©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of Protein Kinase CKII Isoforms in HeLa Cells
ISOFORM-SPECIFIC DIFFERENCES IN RATES OF ASSEMBLY FROM CATALYTIC AND REGULATORY SUBUNITS (*)

(Received for publication, October 25, 1994; and in revised form, January 17, 1995)

Nicholas Chester (1) (2) Il Je Yu (1)(§) Daniel R. Marshak (1) (2)(¶)

From the  (1)W. M. Keck Structural Biology Laboratory, Arnold and Mabel Beckman Neuroscience Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 and the (2)Graduate Program in Molecular and Cellular Biology, State University of New York, Stony Brook, New York 11794

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein kinase CKII (formerly casein kinase II) can be isolated as a heterotetramer, containing two catalytic (alpha or alpha`) and two regulatory (beta) subunits. We have characterized the forms of CKII in HeLa cells using antibodies specific for the alpha or alpha` subunits. Following metabolic labeling with [S]methionine, whole cell soluble extracts were analyzed by immunoprecipitation and gel electrophoresis. Both alpha and alpha` coprecipitate with beta and with each other. However, when extracts are depleted of alpha, a pool of CKII containing only alpha` and beta is identified. Similarly, depletion of alpha` revealed a pool exclusively of alpha and beta. Therefore, we propose that there are three distinct isoforms of CKII within HeLa cells with different catalytic subunit stoichiometries (alpha(2)beta(2), alphaalpha`beta(2), and alpha`(2)beta(2)). With our immunodepletion procedure we have characterized the isoforms by activity analysis, turnover of pulse-labeled subunits, and by localization in subcellular fractions obtained from labeled cells. We have also analyzed complex formation between the catalytic and regulatory subunits by examining the differences in the rate of signal incorporation into subunits in immunoprecipitates obtained from continuously labeled and pulse-labeled cells. We have found that the alpha(2)beta(2) and alphaalpha`beta(2) isoforms assemble relatively slowly (12-16 h), whereas complex formation of the alpha`(2)beta(2) isoform occurs more rapidly (2-4 h). Analysis of isoform complex formation in subcellular fractions from pulse-labeled cells revealed that the majority of nuclear CKII is assembled in the nucleus from free catalytic and regulatory subunit polypeptides.


INTRODUCTION

Protein kinase CKII (^1)(casein kinase II) is a ubiquitous protein serine/threonine kinase present in all eukaryotic cells (for reviews, see Hathaway and Traugh, 1982; Edelman et al., 1987; Tuazon and Traugh, 1991). In mammalian cells, CKII has been implicated in signal transduction pathways, and increased kinase activity has been observed in a variety of cell types after serum (Carroll and Marshak, 1989) or growth factor stimulation (Sommercorn and Krebs, 1987; Klarlund and Czech, 1988; Ackerman and Osheroff, 1989; DeBenedette and Snow, 1991; for review, see Issinger, 1993). Also, mitogenic stimulation of quiescent fibroblasts can be reduced or eliminated by antisense oligonucleotides complementary to CKII mRNAs or by microinjection of CKII antibodies, indicating that CKII is necessary for cell cycle progression (Pepperkok et al., 1991, 1994; Lorenz et al., 1993). The observations that p34 kinase and CKII are targets for one another and that phosphorylations are cell cycle-dependent further imply a regulatory role for CKII in the cell cycle (Russo et al., 1992; Litchfield et al., 1992). Consistent with this has been the identification of a number of nuclear substrates, including oncogene products such as c-myc and c-myb; transcription factors including SRF, c-erbA, c-jun, Max, CREB; and the tumor suppressor p53 (Lüscher et al. 1989, 1990; Manak et al., 1990; Glineur et al., 1989; Baker et al., 1992; Lin et al., 1992; Berberich and Cole, 1992; Lee et al., 1990; Meek et al., 1990). However, in most cases, the exact function of the phosphorylation is not known.

