprinted from Molecular and Cellular Biochemistry, Volume 191 (1999)
Springer-Science+Business Media, B.V.
Library of Congress Cataloging-in-Publication Data
A molecular and cellular view of protein kinase CK2 / edited by Khalil
Ahmed: E. Chambaz, 0.0. Issinger, co-editors
p. cm. --(Developments in molecular and cellular
biochemistry )
ISBN 978-1-4613-4648-7 ISBN 978-1-4419-8624-5 (eBook)
DOI 10.1007/978-1-4419-8624-5
1. Protein kinase CK2--Congresses. 1. Ahmed, Khalil, 1934-
II. Chambaz E. M. III. Issinger, o. O. IV Series
QP606.P76M65 1998
572'.792--dc21 98-8321
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© 1999 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 1999
Softcover reprint ofthe hardcover Ist edition 1999
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Molecular and Cellular Biochemistry:
An International Journal for Chemical Biology in Health and Disease
CONTENTS VOLUME 191, Nos. 1 & 2, January (II) 1999
A MOLECULAR AND CELLULAR VIEW OF PROTEIN KINASE CK2
Drs. Khalil Ahmed, E. Gambaz and O.G. Issinger
Preface 1-2
G. Dobrowolska, FJ. Lozeman, D. Li and E.G. Krebs: CK2, a protein kinase of the next millennium 3-12
S. Sarno, P. Vaglio, L. Cesaro, O. Marin and L.A Pinna: A multifunctional network of basic residues confers unique properties to
protein kinase CK2 13--19
A Krehan and W. Pyerin: Intermolecular contact sites in protein kinase CK2 21-28
M.J. Benitez, G. Mier, E Brione, EJ. Moreno and J.S. Jimenez: Binding a polylysine to protein kinase CK2, measured by Surface
Plasmon Resonance 29-33
E Meggio, O. Marin, S. Sarno and L.A Pinna: Functional analysis ofCK2~-derived synthetic fragments 35-42
D. Leroy, O. Filhol, N. Quintaine, D. Sarrouilhe, P. Loue-Mackenbach, E.M. Chambaz and C. Cochet: Dissecting subdomains involved
in multiple functions of the CK2~ subunit 43--50
M. Kusk, R. Ahmed, B. Thomsen, C. Bendixen, O.-G. Issinger and B. Boldyreff: Interactions ofp rotein kinase CK2~ subunit within
the holoenzyme and with other proteins 51-58
U. Wirkner and W. Pyerin: CK2a loci in the human genome: Structure and transcriptional activity 59-64
X. Xu, E. Landesman-Bollag, P.L. Channavajhala and D.C. Seldin: Murine protein kinase CK2: Gene and oncogene 65-74
I. Korn, S. Gutkind, N. Srinivasan, T.L. Blundell, C.C.AlIende and lE.AlIende: Interactions of protein kinase CK2 subunits 75-83
D. Leroy, G.C. Alghisi, E. Roberts, O. Filhol-Cochet and S.M. Gasser: Mutations in the C-terminal domain of to poi some rase II
affect meiotic function and interaction with the casein kinase 2~ subunit 85-95
M. Riera, N. Roher, E Mira, C. Gil, R. Trujillo, lAguilera, M. Plana and E. Itarte:Association of protein kinase CK2 with eukaryotic
translation initiation factor eIF-2 and with grp94/endoplasmin 97-104
S. Grein, K. Raymond, C. Cochet, W. Pyerin, E.M. Chambaz and O. Filhol: Searching interaction partners of protein kinase CK2~
subunit by two-hybrid screening 105-109
C. G6tz, P. Scholtes,A Prowald, N. Schuster, W. Nastainczyk and M. Montenarh: Protein kinase CK2 interacts with a multi-protein
binding domain ofp 53 111-120
S. Grein and W. Pyerin: BTF3 is a potential new substrate of protein kinase CK2 121-128
K. Ackermann and W. Pyerin: Protein kinase CK2a may induce gene expression but unlikely acts directly as a DNA-binding
transcription-activating factor 129-134
C. Guo,AT. Davis, S. Yu, S. Tawfic and K.Ahmed: Role of protein kinase CK2 in phosphorylation ofnucleosomal proteins in
relation to transcriptional activity 135-142
A. Ghavidel, D J. Hockman and M. C. Schultz: A review of progress towards elucidating the role of protein kinase CK2 in polymerase
III transcription: Regulation ofthe TATA binding protein 143--148
E. Egyhlizi, A Ossoinak, O. Filhol-Cochet, C. Cochet and A. Pigon: The binding ofthe a subunit ofp rotein kinase CK2 and RAP74
subunit ofTFIIF to protein-coding genes in living cells is DRB sensitive 149-159
