©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interactions between the Subunits of Casein Kinase II (*)

R. Daniel Gietz (1), Kevin C. Graham (1), David W. Litchfield (§)

From the (1) Manitoba Institute of Cell Biology, Manitoba Cancer Treatment and Research Foundation, the Department of Biochemistry and Molecular Biology Department of Human Genetics, University of Manitoba, Winnipeg R3E 0V9, Manitoba, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Casein kinase II (CKII) is a protein serine/threonine kinase known to control the activity of a variety of regulatory nuclear proteins. This enzyme has a tetrameric structure composed of two catalytic ( and/or `) subunits and two subunits. We have examined the subunit composition of tetrameric complexes of purified bovine CKII by immunoprecipitation using , `, or subunit-specific antibodies. These experiments indicate that the enzyme can exist as homotetramers (i.e. or `) as well as heterotetramers (i.e. `). To further examine subunit interactions between the , `, or subunits of CKII, we have utilized the yeast two-hybrid system (Fields, S. and Song, O.(1989) Nature 340: 245-246). For these studies, each subunit of human CKII was expressed in yeast as a fusion with the DNA binding domain or with the transcriptional activation domain of the yeast GAL4 transcriptional activator. These studies demonstrate that the or ` subunits of CKII can interact with the subunits of CKII, but not with other or ` subunits. By comparison, the subunits of CKII can interact with , `, or subunits. These results indicate that the CKII holoenzyme forms because of the ability of subunits to dimerize, bringing two heterodimers ( or `) into a tetrameric complex.


INTRODUCTION

Casein kinase II (CKII)() is a protein serine/threonine kinase that is ubiquitously distributed in mammalian tissues and is likely to play an important role in the control of proliferative events (for reviews, see Refs. 1-3). Although its exact biological functions remain poorly understood, genetic studies in Saccharomyces cerevisiae(4, 5) , in Schizosaccharomyces pombe (6, 7), and in Dictyostelium discoideum(8) all suggest that CKII is essential for viability. Immunofluorscence localization studies have demonstrated that the enzyme is localized within the nucleus and the cytoplasm of cells (9, 10) , observations that are consistent with the demonstration that CKII phosphorylates a variety of proteins in each of these compartments. Of particular relevance to a role in the regulation of proliferation is the demonstration that CKII phosphorylates a host of regulatory nuclear proteins including transcription factors such as the serum response factor, c-Myc, N-Myc, c-Myb, c-Jun, and the tumor suppressor p53 (reviewed in Refs. 3 and 11).

CKII is a tetrameric enzyme composed of two (and/or `) subunits and two subunits. The and ` subunits contain all of the conserved consensus motifs of protein kinase family members (12) and have kinase activity (13, 14, 15, 16, 17) . The precise functions of the subunit are not known, but does appear to have a role in regulating the kinase activity of the subunits (18, 19, 20) . Although enhances protein kinase activity exhibited by toward most protein substrates, it actually inhibits the phosphorylation of calmodulin (21). Complementary DNAs encoding the , `, and subunits of CKII have been isolated from human and chicken cDNA libraries (13, 14, 22, 23, 24) . These studies indicate that the and ` subunits are closely related proteins that are encoded by distinct genes. Between mammals and birds, the and ` subunits are very highly conserved with the deduced amino acid sequences of human and chicken and ` exhibiting 98 and 97% identity, respectively. The subunit of CKII exhibits an even greater degree of conservation. Remarkably, the deduced amino acid sequences of human and chicken are identical and differ from the deduced sequence of frog by only a single amino acid (14, 22, 24, 25) . Collectively, these studies suggest that the functional properties of CKII are highly conserved throughout evolution. Furthermore, these results obviously indicate that the tetrameric structure of CKII is conserved between species.

In the present study, we have demonstrated by immunoprecipitation using - and `-specific antibodies that mammalian CKII exists as homotetramers ( or `) and as heterotetramers (`). Furthermore, using a yeast two-hybrid system originally described by Fields and Song (26) , we have demonstrated that the or ` subunits of CKII can interact with the subunits of CKII, but not with other or ` subunits. By comparison, the subunits of CKII can interact with , `, or subunits. These results indicate that the formation of the CKII holoenzyme involves dimerization of subunits to bring two or ` heterodimers into a tetrameric complex.