In most organisms and tissue types, it has been proposed that there are two forms of the enzyme of heterotetrameric subunit stoichiometry: alpha(2)beta(2) and alphaalpha`beta(2) (for review, see Pinna, 1990). Subunits alpha and alpha` are thought to be catalytic based on homology to conserved subdomains in other kinases (Lozeman et al., 1990; for review, see Hanks et al., 1988). It has been shown by in vitro reconstitution studies that the alpha and alpha` subunits have phosphotransferase activity in the absence of the beta subunit (Cochet and Chambez, 1983; Hu and Rubin, 1990; Jakobi and Traugh, 1992) and in vivo, by overexpression of subunits in mammalian and insect cells, that the beta subunit enhances activity of the catalytic subunit 5-10-fold (Grankowski et al., 1991; Filhol et al., 1991; Heller-Harrison and Czech, 1991).

In addition to activity characterization, the subcellular distribution of CKII in mammalian cells has also been investigated. CKII has been shown to be present both in the nucleus and cytosol in all tissue types examined to date (Inoue et al., 1984; Meggio and Pinna, 1984; Filhol et al., 1990a) and cultured cell lines (Yu et al., 1991; Pepperkok et al., 1991; Serrano et al., 1987). It has also been found in mitochondrial and microsomal fractions (Edelman et al., 1987; Tuazon and Traugh, 1991) and has been identified as an extracellular matrix-associated kinase or ecto kinase (Kübler et al., 1983). It is described as having mainly nuclear localization, although the cytosolic to nuclear protein ratio may depend on the cellular growth state (Filhol et al., 1990a; Lorenz et al., 1993). The widespread dissemination of CKII in a cell and its association with different cellular proteins such as HSP90 (Miyata and Yahara, 1992) and p53 (Filhol et al., 1992) and heterochromatin (Filhol et al., 1990b), suggest that the enzyme exists in a number of pools or states in a cell and that different enzyme pools may have different activity or function. However, it is important to distinguish between identification of different enzyme pools based on criteria such as localization and cellular protein association, and identification of different forms of the enzyme based on subunit stoichiometry.

In both yeast and higher organisms, the alpha and alpha` subunits are encoded by separate genes and are highly homologous to each other, sharing 85% amino acid identity in human (Lozeman et al., 1990; Padmanabha et al., 1990). However, it is not known if there are biochemical differences between the catalytic subunits. Furthermore, to date it has not been possible to determine isoform composition for a preparation of CKII; SDS-PAGE and sedimentation velocity analysis indicate only the presence of subunits (typically, major alpha and beta bands with a minor abundance alpha` band are seen), and polypeptide association results in the formation of tetrameric complex(es) (for review, see Pinna, 1990). There is evidence that different tissue types may express the catalytic subunits at varying levels (for reviews, see Tuazon and Traugh, 1991; Issinger, 1993); however, if all three subunits are present simultaneously, it is not possible to tell which forms of CKII are present.

In this report we describe an immunodepletion assay using catalytic subunit specific antibodies that allowed us to identify CKII isoforms in HeLa cells. Using this immunoprecipitation technique enabled us to engage in isoform characterization, in which we set out to determine if there were any biochemical differences between isoforms based on turnover, subcellular localization, and complex formation. Our results indicate that marked isoform-specific differences exist in the rate of complex formation between newly synthesized polypeptides.


MATERIALS AND METHODS

Synthetic Peptides

Peptides CSH 150 H-RRREEETEEE-OH and CSH 353 H-RRRDDDSDDD-OH were used as substrates in the activity assay. Peptides CSH 175 H-CTPSPLGPLAGSPVIAAANPLGMPV-NH(2) (spanning from Thr to Val of the carboxyl terminus of human CKII alpha subunit), and CSH 244 H-CEQSQPCADNAVLSSGLTAAR-OH (spanning from Glu to Arg of the carboxyl terminus of human CKII alpha` subunit) were used as antigens. Peptides were synthesized as described (Barany and Merrifield, 1979; Marshak and Carroll, 1991) using an Applied Biosystems/Perkin-Elmer model 430A automated peptide synthesizer. A cysteine residue was added to these sequences used as antigens to facilitate coupling to hemocyanin as described below. The final structure of the peptides was verified by amino acid analysis, automated sequence analysis, plasma desorption mass spectrometry, and analytical microbore high performance liquid chromatography (Marshak and Carroll, 1991).