K.A Tenney and C.Y.C. Glover: Transcriptional regulation of the S. cerivisiae ENAI gene by casein kinase II 161-167
R. Lin and J. Hiscott: A role for casein kinase II phosphorylation in the regulation ofIRF -1 transcriptional activity 169-180
G.-T. Sheu and I.A. Traugh: A structural model for elongation factor 1 (EF-l) and phosphorylation by protein kinase CKII 181-186
L. McKendrick, D. Milne and D. Meek: Protein kinase CK2-dependent regulation of p53 function: Evidence that the phosphorylation
status ofthe serine 386 (CK2) site ofp53 is constitutive and stable 187-199
FJ. Moreno, 1 Diaz-Nido, lS. Jimenez and lAvila: Distribution ofCK2, its substrate MAPlB and phosphatases in neuronal cells 201-205
E Lebrin, L. Bianchini, T. Rabilloud, E.M. Chambaz and Y. Goldberg: CK2a -protein phosphatase 2Amolecular complex: Possible
interaction with the MAP kinase pathway 207-212
D.G. Bosc, B. Liischer and D.W. Litchfield: Expression and regulation of protein kinase CK2 during the cell cycle 213--222
D. Li, G. Dobrowolska and E.G. Krebs: Identification of proteins that associate with protein kinase CK2 223--228
1 Roig,A Krehan, D. Colomer, W. Pyerin, E. Itarte and M. Plana: Multiple forms of protein kinase CK2 present in leukemic cells: 229-234
In vitro study of its origin by proteolysis
235-237
Index to Volume 191
Molecular and Cellular Biochemistry 191: 1-2, 1999.
© 1999 Kluwer Academic Publishers.
Preface
It is now generally recognized that protein kinase signaling proved to be exceptionally problematic. In addition to this
is involved in virtually every aspect of cell function, including retrospective, Dr. Krebs discussed the recent advances from
growth and proliferation. The field of protein phosphoryla his laboratory dealing with the characterization of CK2
tion, including the enzymes involved in this posttranslational isoforms and association of other proteins with this kinase,
modification, continues to advance at a fascinating pace. Of which might have implications for its cellular regulation.
note, the general symposium on the topic of signal trans The first session (chaired by W. PyerinlL.A. Pinna) was
duction entitled 'Cellular regulation by protein phosphoryla devoted to 'CK2 molecular organization; structure-activity
tion: Forty years of progress' was held in Seattle as an relationship.' The session was opened by L.A. Pinna (Padova,
ASBMB Satellite Meeting of the International Congress of Italy) who spoke on the structural features underlying
Biochemistry (August 1997). The special meeting on protein substrate specificity and regulation of the CK2a subunit. This
kinase CK2 (formerly known as casein kinase 2 or II) may was followed by a contribution by A. Krehan (Heidelberg,
be regarded as complementary to that theme. Mounting Germany) dealing with the analysis of CK2~ domains that
evidence continues to suggest that this kinase is involved in interact closely with the CK2a. J. Jimenez (Madrid, Spain)
signal transduction related to many cellular functions and to described surface plasmon resonance as an approach to
point the role of CK2 in normal and abnormal cell growth determining the molecular interaction in CK2 as related to
and proliferation. The localization ofCK2 in the cytoplasmic its regulation. The talk by F. Meggio (Padova, Italy) was
and nuclear compartments accords with its multifunctional concerned with the functional analysis of CK2~-derived
role in the cell. Because CK2 belongs to the family of serine/ synthetic fragments. C. Cochet (Grenoble, France) outlined
threonine kinases that apparently do not require a second an analysis of the sub domains involved in multiple functions
messenger or activator, delineation of the regulation of its of the CK2~ subunit. The presentation by B. Boldyreff
functional activity remains a challenge. (Odense, Denmark) was on the interactions ofCK2~ within
Since the first international meeting on this topic, held in the holoenzyme, as well as with other proteins. Finally, B.