EXPERIMENTAL PROCEDURES

Materials

CKII was purified from bovine testis (27) . Antisera specific to the , `, or subunits of CKII were previously described (28, 29) and were affinity-purified on peptide affinity columns as described elsewhere (30) . The cDNAs encoding the human CKII and CKII ` subunits were isolated from a human T-cell library (13) . The cDNA encoding the subunit of human CKII was similarly isolated from a human T-cell library and was a generous gift of Dr. F. Lozeman and Dr. E. Krebs (University of Washington, Seattle). Each of these cDNAs had been cloned into the BamHI site of pBluescript (sk) to make the following constructs sk/CKII , sk/CKII `, and sk/CKII . Plasmids encoding the GAL4 DNA binding domain (pAS1-CYH2) or the GAL4 transcriptional activation domain (pACTII) were obtained from Dr. Stephen Elledge (Baylor College of Medicine, Houston, TX) (31, 32) . Oligonucleotides were synthesized on an Applied Biosystems model 380B DNA synthesizer by Dr. Mike Mowat (Manitoba Institute of Cell Biology). Heat-killed, formalin-fixed Staphylococcus aureus cells (Pansorbin) was obtained from Calbiochem. The Bst sequencing kit for DNA sequencing was purchased from Bio-Rad.

Immunoprecipitations

Purified CKII (0.25 µg) was immunoprecipitated using CKII subunit-specific antibodies by incubating the enzyme for 1 h on ice in phosphate-buffered saline with affinity-purified antibodies (5 µg). Immune complexes were isolated by the addition of 40 µl of Pansorbin (10% v/v) and further incubation on ice for 30 min. Immunoprecipitates were collected by centrifugation and were washed twice with radioimmune precipitation buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate) and once with 50 mM Tris-Cl, pH 7.5, prior to solubilization by the addition of Laemmli (33) sample buffer and incubation in a boiling water bath for 3 min. Samples were subjected to SDS-polyacrylamide gel electrophoresis (33) followed by transfer to nitrocellulose (34) using transfer buffer (25 mM Trizma (Tris base), 192 mM glycine, 20% methanol) containing 0.005% SDS. CKII subunits were detected using CKII subunit-specific antiserum (each added at a concentration of 1:1000) followed by the addition of I-protein A (1 µCi/ml, final concentration) as described previously (28) . After extensive washing of the filter and drying, filters were subjected to autoradiography. For quantitation, individual I-containing bands were excised and analyzed by counting.

Plasmid Constructs

To facilitate cloning of the cDNAs encoding CKII , `, or into the pAS1-CYH2 and pACTII vectors, a BamHI site was introduced at the 5` end of the coding region for each cDNA using the polymerase chain reaction (35) . A 718-base pair fragment of CKII was amplified from an sk/CKII template using the following primers: 5` CGC GGG ATC CTG TCG GGA CCC GTG CCA 3` (sense primer) and 5` ATC ATA ATT GTC ATG TCC ATG GAA 3` (antisense primer). The fragment was digested with BamHI and NcoI and used to replace a corresponding BamHI/NcoI fragment of the sk/CKII construct. A 383-base pair fragment of CKII ` was similarly amplified from an sk/CKII ` template using the following primers: 5` GCG GGG ATC CTG AGC AGC TCA GAG GAG 3` (sense primer) and 5` CAG GAT CTG GTA GAG TTG C 3` (antisense primer). The fragment was then digested with BamHI and PpuM1 and used to replace a corresponding BamHI/PpuM1 fragment of sk/CKII `. A 673-base pair fragment containing the entire coding region of CKII was amplified from an sk/CKII template using the following primers to introduce BamHI sites at each end of the cDNA: 5` GCG GGG ATC CTG AGC AGC TCA GAG GAG 3` (sense primer) and 5` CGG GGG ATC CAG ACT GCA GGA CAG GTG 3` (antisense primer). The fragment was digested with BamHI and cloned into the BamHI site of sk. The sequence of all amplified fragments was confirmed by sequencing of double-stranded Bluescript templates using the method of Sanger et al.(36) , with a Bst sequencing kit. Each of the cDNAs was subsequently cloned into the BamHI site of pAS1-CYH2 and of pACTII. The pAS1-CYH2/CKII and pACTII/CKII constructs each encode fusion proteins containing the entire CKII with the exception of Met which is lost through fusion to the respective domains of GAL4. Similarly, the pAS1-CYH2/CKII ` and pACTII/CKII ` constructs each encode fusion proteins containing the complete sequence of CKII ` beginning at Pro. The entire sequence of CKII starting with Ser is contained within the fusion proteins that are encoded by the pAS1-CYH2/CKII and pACTII/CKII plasmids. All pAS1-CYH2 and pACTII plasmids were routinely maintained in Escherichia coli DH5.