Antibody Preparation

Antibodies against peptides CSH 175 and CSH 244 were prepared as described (Yu et al., 1991). Briefly, the peptides were coupled to keyhole limpet hemocyanin (Sigma) via maleimidobenzoyl-N-hydroxysuccinimide (Pierce Chemical Co.) as described (Harlow and Lane, 1988). Antisera against the complexes were raised as follows. The complexes were mixed with 0.5 ml of Freund's complete adjuvant and injected subcutaneously. Booster injections were given with incomplete adjuvant at 2-week intervals. Additional booster injections were given at 2-week intervals until maximum serum titer was reached. Serum antibody titer was determined by solid phase radioimmunoassay.

Cell Culture

HeLa cells were grown at 37 °C on 10-cm dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.

Cell Labeling

For metabolic labeling of proteins, HeLa cells were split at 2.5-4.0 times 10^6 cells/dish 36 h prior to the addition of label. Cells were incubated in the presence of 0.5-1.0 mCi of TranS-label (ICN Biomedicals Inc., Lisle, IL) in methionine-free medium. For pulse-chase experiments, cells were labeled for either 2 or 12 h, washed with phosphate-buffered saline (PBS), and replenished with complete medium. For continuous labeling, the labeling medium was supplemented with complete medium to 5% at 6 h and to 10% at 12 h of the initial medium volume. For both types of labeling procedures, cells were harvested at the indicated time points by several washes in PBS and were removed from the dishes with a plastic scraper. Cell pellets were stored at -80 °C prior to lysis and immunoprecipitation.

Subcellular Fractionation

Subcellular fractionation was performed essentially as described (Stallcup et al., 1978). For fractionation of unlabeled cells, 1-2 times 10^8 PBS-washed cells (typically 10 confluent 10-cm dishes) were allowed to swell on ice in 2-3 ml of hypotonic buffer containing 20 mM Tris-HCl, pH 7.80, 6 mM MgCl(2), 4 mM CaCl(2), 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml pepstatin A, 1 mM phenanthroline for 5 min; an equal volume of 0.6 M sucrose, 0.2% Nonidet P-40, 0.5 mM DTT was added, and the cells were broken by 15-20 strokes of a Kontes type B Dounce homogenizer. Completeness of breakage was verified by light microscopy. Crude nuclei were pelleted by centrifugation for 10 min at 1,500 times g, and the supernatant was removed and respun at 100,000 times g for 30 min. The resulting supernatant was the source of soluble cytosolic extract used for immunoprecipitation. Nuclei were purified by resuspending the 1,500 times g pellet in buffer containing 0.25 M sucrose, 20 mM Tris-Cl, pH 7.80, 10 mM MgCl(2), 0.5 mM DTT, and protease inhibitors. The crude nuclear suspension was layered over 2.0 M sucrose containing 20 mM Tris-HCl, pH 7.80, 10 mM MgCl(2), 0.5 mM DTT and was centrifuged for 45 min at 48,000 times g. The nuclear pellets were drained and resuspended in nuclei wash buffer containing 10 mM Tris-HCl, pH 7.80, 15 mM NaCl, 60 mM KCl (Bresnick et al., 1992), and nuclei were recentrifuged at 3,000 times g for 5 min. Light phase microscopic examination of purified nuclei showed preparations to be free of debris and whole cells. Nuclei were either stored at -80 °C prior to lysis or lysed directly in lysis buffer prior to protein assay and immunoprecipitation as outlined below. Fractionation of S-labeled cells was performed as described above, except the procedure was scaled down to accommodate fewer cells. Time points from labeling experiments were represented by one or two dishes (split at 2.5-4.0 times 10^6 cells/10-cm dish) and were subjected to individual subcellular fractionation. This required the following modifications. No DTT was included in the buffers used for fractionation, and nuclei were spun through a 1.8 M sucrose cushion to maximize yields.