Heidelberg in 1994, several new avenues of CK2 research Guerra (Odense, Denmark) introduced data on the crystal
have emerged despite the persisting deficiencies in our structure of the catalytic subunit of CK2 (CK2a) with
understanding of the regulation of its activity. Among the potential implications for its modes of activation.
significant new directions are the studies related to the The second session (chaired by O.-G. Issinger) dealt with
structure of the enzyme, especially its crystal structure, as 'CK2 genes.' The first talk was by U. Wirkner (Heidelberg,
well as an interesting aspect of CK2 function that involves Germany), who gave an account of her analysis of the
its subunits as binding partners of several other proteins. In structure ofthe gene encoding the human CK2 a subunit. In
addition, new data have been gathered on the role of CK2 in the same vein, D. Seldin (Boston, USA) presented structural
transcription as well as certain other cellular functions. To information on the murine CK2a' gene and its chromosomal
address these various aspects of the progress on CK2, a mapping.
number of key scientists from different parts of the world The third session (also chaired by O.-G. Issinger) was
came together at the second international meeting on 'A devoted to 'CK2 and interacting partners.' The session was
Molecular and Cellular View of Protein Kinase CK2', held opened by J. Allende (Santiago, Chile), who gave a review
near Grenoble at Villard de Lans on September 24--26, 1997. of the studies on site-directed mutagenesis of the CK2a
The meeting was attended by nearly 50 participants and subunit and its implications for interaction with the ~ subunit.
included 28 presentations. D. Leroy (Lausanne, Switzerland) gave an account of this
The meeting opened with a plenary lecture by Edwin G. group's continuing studies on the interaction of CK2 with
Krebs (Seattle, USA) under the poetic title 'CK2, a protein topoisomerase II. E.ltarte (Bellaterra, Spain) reviewed studies
kinase for the next millenium.' Professor Krebs recounted the on the association ofCK2 with eukaryotic translation initiation
historical development of the studies of CK2 and provided factor eIF-2 and grp94/endoplasmin. The talk by S. Grein
thoughtful insights into why certain aspects of CK2 have (Heidelberg, Germany) also focused on the identification of
2
partners of CK2~. The final talk in the session was by M. regulation of the MAP kinase pathway by CK2. The talk by
Montenarh (Homburg, Germany), who reviewed the recent D. Litchfield (London, Canada) focused on expression and
studies in his laboratory on the role of CK2 in interactions regulation ofCK2 during the cell cycle. Along the same lines,
of p53 with cellular partners. D. Li (Seattle, USA) presented evidence for the role of CK2
The fourth session (chaired by D. Meek/C. Cochet and Y. in the control of cell growth and progression of the cell cycle.
Goldberg/K.Ahmed) was devoted to 'CK2 and transcriptional The final talk was given by M. Plana (Bellaterra, Spain), who
translational activities.' The first talk was by W. Pyerin described the data on CK2 forms and expression in human
(Heidelberg, Germany) dealing with a possible role of myeloid leukemia cells.
unscheduled CK2a expression in induction of gene expres The above general description of the talks in the various
sion in cultured human cells. K. Ahmed (Minneapolis, USA) sessions of the meeting highlights the recent developments
presented data suggesting differential translocation of in the studies ofCK2. The 28 papers in this issue ofM olecular
functional CK2 activity to the active and inactive nucleo and Cellular Biochemistry are the materials presented by the
somes in relation to transcriptionaJ activity. M. Schultz authors at the meeting. We believe they provide a view ofthe
(Edmonton, Canada) summarized a role of CK2 in tran latest progress on protein kinase CK2. To that end, we express
scription by RNA polymerase III. The studies discussed by our sincere gratitude to all the participants in the meeting for