Transformation and Maintenance of Yeast

Yeast strains were grown and manipulated according to standard methods (37) . For two-hybrid experiments, the yeast strain DGY63::171 (MAT a, ade2 trp1-901 leu2-3, 112 his3-200 gal4 gal80::pRY171[GAL1lacZ](38) was transformed with equal quantities of various pAS1-CYH2 and pACTII plasmids using the highly efficient method of Gietz et al. (39). Transformants were selected on synthetic complete minus Trp-Leu plates at 30 °C and grown for 3-5 days. Colonies were then replicated onto synthetic complete minus Trp-Leu plates containing 50 mM KHPO, 2% sucrose as a carbon source, and 1 mg/ml 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal) to visualize colonies expressing -galactosidase.

Measurement of -Galactosidase Activity

Assays for -galactosidase activity were performed essentially as described previously (40) . Briefly, yeast were grown on synthetic complete minus Trp-Leu plates for 3-5 days at 30 °C. Several colonies from each plate were scraped from the plate with an inoculating loop, transferred to 1 ml of distilled water and thoroughly mixed. OD measurements were taken so that -galactosidase activities could be normalized according to the density of yeast cells. An aliquot (200 µl) of each suspension of yeast was transferred to a microcentrifuge tube and centrifuged briefly to pellet the yeast cells. After removing the supernatant, the cells were resuspended in 500 µl of Z buffer (100 mM NaPO, pH 7.0, 10 mM KCl, 1 mM MgSO, 38 mM -mercaptoethanol). A 50-µl aliquot of 0.1% SDS was added, and the cells were vortexed vigorously for 15 s. Chloroform (50 µl) was then added, and the cells were vortexed for an additional 15 s. Assays for -galactosidase were immediately initiated by the addition of 100 µl of 4 mg/ml o-nitrophenyl -D-galactopyranoside. Following incubation for 6 min at 37 °C, reactions were terminated by the addition of 500 µl of 1 M NaCO. Reaction mixtures were centrifuged for 1 min, and the OD was measured. -galactosidase activities in Miller units were calculated according to the following formula: units = OD 1000/(volume)(time)(OD) (41) .


RESULTS

Immunoprecipitation of CKII with Subunit-specific Antibodies

CKII is a tetrameric enzyme composed of two catalytic ( and/or `) subunits and two subunits (13, 14) . As we had previously noted (27) , purified bovine testis CKII consists of comparable amounts of and ` subunits (Fig. 1A). Direct sequence analysis and molecular cloning studies have demonstrated that CKII and CKII ` are very closely related proteins that have distinct carboxyl-terminal domains (13, 14, 27). Antipeptide antibodies raised against carboxyl-terminal peptides of CKII or CKII ` do not exhibit any cross-reactivity on immunoblots (Fig. 1B, lanes 1-4). Similarly, antibodies directed against do not react with or ` subunits (Fig. 1B, lanes 5 and 6). On immunoblots the anti- antisera recognizes CKII more efficiently than anti-` antisera recognizes its respective antigen (compare lanes 1 and 2 with lanes 3 and 4).


Figure 1: Electrophoretic and immunoblot analysis of purified CKII. A, purified bovine testis CKII (2 µg) was subjected to electrophoresis on a 12% SDS-polyacrylamide gel and visualized by staining with Coomassie Blue. The (M 45,000), ` (M 40,000), and subunits (M 26,000) of CKII are marked on the right side of the lane (from top to bottom). B, purified CKII (0.05 µg, lanes 1, 3, and 5; 0.25 µg, lanes 2, 4, and 6) was subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Individual lanes were incubated with antipeptide antiserum specific for the (lanes 1 and 2), ` (lanes 3 and 4), or (lanes 5 and 6) subunits of CKII. Each antiserum was utilized at a dilution of 1:1000. Immune complexes were detected with I-protein A and visualized by autoradiography as described under ``Experimental Procedures.'' The positions of CKII , CKII `, and CKII are indicated as in Panel A.