Immunoprecipitation

Whole cell extracts (labeled or unlabeled) were prepared by lysis of PBS-washed cell pellets in 0.5 ml of lysis buffer containing 50 mM Tris-Cl, pH 7.50, 0.5 M NaCl, 1% Nonidet P-40, 10% glycerol, 10 mM EDTA, 2 mM EGTA, and protease inhibitors as described under ``Subcellular Fractionation.'' After incubation on ice for 5-10 min, extracts were centrifuged at 15,000 times g for 3 min, and aliquots of supernatants were taken for protein quantitation using the method of Bradford(1976). Nuclear extracts (labeled or unlabeled) were prepared similarly by adding 5 volumes of lysis buffer to pelleted and washed nuclei. All sample extracts, either whole cell, cytosolic or nuclear, were normalized to a standard amount of protein in the extract for each immunoprecipitation in a given experiment, and 100-500 µg of protein in the extract was used in each immunoprecipitation. Reaction volumes were normalized to 0.5 ml with lysis buffer prior to the addition of antibody. For immunoprecipitation reactions, samples were first incubated with 50-100 µg of purified normal rabbit IgG. Pure IgG was obtained by protein A affinity purification of whole rabbit serum (Cappel, West Chester, PA) as described (Harlow and Lane, 1988), and incubated for 60 min on ice. Samples were then preadsorbed one or two times with 0.5 ml of 10% (w/v) fixed and killed Staphylococcus aureus (Zysorbin, Zymed Laboratories, South San Francisco, CA) followed by incubation with primary antibody for 90 min. Antisera to the catalytic subunits were titrated based on a fixed amount of protein in the extract so that immunoprecipitation reactions were quantitative. Typically, 3 µl of alpha antiserum and 10 µl of alpha` antiserum were required for 200 µg of protein in the immunoprecipitation. Precipitation of the antibody-antigen complex was accomplished by rotation for 60 min in the presence of protein A beads (Pierce). For [S]methionine immunoprecipitation, beads were washed extensively in lysis buffer, water, 0.5 M LiCl, pH 7.50, and lysis buffer using multiple tube transfers. An aliquot (100 µl) of sample buffer (Laemmli, 1970) was added to aspirated bead pellets, and samples were heated for 10 min at 85 °C prior to gel electrophoresis. For immunoprecipitation of unlabeled lysates for activity assay, beads were washed first in lysis buffer and second in activity wash buffer containing, 50 mM MOPS, pH 7.0, 10 mM MgCl(2), 60 mM NaCl prior to activity assay.

Multistep Immunoprecipitation

Supernatants from protein A bead pellets generated from immunoprecipitation reactions (above) were used as the source of extract for immunoprecipitation with alternate CKII antibodies. First, the clearing step was repeated two or three times with adsorption of immune sera with S. aureus cell pellets that were subsequently discarded. Extracts that had been cleared a total of three or four times were used for standard immunoprecipitation reactions with alternate CKII antibodies. Immunoprecipitation of CKII beta subunit from cleared extracts required that prior immunoprecipitation steps be carried out in a volume of 100 µl; denaturing immunoprecipitation was performed as described below.

Antibody to CKII beta was made as described (Yu et al., 1991). To immunoprecipitate the beta subunit with beta antiserum, denaturation of extracts was required. Labeled extracts were heated to 90 °C for 15 min in lysis buffer with SDS added to a final concentration of 1%. After heating, SDS-containing extracts were diluted 10-fold in 50 mM Tris-Cl, pH 7.5, 50 mM NaCl, and either beta antiserum alone was added, or a combination of alpha, alpha`, and beta antisera was added to the immunoprecipitation. All subsequent steps in the immunoprecipitation prior to gel electrophoresis were carried out as described above.