E. Egyhazi (Stockholm, Sweden) focused on the role of their efforts.
CK2a in the activity of RNA polymerase II. C. Glover This meeting was sponsored by INSERM (Institut National
(Athens, USA) considered the role of CK2 in the tran de la Sante, et de la Recherche Medicale), and by European
scriptional regulation of the S. cerevisiae ENAl gene. l Biomed-2 N° BMH4-CT96-0047. Additional support was
Hiscott (Montreal, Canada) discussed the role ofCK2 in the provided by BioRad, Boehringer Mannheim, Life Tech
regulation of transcription factors controlling cytokine gene nologies, Medgene Science, NEN, Neosystem, Polylabo,
activation. The final talk in this session was by lA. Traugh Promega, Sigma, Zeiss, l'Institut de Recherche Pierre Fabre,
(Riverside, USA), who presented data on the phosphorylation Ie Laboratoire F oumier, and la Region Rh6ne-Alpes (Commis
of elongation factor 1 (EF-l) by CK2. sion de la Recherche), for which we are very thankful. The
The final session (chaired by B. Boldyreff/E. Itarte) was support from these various sources was critical to the meeting.
devoted to 'CK2 and specific cellular functions.' D. Meek We express our special thanks to Ms. S. Lidy and to Drs. C.
(Dundee, UK) reviewed the evidence on phosphorylation and Cochet, O. Filhol, andY. Goldberg for their outstanding efforts
control of the p53 tumor suppressor product by CK2. The in making the many local arrangements and in providing an
presentation by l Avila (Madrid, Spain) gave an account of amicable environment, which contributed to the success of
studies on the phosphorylation by CK2 of proteins related to the meeting. Finally, we also wish to acknowledge Dr. Brigitte
microtubules and its functional consequences. Y. Goldberg Boldyreff (Odense University) for her contributions during
(Grenoble, France) submitted evidence in support of the the editorial review of the papers.
Guest Editor: Khalil Ahmed, Minneapolis, USA
Guest Co-editors: Edmond M. Chambaz, Grenoble, France
Olaf-Georg Issinger, Odense, Denmark
Molecular and Cellular Biochemistry 191: 3-12, 1999.
© 1999 Kluwer Academic Publishers.
CK2, a protein kinase of the next millennium
Grazyna Dobrowolska, Fred 1. Lozeman, Dongxia Li and Edwin G.
Krebs
Department ofP harmacology, University of Washington, Seattle, WA, USA
Abstract
Protein kinase CK2 is a ubiquitous eukaryotic protein kinase composed of two catalytic subunits, a and/or a', and two regulatory
subunits, ~. In order to define similarities and dissimilarities between the a and a' catalytic subunits, which might account for
their particular cellular functions, different forms of the enzyme were expressed in Sf9 cells and their properties determined.
Both catalytic subunits were expressed separately, and also along with the regulatory ~ subunit, in order to obtain free a and
a', as well as a2~2 and a' 2~2 forms. Our results confirm that the ~ subunit acts to stabilize the a and a' subunits and also influences
the substrate specificity and kinetic properties of the enzyme. Although significant differences between the specificities of
holoenzymes a2~2 and a' 2~2 as determined using a number of substrates were not detected, autophosphorylation studies on
a2~2 and a/2~2 revealed significant differences in this property. The regulatory subunit ~ was phosphorylated less rapidly by
the a' subunit than by the a subunit, and the extent of phosphorylation of ~ by a was also greater than that of ~ by a'. It was
also noted that the thermo-stability and the extent of its activation by NaCl were greater for a2~2 than for a' 2~2' These different
properties may relate to distinct functions of the two form ofCK2. (Mol Cell Biochem 191: 3-12, 1999)
Key words: protein kinase CK2, Sf-9 cells, baculovirus expression system, autophosphorylation
Introduction
their source of the enzyme, and it would seem likely that a
few nuclei, a rich source of CK2, might have been present.