To determine whether CKII and CKII ` exist in the same tetrameric complex, we subjected purified bovine testis CKII to immunoprecipitation using these subunit-specific antibodies (Fig. 2). In an initial round of immunoprecipitation (Fig. 2A), purified CKII was immunoprecipitated with antibodies directed against CKII , CKII `, or CKII , respectively. Qualitatively, all immunoprecipitations are similar since , `, and are present in each immunoprecipitate. This result indicates that some of the CKII exists in a heterotetrameric complex composed of one subunit, one ` subunit, and two subunits. However, there are significant quantitative differences between the immunoprecipitates obtained with the different antibodies. All subunits of the purified enzyme are quantitatively immunoprecipitated with anti- antibodies (Fig. 2A, lane 9) since none of any of the subunits can be detected in the immunoprecipitation supernatant (Fig. 2C, lane 10). The quantitative immunoprecipitation of CKII with anti- antibodies confirms that the tetrameric complex was intact under our immunoprecipitation conditions. Quantitation of the I content resulting from the presence of I-protein A attached to immune complexes for the and ` bands indicates that the relative ratio of :` is 2.4:1 for anti- immunoprecipitates. Since comparable amounts of and ` are present in the testis enzyme (Fig. 1), it is apparent from this experiment that the antipeptide antibodies raised against ` recognize ` less efficiently than antibodies directed against recognize . By comparison with anti- immunoprecipitations, all subunits are not quantitatively isolated when immunoprecipitates are performed with anti- or with anti-` antibodies (Fig. 2A, lanes 1-8). Furthermore, alterations in the relative intensity of CKII to CKII ` are observed when the different antibodies are used for immunoprecipitation. While the amount of that is isolated by immunoprecipitation using or antibodies is very similar (Fig. 2A, lanes 1-4 compared with lane 9), significantly less is isolated using anti-` antibodies. Also, the signals observed for and ` in immunoprecipitates using anti-` antibodies are very similar (Fig. 2A, lanes 5-8). However, when it is considered that ` is detected less efficiently than , this result indicates that ` is more abundant than in immunoprecipitates performed with anti-` antibodies. The :` ratio is 5.3:1 for anti- immunoprecipitates and is only 0.67:1 for anti-` immunoprecipitates. These data reflect an enrichment of relative to ` in anti- immunoprecipitates and a corresponding enrichment of ` relative to in anti-` immunoprecipitates. Collectively, these results indicate that some of the purified CKII is composed of homotetramers (i.e. and `). Analysis of material isolated in a second round of immunoprecipitation supports this conclusion (Fig. 2B) as does the analysis of supernatants obtained following one or two rounds of immunoprecipitation (Fig. 2C).


Figure 2: Immunoprecipitation of purified CKII with subunit-specific antipeptide antibodies. A, pPurified CKII (0.25 µg) was immunoprecipitated using anti- antibodies (lanes 1-4), anti-` antibodies (lanes 5-8), or anti- antibodies (lane 9). Immunoprecipitates were electrophoresed on SDS-polyacrylamide gels and analyzed by immunoblot analysis using a mixture of anti-, anti-`, and anti- antiserum each at a dilution of 1:1000. Immune complexes were detected with I-protein A and visualized by autoradiography. B, supernatants obtained following immunoprecipitation with either anti- antibodies (lanes 1-3) or anti-` antibodies (lanes 4-6) were subjected to a second round of immunoprecipitation with anti- antibodies (lanes 1 and 4), anti-` antibodies (lanes 2 and 5), or anti- antibodies (lanes 3 and 6). Immunoprecipitates were analyzed by immunoblotting as in Panel A. Purified CKII was also analyzed (lane 7, 0.25 µg; lane 8, 0.125 µg). C, supernatants obtained following one or two rounds of immunoprecipitation were subjected to electrophoresis and analyzed by immunoblotting as in Panels A and B. As a standard, purified CKII (0.25 µg) is also analyzed (lane 1). Supernatants are as follows: lane 2, one round of immunoprecipitation with anti-; lane 3, two rounds of immunoprecipitation with anti-; lane 4, immunoprecipitation with anti- followed by anti-`; lane 5, immunoprecipitation with anti- followed by anti-; lane 6, one round of immunoprecipitation with anti-`; lane 7, immunoprecipitation with anti-` followed by anti-; lane 8, two rounds of immunoprecipitation with anti-`; lane 9, immunoprecipitation with anti-` followed by anti-; lane 10; one round of immunoprecipitation with anti-. The positions of the , `, and subunits of CKII are marked as in Fig. 1.