Cross-linking Antibodies to Protein A Beads

For immunoblot analysis of CKII immunoprecipitates, it was necessary to cross-link immune IgG to immobilized protein A. Both alpha and alpha` antisera were affinity purified over a protein A column as described (Harlow and Lane, 1988). Purified IgG was concentrated and dialyzed against PBS and then quantitated by protein assay. Five hundred µg of IgG was bound to 1 ml of 50% protein A bead slurry (Pierce) by rotation at room temperature in PBS for 30 min. After binding, PBS was substituted by 0.2 M sodium borate, pH 9.2, dry dimethyl pimelimidate (Pierce) was added to a final concentration of 20 mM, and the reactions were allowed to proceed for 2 h at room temperature. Reactions were terminated by incubating beads in 0.2 M ethanolamine, pH 8.3, for 10 min. Unlinked IgG was removed by multiple washes in 100 mM glycine, pH 2.5. After equilibration of IgG bead preparations in PBS, cross-linked antibodies were used directly in immunoprecipitation reactions as described below. Immunoprecipitation of CKII with cross-linked antibodies was performed by adding 75 µl of beads to 500 µg of protein in whole cell extract in a volume of 500 µl and rotating overnight at 4 °C. Supernatants were removed, and two more clearing steps were performed with the same cross-linked antibodies. Cleared extracts were used in immunoprecipitation reactions using alternate cross-linked catalytic subunit antibodies. Bead pellets from both steps were washed extensively in lysis buffer using one tube transfer. Elution of antigen from cross-linked beads necessitated heating bead pellets to 110 °C in lysis buffer containing 1% SDS for 15 min. After vortexing, beads were centrifuged, supernatant was removed, and the elution process was repeated two times. Pooled supernatants were subsequently reheated in Laemmli sample buffer prior to gel electrophoresis and immunoblotting as described below.

Gel Electrophoresis, Autoradiography, and Immunoblots

Gel electrophoresis was performed with 12.5% (w/v) SDS-PAGE using the buffer system of Laemmli(1970). For autoradiography and quantitative image analysis, gels were treated with ENTENSIFY (DuPont NEN) autoradiography enhancement reagent, dried, and exposed to a PhosphorImager plate (Fuji) and subsequently to x-ray film (X-Omat, Eastman Kodak) at -80 °C. Gel signals were imaged with a Fuji Bio-Image MacBAS 1000 analyzer, and quantitated signals were defined as PhosphorImager or PSL units.

For immunoblot analysis, proteins were transferred electrophoretically to nitrocellulose filters (Schleicher & Schuell) and blocked in 1% polyvinylpyrrolidone. A combination of beta subunit antiserum and rabbit antiserum anti-CSH124 representing a common internal epitope between alpha and alpha` (Yu et al., 1991), diluted at 1:500, was used as primary antibody. Donkey anti-rabbit immunoglobulin, F(ab`)(2) horseradish peroxidase-conjugated and diluted at 1:2,000 was used as secondary antibody. Signal detection was by enhanced chemiluminescence (ECL; Amersham) with x-ray film (Kodak) exposure.

Activity Assay

Immunoprecipitates in the form of a 50% slurry of protein A beads in activity wash buffer represented the source of CKII enzyme in the activity assay. For activity assay, 5 µl of slurry was incubated in in 30 µl of activity assay buffer containing 10 mM MgCl(2), 10 mM NaCl, 50 mM MOPS, pH 7.0, 100 µM ATP, 60 mM beta-glycerophosphate, 0.5-1.0 mM peptide substrate (RRRDDDSDDD or RRREEETEEE), and 5 µCi of [-P]ATP (3,000 Ci/mmol, Amersham) for 30 min at 30 °C. Reactions were terminated by adding trichloroacetic acid to a final concentration of 10% (w/v), and phosphate incorporation was determined by adsorption to phosphocellulose paper as described (Kuenzel and Krebs, 1985; Marshak and Carroll, 1991). It was determined in control experiments that phosphate incorporation was linear with time to at least 60 min, that linear incorporation kinetics occurred with enzyme dilution (protein A beads), and that the inclusion of the phosphatase inhibitor beta-glycerophosphate was required to give maximal incorporation of phosphate into peptide substrate.