In addition some ofthe properties oftheir enzyme suggested
In this contribution, the early history of CK2 and problems that it might well have been what we now call CK2. These
related to studying its function will be discussed. In addition, studies took place at about the same time as the discovery by
we will present some recent studies on the properties of CK2 E.H. Fischer and me of the phosphorylation and dephos
subunits and its holoenzyme forms expressed in the baculo phorylation of muscle glycogen phosphorylase, and the
virus system. The title of the article, 'CK2, a protein kinase independent work carried out by E. W. Sutherland et al. on
of the next millennium', might be construed to mean that the liver phosphorylase. A few years later, Rabinowitz and
progress being made in our understanding of this kinase is Lipmann [3] described a protein kinase from bovine brain
proceeding at such a snail's pace and that it will take another that phosphorylated phosvitin and casein. They showed,
millennium to learn much about it; but this is not our intent. however, that if casein was first dephosphorylated by
Instead, we wish to simply emphasize the fact that CK2 is treatment with dilute alkali, then it became a very poor
such a rich topic for investigation that research involving this substrate. This suggested to them that perhaps the kinase
enzyme can continue for decades to come! recognizes an acidic environment near the serine or threonine
CK2 is one of the first protein kinases to have been residues that are to be phosphorylated. They showed that
detected, and perhaps it may be the very first known kinase their kinase, which probably was CK2, was distinct from
[1]. Forty years ago Burnett and Kennedy [2] described a phosphorylase kinase. In the late 1960s, at about the same
soluble enzyme that catalyzed the phosphorylation of casein. time as the multifunctional cyclic AMP-dependent protein
In their studies they utilized a mitochondrial preparation as kinase (PKA) was discovered by Walsh et al. [4], a number
Present address: G. Dobrowolska, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego Sa, 02-106 Warsaw, Poland
Address/or offPrints: E.G. Krebs, Department of Pharmacology, University of Washington, Seattle, WA 98195, USA
4
of new reports of casein kinases began to appear; these reports translation factors, and other proteins crucial for cell growth,
stressed the fact that the activities being studied were not proliferation, and differentiation. The potential role of CK2
affected by cyclic AMP. As the 1970's approached, it was with respect to these functions also fits well with genetic data
becoming clear that tissues contained two different cyclic showing that CK2 is essential for cell viability [9]. Further
AMP-independent casein kinases, CKI and CK2 [5]. more, recent work with CK2a transgenic mice has shown that
From the above historical sketch it is clear that CK2 dysregulation of CK2 expression causes an exacerbation of
belongs to a select group of the earliest protein kinases T-cell acute lymphoblast leukemia [10]. In addition, there are
known. Why then, after this many years, don't we know more also several reports [11] describing elevated CK2 activity in
about its exact biological functions and its mode of regula transformed cells.
tion? Perhaps one explanation for this is the fact that for Concerning the third point ofHawthaway and Traugh, i.e.
almost twenty years, investigators always used casein, an our lack of understanding as to the mechanisms responsible
unnatural substrate, in their work. In addition, relatively few for regulating CK2, has significant progress been made? The
laboratories were interested in CK2, i.e. as compared to other early observations with respect to effectors that influence
protein kinases that were 'coming on stream'. CK2 activity in vitro are still valid, e.g. the effects of heparin,
In their classical review of the casein kinases, Hathaway 2, 3-diphosphoglycerate and polyamines, but it has not been
and Traugh [5] suggested that the casein kinases were hard possible to put these effects into a physiological context.
to define from a functional standpoint for several distinct What about the possibility of the regulation of CK2 by
reasons. They were: (1) insufficient information with regard phosphorylation-dephosphorylation, as was once suggested
to their 'real substrates', (2) the fact that the substrates ofCK2 by studies from several laboratory including this one? This
that had been identified were usually found in highly complex question remains open, but more often than not these effects
systems, i.e. as opposed to protein kinases whose substrates have been difficult to confirm [12]. Some investigators feel
consisted of metabolic enzymes, (3) it had been difficult to that CK2 is a constitutively active protein kinase, but others
identify mechanisms for regulating CK2 and CKI, (4) find this difficult to accept. Perhaps it is regulated exclusively
endogenous substrates, as obtained for studying the casein by variations in its cellular concentration.