Supernatants obtained following the first round of immunoprecipitation were subjected to a second round of immunoprecipitation using anti-, anti-`, or anti- antibodies (Fig. 2B) or were analyzed directly on immunoblots together with supernatants obtained following two rounds of immunoprecipitation (Fig. 2C). When the supernatant from an anti-` immunoprecipitate is subjected to immunoprecipitation with anti- (Fig. 2B, lane 4), the immunoprecipitate contains primarily and subunits. The :` ratio of this immunoprecipitate based on I content is 5.9:1. Similarly, the supernatant obtained following two rounds of immunoprecipitation with anti-` (Fig. 2C, lane 8) demonstrates that is much more abundant than ` (:` ratio of 7.5:1). A corresponding enrichment of ` relative to is observed in the supernatant obtained following one or two rounds of anti- immunoprecipitation (Fig. 2C, lane 3; :` ratio of 0.3:1) and when the supernatant from an anti- immunoprecipitation is subjected to immunoprecipitation with anti-` in the second round (Fig. 2B, lane 2; :` ratio of 0.1:1.0).

Analysis of Subunit Interactions with the Two-hybrid System

To examine subunit interactions between the various subunits of CKII, we utilized the yeast two-hybrid system developed by Fields and Song (26) . For these experiments, cDNAs encoding the , `, or subunits of human CKII were each ligated into the pAS1-CYH2 and pACTII vectors described by Durfee et al.(31) in order to express each subunit as a fusion with the DNA binding domain (pAS1-CYH2) or with the transcriptional activation domain (pACTII) of the yeast GAL4 transcriptional activator. The yeast strain DGY63::171, which expresses -galactosidase under the control of the GAL1 promoter that is regulated by the GAL4 transcription factor was then cotransformed with different combinations of pAS1-CYH2 and pACTII constructs. Initial experiments were performed to verify that interactions between fusion proteins containing CKII (or CKII `) and CKII could be detected in yeast and to determine whether any of the DNA-binding domain fusions encoded by pAS-CYH2/CKII , pAS1-CYH2/CKII `, pAS1-CYH2/CKII nonspecifically stimulated -galactosidase expression. Interactions between hybrid proteins are indicated when yeast that are grown on media containing X-gal turn blue because they express -galactosidase. Yeast that had been cotransformed with combinations of (or `) and hybrids turned blue when grown in the presence of X-gal, indicating that interactions between the CKII subunits can be detected with the two-hybrid system (data not shown). Importantly, -galactosidase expression was not evident in those yeast expressing only one of the CKII subunit hybrids demonstrating that none of the CKII subunit hybrids can stimulate transcription in the absence of a binding partner (data not shown). To assess interactions between all of the subunits of CKII, yeast were cotransformed with all combinations of pAS1-CYH2 and pACTII constructs encoding each of the CKII subunits (). An indication of interaction between the various fusion proteins was obtained by measuring -galactosidase activity in extracts prepared from various transformants. All transformants obtained using various combinations of subunits together with either or ` subunits express significant amounts of -galactosidase activity. The highest -galactosidase expression is observed when GAL4 DNA binding hybrids of the subunit are cotransformed with GAL4 transcriptional activation hybrids of either or `. Less significant -galactosidase expression is observed when these hybrid combinations are reversed such that the GAL4 DNA binding hybrids of or ` are cotransformed with GAL4 transcriptional activation hybrids of . Although we do not know why different levels of -galactosidase expression are achieved when the hybrid combinations are reversed, these results provide as expected, a clear indication that the or ` subunits of CKII interact with the subunit of CKII in the two hybrid system. Interestingly, when yeast are cotransformed with the subunit hybrid of the GAL4 DNA binding domain together with the subunit hybrid of the GAL4 transcriptional activation domain, measurements of -galactosidase expression are comparable to those observed when the GAL4 transcriptional activation hybrid of is cotransformed into yeast with the GAL4 DNA binding domain hybrids of or `. By comparison, minimal -galactosidase activity was expressed in yeast transformed with various combinations of hybrids encoding and/or `. Of these combinations, only yeast cotransformed with GAL4 DNA binding domain and GAL4 transcriptional activation domain hybrids of expressed measureable activity. However, the activity of this combination was more than 20-fold less than observed for any of the combinations involving hybrids of .