RESULTS

Immunoprecipitation Analysis of HeLa Whole Cell Extracts Using Catalytic Subunit-specific Antibodies

Rabbit antiserum was raised against synthetic peptides made to the carboxyl termini of human catalytic subunit (alpha and alpha`) sequences (Lozeman et al., 1990) as described under ``Materials and Methods.'' The antisera were evaluated by immunoprecipitation assay of HeLa whole cell extracts generated by high salt lysis of intact cells. Asynchronous cells labeled metabolically with [S]methionine for 6 h were lysed, and aliquots containing equal amounts of protein in the extracts were immunoprecipitated using catalytic subunit antibodies as well as separately with a nonimmune control rabbit antibody. The immune complexes were subjected to gel electrophoresis followed by autoradiography. Fig. 1A shows that anti-alpha antibody immunoprecipitated, in addition to a putative alpha subunit band of 43 kDa, bands that appeared at 28 and 38 kDa, which corresponded to alpha` and beta subunit polypeptides (Fig. 1A, lane 2). Similarly, anti-alpha` antibody precipitated a 38-kDa species that corresponded to alpha` and, in addition, coprecipitated polypeptides that corresponded to alpha and beta subunits (Fig. 1A, lane 3). To verify that the three polypeptides were CKII subunits, in a separate experiment immunoblot analysis was performed on CKII immunoprecipitates. Similar immunoprecipitation reactions were carried out with unlabeled whole cell extract. Equal amounts of protein in the extract were immunoprecipitated using cross-linked catalytic subunit antibodies and cross-linked nonimmune rabbit IgG. Following electrophoresis of proteins eluted from protein A beads and electrophoretic transfer to nitrocellulose, immunoblot analysis was performed. A mixture of two rabbit antibodies was used simultaneously for immunoblotting. They have been described previously (Yu et al., 1991). Antibody 276 recognized a common epitope between the alpha and alpha` subunits, and antibody 278 recognized subunit beta; therefore all three subunits could be detected in this assay. The immunoblot result in Fig. 1B shows that the alpha and alpha` subunit immunoprecipitation reactions yielded a pattern similar to that of [S]methionine immunoprecipitations for alpha and alpha` CKII subunits. Our results indicate that in addition to immunoprecipitation of cognate subunits, the alpha and alpha` antibodies in each case coimmunoprecipitated the two additional subunits. From these data, it is possible to determine that a form of CKII is present which contains, in addition to the regulatory subunit, both catalytic subunits. The inferred subunit stoichiometry of this holoenzyme isoform is alphaalpha`beta(2).


Figure 1: Immunoprecipitation of CKII from HeLa cells using catalytic subunit-specific antibodies. CKII was immunoprecipitated from unlabeled cells or cells labeled with [S]methionine for 6 h. Whole cell extracts were prepared and divided so that equal protein was used for immunoprecipitation reactions with either nonimmune sera (NRS, lane 1), alpha subunit antiserum (lane 2), or alpha` subunit antiserum (lane 3). Panel A, autoradiogram of SDS-PAGE of immunoprecipitates from labeled extract. Panel B, immunoblot of immunoprecipitates from unlabeled extract. Cross-linked antibodies were used for three immunoprecipitation reactions (lanes 1-3). After electrophoresis and transfer to nitrocellulose, the blot was probed simultaneously with a combination of anti-alpha and alpha`, and anti-beta antibodies. Molecular mass standards are indicated (kDa). The positions of CKII alpha, alpha`, and beta subunits are shown in panels A and B. Panel B also contains immunoglobulin heavy chain (arrows).