kinases may have already been fully phosphorylated and (5) In the experimental work to be reported in this paper, we
the casein kinases were often tightly bound to their substrates. have examined the catalytic properties of the two types offree
Of these difficulties, the first three seem to us to be the catalytic subunits, CK2a and CK2a' and the two holo
particularly important. How well have investigators been able enzymes, CK2a2~2 and CK2a'2~2' For this work we used
to get around these problems? recombinant enzymes expressed in baculovirus. The baculo
Insofar as the identification of physiologically significant virus expression system was chosen, since it is often the best
substrates for CK2 is concerned, great progress has been way for producing large quantities of properly folded
made. In their 1982 review Hathaway and Traugh mentioned proteins. The results that are presented show small but
about a dozen proteins that could serve as substrates ofCK2, nonetheless significant differences between the various
and several of them were deemed to be significant from a enzyme forms.
functional standpoint. In a second review about nine years
later, Tuazon and Traugh [6] cited 42 potentially important
substrates. In a review by Allende and Allende [7] the number Materials and methods
had risen to around 100. Finally in a very recent review by
Pinna and Meggio [8] 160 substrates are mentioned. Clearly,
then, insofar as to total number of substrates is concerned, Materials
there no longer seems to be a problem. In fact, it seems
probable that CK2 may have more substrates than any other Synthetic peptides (RRRDDDSDDD and the cMyc peptide,
known protein kinase. RRRPPTTSSDSEEEQEEDEE) were synthesized by Dr. P.
Has the identification of a host of substrates for CK2 Chou and H. Zebrowski (Chemical Synthesis Facility of the
helped us in defining its functional role? The answer to this Howard Hughes Medical Institute, University ofWashington).
question is yes and no. On the one hand, each new substrate Bac PAK 6 viral DNA and PVL941 transfer vector were
that is added to the list creates one additional problem of obtained from Clontech; pGEX-2T expression vector, gluta
defining the effect of phosphorylation on protein function. thione-Sepharose and Sephacryl S-200 were from Pharmacia
On the other hand, the identification of numerous substrates LKB Biotechnology Inc., Heparin-agarose, polylysine
for CK2 permits their being grouped according to function agarose, phosphocellulose, ~ casein, heparin,ATP, GTP and
and this grouping suggests functional roles for the enzyme. all other reagents and chemicals were purchased from
Thus, one large set of subtrates for CK2 includes enzymes Sigma.
that control DNA and RNA synthesis, transcription and
5
Cells finally the enzymes were eluted with Buffer B. Fractions
containing CK2 activity were concentrated and applied to a
Spodoptera frugipedra (Sf9) cells were maintained in TNM Sephacryl S-200 (2.4 x 92 cm) column equilibrated with Buffer
FH medium GIBCO) supplemented with 10% fetal calf serum. C (IOOmMTris-HCl, pH 7.5, 500mMNaCl, 5 mM EDTA, 5
mM EGTA, 10% glycerol, 0.1 mM PMSF, 1I1gimlleupeptin).
Gel filtration was performed in Buffer C at a flow rate of 10
Expression of different forms ofh uman CK2 mllh. Fractions with Mr 135000 containing CK2 activity were
collected. The enzymes were concentrated, dialyzed against
CK2a, a' and ~ subunits were expressed in Sf9 cells using the Buffer A without NaCl, and stored in small aliquots at -70°C.
baculovirus expression system. Standard techniques were used
to generate the expression constructs [13]. Each of the cDNA
clones encoding human CK2a, a' or ~ subunit was subcloned Purification oft he recombinant human catalytic subunits
into the transfer vector pVL 941 from the BamHI site. The CK2 a and CK2 a'
transfer vectors carrying CK2a or CK2a' or CK2~ cDNA were
separately co-transfected with Bsul-digested BacPAK viral The procedure applied for purification of the free catalytic
DNA into insect Sf9 cells. Generation and purification of subunits was basically the same as for the oligomeric enzymes
recombinant viruses were carried out according to Clontech's except for two differences. Since the free catalytic subunits
protocol. Viruses with the best ability to produce the recom (ala' do not bind to polylysine-agarose under the experimental
binant proteins were selected for enzymes production. Sf9 cells conditions (250 mM NaCl), the flow through from these
(5 x 107 cells) were infected with either the CK2a or CK2a' columns was collected and applied directly to heparin
virus separately to obtain the free catalytic subunits, or were agarose columns. The second difference was that CK2ai
coinfected with the CK2~ virus to obtain the oligomeric CK2a' from Sephacryl S-200 column were eluted as single
enzymes, CK2a2~2 ora'2 ~2' at a multiplicity of infection (MOl) peaks of CK2 activity at positions Mr 40000-45000.