DISCUSSION

It is a general feature, although not necessarily a universal feature (i.e. no subunit has been identified in Dictyostelium) (8) , that CKII is a tetrameric enzyme composed of two subunits and two and/or ` subunits. Data obtained in this study using the yeast two-hybrid system indicate that the subunit can interact with another subunit, suggesting that the tetrameric CKII complex forms through association between subunits which brings two and/or ` heterodimers into a tetrameric complex (Fig. 3). A low level of -galactosidase activity is observed in yeast cells cotransformed with GAL4 DNA binding domain fusions of CKII and with GAL4 transcriptional activation domain fusions of CKII . However, it is unlikely that interactions are responsible for tetramer formation since no activity is observed when any other combinations of CKII or CKII ` are analyzed. If one subunit were unable to interact with another subunit, we would predict that CKII would exist as a heterodimer rather than a tetramer.


Figure 3: Model of tetrameric CKII complexes. Immunoprecipitations indicate that CKII exists in homotetrameric complexes ( and `) and in heterotetrameric complexes (`). Results obtained with the yeast two-hybrid system indicate that tetrameric CKII complexes form through interactions of subunits.



We have previously demonstrated that the bulk of newly synthesized subunit is rapidly incorporated into complexes with subunits (42) . By comparison, the subunit is synthesized in excess of and incorporates slowly with . These observations imply that CKII complex formation would be governed by the availability of subunit. From data presented here, it appears that formation of the tetrameric CKII complex is mediated through the subunit and that there are only modest differences (i.e. approximately 2-fold) in the interactions of the or ` subunits of CKII with . Therefore, the incorporation of or ` subunits into the tetramer would be determined by the availability of the respective proteins. We do not know whether the assembly of tetrameric CKII is an ordered process. However, since the two hybrid data indicates that subunits can interact directly, presumably in the absence of subunits, then formation of dimers could precede incorporation of or ` subunits.

At present, distinct functions for CKII and CKII ` remain poorly defined. The intracellular localization of the two isozymic forms has been examined by immunofluorescence techniques (9, 10) . In chicken DU249 cells, Krek et al.(10) suggest that there is a great deal of overlap between the localization of and ` and that both isozymes are predominantly nuclear. By comparison, Yu et al. (9) suggest that there are dramatic differences between the localization of CKII and CKII `. Discrepancies between the two studies are difficult to resolve, but may result from the different experimental systems under examination. The results presented in this study demonstrate that bovine testis CKII exists in homotetrameric complexes (, `) and in heterotetrameric complexes (`) (see Fig. 3). We have previously noted that CKII ` is much less prominent in other cell lines than it is in testis (28, 42) . Therefore, it is possible that, in systems in which ` is less abundant than , ` is primarily in heterotetrameric (`) complexes. By comparison, in systems such as testis, where ` is as abundant as , then ` also exists in homotetrameric ` complexes. Although we do not know the tetrameric composition of the CKII under examination in the studies or Krek et al.(10) or Yu et al.(9) , differences in the tetrameric composition of the CKII could determine whether or not and ` are actually colocalized. Clearly, it will be of interest to examine the localization of and ` in a cell system in which CKII ` is abundant and a significant proportion of the enzyme is in homotetrameric ` complexes. A systematic analysis of the unique properties of CKII and CKII ` will undoubtedly yield a more complete understanding of the functions of the different tetrameric CKII complexes.

  
Table: Transcriptional activation by hybrid proteins



FOOTNOTES

*
This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and from the Terry Fox Foundation (to D. W. L.), the Medical Research Council of Canada (to R. D. G.), and the Manitoba Health Research Council (to R. D. G. and D. W. L). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Research Scientist of the National Cancer Institute of Canada. To whom correspondence should be addressed: Manitoba Institute of Cell Biology, 100 Olivia St., Winnipeg, Manitoba R3E 0V9, Canada. Tel.: 204-787-2177; Fax: 204-787-2190; E-mail: litchfi@cc.umanitoba.ca.

The abbreviations used are: CKII, casein kinase II; X-gal, bromo-4-chloro-3-indolyl -D-galactoside.


ACKNOWLEDGEMENTS

We thank Sharon Rennie and Elzbieta Slominski for helpful assistance during the course of this work and Dr. Michael Mowat for synthesis of oligonucleotides and helpful discussions. We also thank Dr. Stephen Elledge (Baylor College of Medicine) for providing plasmids used in the two-hybrid studies.


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