In addition to including a negative control for the immunoprecipitation in each of the two types of immunoprecipitation procedures, we also performed two additional experiments to assay for specificity of the two catalytic subunit antibodies. In one experiment, dicistronic bacterial expression strains that expressed alpha and beta, or alpha` and beta subunits together (dicistronic recombinant plasmid was a gift of Joan Brooks at New England Biolabs) were used to test for catalytic subunit antibody cross-reactivity. When extracts from strains that express simultaneously either alpha and beta subunits, or alpha` and beta together, were used for immunoprecipitation, we determined that it was only possible to precipitate alpha and beta with alpha antiserum, and to precipitate alpha` and beta with alpha` antiserum (data not shown). In an additional experiment, it was determined that the two peptides used to raise alpha and alpha` antisera blocked corresponding HeLa cell extract immunoprecipitation reactions, and peptide to alpha did not block precipitation of alpha` with alpha` antibody, nor did peptide to alpha` block precipitation of alpha with alpha antibody (data not shown).

Immunoprecipitation of Additional CKII Isoforms by Immunodepletion Assay

Comparison of anti-alpha and anti-alpha` immunoprecipitation reactions in Fig. 1, A and B, shows differences between the two types of immunoprecipitations. More alpha subunit was precipitated in the anti-alpha than in the anti-alpha` immunoprecipitation; in contrast, an approximately equal amount of the alpha` subunit was precipitated with each antibody. Therefore, the question is raised as to whether just the alphaalpha`beta(2) form of CKII is immunoprecipitated by the two antibodies, or if additional form(s) are immunoprecipitated as well. To address this issue, we designed an immunodepletion assay that utilized a two-step immunoprecipitation procedure: (i) extracts were cleared with either alpha or alpha` antibody; (ii) cleared extracts were used in immunoprecipitation reactions with antibody against the alternate catalytic subunit. Whole cell extract was prepared from cells labeled with [S]methionine for 6 h, and protein in the extract was quantitated. The extract was divided equally into seven immunoprecipitations (Fig. 2A, 1st IP) that were cleared three times with either nonimmune, alpha, or alpha` antiserum, using S. aureus cell pellets that were subsequently discarded. Cleared extracts were then used for immunoprecipitation with alternate catalytic subunit antibodies, and products of these reactions were subjected to electrophoresis. The autoradiogram in Fig. 2A shows the products of the second immunoprecipitation step (Fig. 2A, 2nd IP). Present in this experiment were controls to assay for antigen that remained in the extracts after clearing. Comparison of lanes 2 and 3, and lanes 5 and 6 indicates that cognate or associated CKII subunits were not present in either of the two cleared extracts. Comparison of lanes 6 and 7 shows that alpha is immunoprecipitated with beta when the extract has been cleared of alpha` and associated subunits. Likewise, comparison of lanes 3 and 4 shows immunoprecipitation of alpha` and beta subunits in extract cleared of alpha and associated subunits. Our results show that in addition to the alphaalpha`beta(2) form present in HeLa extracts, there are two additional forms of CKII with inferred subunit stoichiometries of alpha(2)beta(2) and alpha`(2)beta(2). Furthermore, the assay shows what forms of enzyme are recognized by each antibody: antibody to alpha catalytic subunit immunoprecipitates alpha(2)beta(2) and alphaalpha`beta(2) (Fig. 1A, lane 2, and Fig. 2A, lane 2), and antibody to alpha` immunoprecipitates alpha`(2)beta(2) and alphaalpha`beta(2) (Fig. 1A, lane 3, and Fig. 2A, lane 5).