of 10 and grown in 100 ml culture at 27°C. After 48 h, cells
were centrifuged at 2000 x g for 10 min., washed two times
with 10 ml of ice-cold PBS and frozen at-70°C. Expression in E. coli and purification of the human CK2 f3
subunit
Purification of holoenzyme forms of CK2 ai32 and a'i32 Expression and purification were performed as described in
Li et al. [14]. Removal ofGST (cleavage by thrombin) was
Collected Sf9 cells in which the ~ subunit as well as the a or done according to the Pharmacia protocol. Approxi-mately
a' subunits was expressed, were resuspended in 5 ml of 10 mg of 90% pure GST-CK2~, as judged by SDS-PAGE
homogenization buffer (50 mM Tris-HCI, pH 8.5, 250 mM followed by Coomassie Blue staining, was obtained from 1
NaCl, 5 mM EDTA, 50 mM NaF, 250 mM sucrose, 2 mM DTT, I ofLB culture. Experiments were performed on GST -CK2~,
1 mM PMSF, 10% glycerol, 0.05% Triton X-IOO, 10 I1giml as well as on non-fusion protein CK2~.
leupeptin) and were sonicated 3 times for 20 sec on ice. The
sonicates were centrifuged at 12,000 x g at 4°C for 30 min
and were then applied to 20 ml phosphocellulose columns CK2 assays
previously equilibrated with Buffer A (50 mM Tris-HCl, pH
7.5,5 mM EDTA, 5 mM EGTA, 0.1 mM PMSF, 10% glycerol, CK2 activity was measured routinely as described by Kuenzel
1l1gimlleupeptin, 250 mM NaCl). The columns were washed et al. [15], in a final volume of 25 ml containing 20 mM
with 200 ml of equilibration buffer. The enzymes were step Hepes, pH 7.4, 20 mM MgCI2, 100 mM [y2P] ATP (specific
eluted by Buffer B (Buffer A containing I M NaCl, instead activity 2000 cpm/pmol), 20 mM NaCI (for the free catalytic
of 250 mM NaCl). Fractions with the highest CK2 activity subunits) or 150 mM NaCI (for the oligomeric holoenzymes),
were pooled, diluted to a concentration of250 mM NaCI and 100 mM RRRDDDSMD peptide substrate and an aliquot of
applied to 5 ml polylysine-agarose columns preequilibrated the CK2 preparation. Incubation was carried out for 10 min
with Buffer A. The columns were washed with 50 ml of at 30°C and phosphate incorporation was evaluated by the
Buffer A and 50 ml of Buffer A containing 500 mM NaCI. phosphocellulose paper procedure. With peptides that do
Enzymes were eluted by Buffer B. Fractions with CK2 not bind to phosphocellulose paper, because they are devoid
activity were pooled, diluted to a concentration of 250 mM of basic residues, the isolation of radioactive amino acids
NaCI and applied to 2 ml heparin-agarose columns equilibrated after partial acid hydrolysis of phosphorylated peptide (6N
with Buffer A. The columns were washed first with 20 ml of HCl, 105°C, 4 h) was performed as previously described in
Buffer A, followed by Buffer A containing 400 mM NaCI and Perich et al. [16]. After high voltage electrophoresis and
6
autoradiography the amount of incorporated phosphate was obtain the holoenzyme forms, a2~2 and a'2 ~2' The expressed
estimated by counting radioactive phospho serine spots. When proteins were purified to about 90% homogeneity using
proteins were used as the substrates incubation under the standard chromatographic procedures (Table 1 and Fig. lA).
same conditions as above was performed. Protein concentra Biochemical characterization of the pure enzymes (CK2a,
tions were 10 mg/ml unless described differently. Reactions CK2a', CK2a2~2 and CK2a'2~2) was then performed. By
were stopped by 10% TCA and the incorporated phosphate selecting recombinant viruses that gave the highest levels of
in the pellets was measured. In some instances, e.g. the transfected proteins, it was possible to minimize contamina
phosphorylation of the CK2~ subunit, SDS-PAGE followed tion with insect CK2 to less than 1% of the total. The
by autoradiography was employed. Phosphate incorporation holoenzyme forms, a2~2 and a'2 ~2' were also prepared in vitro
was estimated by counting excised radioactive bands. CK2 by mixing purified CK2 subunits a and ~ or a' and ~,
activity is presented as an amount of phosphate transferred respectively (see above). In this instance, the formation of
to the proteinlpeptide substrate per min. tetramers with an Mr around 135000 was confirmed by gel
filtration on Sephacel G200 or Superose 12 columns (data not
shown). Regardless ofthe method of preparation, the specific
Autophosphorylation assays activities of the recombinant enzymes were similar and
comparable to the specific activity of CK2 holoenzyme
Autophosphorylation experiments were performed in 20 mM purified from bovine testis. For example, the specific activity
Tris-HCl, pH 7.5, 20 mM MgC12, 100 mM [y32_P]ATP of the recombinant a2~2 as determined from several experi
(specific activity 4000 cpm/pmol) for 30 min (unless described ments was in the range of 1.9-2.4 Ilmole/minlmg and that of
differently) at 30DC and stopped by addition of Laemmli a'2~2 was in the range of2.2-2.6Ilmole/minlmg, whereas the
sample buffer. Analysis was performed by SDS-PAGE specific activity ofCK2 purified from bovine testis was 3.0
followed by autoradiography. Ilmole/min/mg. These findings are of interest, inasmuch as
variable results have been reported by others for CK2
obtained by mixing recombinant CK2a and CK2~ subunits
Other procedures
expressed in Sf9 cells. Our data are in agreement, however,
with the results of Glover's group [17] using Drosophila CK2
In order to determine thermal stability, approximately 5 ng
expressed in Sf9 cells, but data presented by Filholet al. [18],
of different forms of human CK2 expressed in Sf9 cells were
who studied an identical system to that of Glover, suggested
incubated at 45DC for 0-30 min in 25 ml of Buffer A without that the fully active tetrameter of Drosophila CK2 could be
NaCl. After incubation, CK2 activity was measured as
obtained only by co-infection of Sf9 cells with viruses
described above using RRRDDDSDDD as the substrate. In
expressing CK2a and ~ subunits and not by the in vitro
determining kinetic constants for the various forms of CK2,
recombination of previously expressed proteins.
Lineweaver-Burk plots by linear regression analysis from at
least 3 independent experiments was applied.
Autophosphorylation
Results
CK2 has been known to undergo autophosphorylation on its
~ subunit and also on its a and a' subunits [11]. The auto
Preparation of CK2 subunits and holoenzyme forms phosphorlation sites on the ~ subunit were determined to be
Ser 2 and/or Ser 3 [19]. However, in these studies, the CK2
In general, it has been difficult for investigators to obtain the used was isolated from animal tissues and represented a
requisite materials needed to study the different isoforms of mixture of several forms of the enzyme, i.e. a2~2' a'2~2' and
CK2 and its individual subunits. As obtained from animal aa'~2' Information concerning autophosphorylation of each
tissues holoenzyme preparations of CK2 consist of mixtures individual holoenzyme form was not obtained. This was
ofa2~2' a'2~2' and, probably, aa'~2; conventional fractiona achieved in the present study for a2~2 and a'2 ~2 which were
tion procedures have not been satisfactory for their separa isolated using the baculovirus expression system by co
tion. In addition, the free a and ~ subunits of CK2 are not infecting either a with ~ or a' with ~. As illustrated in Fig.
ordinarily found in tissues, at least in preparative amounts, lB, autophosphorylation of recombinant human CK2a2~2
so that they likewise cannot be obtained from this source. For occurred mostly on the ~ subunit, whereas autophosphoryla
these reasons recombinant methods constitute the procedures tion of the CKla2~2 occurred prominently on the a' subunit
of choice for obtaining the various forms ofCK2. The human as well as on the ~ subunits. Noteworthy was the finding that
CK2 catalytic subunits, a and a' were expressed in insect phosphate incorporation on the ~ subunit of CK2a'2~2 was
cells either alone, or along with the ~ subunit in order to distinctly less than that seen with CK2a'2~2 (Fig. IB).