Figure 2: Immunoprecipitation of CKII isoforms by immunodepletion assay. Whole cell extracts were prepared from unlabeled cells or cells labeled with [S]methionine for 6 h, and equal protein was used in each of seven two-step immunoprecipitation reactions. The first immunoprecipitation reactions (1st IP) represent the immunodepletion step carried out with either nonimmune (NRS, lanes 1, 2, and 5), anti-alpha (lanes 3 and 4), or anti-alpha` (lanes 6 and 7) antibodies. The second immunoprecipitation reactions (2nd IP) were performed with supernatants from the first immunoprecipitation reactions (cleared extracts) and were carried out with either nonimmune (lane 1), anti-alpha (lanes 2, 3, and 7), or alpha` (lanes 4, 5, and 6) antibodies and subjected to electrophoresis. Panel A, autoradiogram of second immunoprecipitation reactions from labeled immunodepleted extracts (2nd IP). Panel B, immunoblot of second immunoprecipitation reactions from unlabeled immunodepleted extracts probed with anti-alpha and alpha`, and anti-beta antibodies. Both immunoprecipitation steps were performed with cross-linked antibodies. Molecular mass standards are indicated (kDa). The positions of CKII alpha, alpha`, and beta subunits are indicated in panels A and B. Panel B also contains immunoglobulin heavy chain (arrows).



The [S]methionine label result was verified by performing a similar immunodepletion experiment with unlabeled extract and cross-linked antibodies. Equal amounts of extract were divided into seven immunoprecipitation reactions, and three consecutive clearing steps were performed using cross-linked antibodies. Products from immunoprecipitations with alternate catalytic subunit antibodies were subjected to electrophoresis, transferred to nitrocellulose, and probed with antisera in a manner identical to the experiment in Fig. 1B. The immunoblot in Fig. 2B shows a result similar to that obtained with [S]methionine-labeled lysates. The alpha and beta subunits coimmunoprecipitated in the absence of the alpha` subunit (lane 7), and free alpha` subunit was detected, independent of the alpha subunit (lane 4). In this reaction, the presence of small amounts of contaminating IgG light chain that eluted from the protein A beads and migrated in the same region of the gel rendered detection of beta subunit difficult.

Activity Assay of Immunodepleted Immunoprecipitation Reactions

To determine if different CKII isoforms are associated with activity and to determine the specific activity of each isoform in whole cell extract, kinase activity was measured in immunoprecipitations derived from immunodepletion assays. Whole cell extract was prepared and divided so that 500 µg of protein in the extract was used for each of seven immunoprecipitations, and two-step immunodepletion assays were carried out as for labeled extracts, described above. The precipitates generated from the second immunoprecipitation reactions were used for CKII activity assay with either RRREEETEEE (ETE) or RRRDDDSDDD (DSD) synthetic peptide substrate as outlined under ``Materials and Methods.'' The peptides represent specific casein kinase II substrates, and the K of DSD peptide is approximately 40-fold greater than that of ETE peptide using purified bovine lung CKII (Kuenzel et al., 1987). In Fig. 3is a representative experiment that shows the activity associated with each type of immunodepleted immunoprecipitation. Activity is expressed as pmol of phosphate incorporated/min/mg of protein for each type of immunoprecipitation reaction for the two peptide substrates. For DSD peptide, the alpha(2)beta(2) isoform catalyzed incorporation of 115 pmol of phosphate/min, and alpha`(2)beta(2) incorporated 38 pmol of phosphate/min; for ETE peptide, alpha(2)beta(2) had an activity of 22 pmol/min, and alpha`(2)beta(2), 4 pmol/min/ mg of protein in the extract. To calculate the activity associated with the alphaalpha`beta(2) isoform, the activity associated with the alpha(2)beta(2) and alpha`(2)beta(2) isoforms was subtracted from the sums of alphaalpha`beta(2) and alpha(2)beta(2), and alphaalpha`beta(2) and alpha`(2)beta(2) isoform activities, respectively, and averaged. For DSD and ETE peptides, respectively, the activity was 85 and 9 pmol/min/mg of protein in the extract. Relative activity contributions of each isoform expressed as a percentage of total activity are summarized in Table 1and were determined by DSD peptide. The activity contributions for alpha(2)beta(2) and alphaalpha`beta(2) isoforms were approximately 45% each, and alpha`(2)beta(2) 10% of the total activity in whole cell extract. We have also obtained comparable activity contribution values by ETE peptide (data not shown).


Figure 3: Activity assay of isoforms associated with immunodepleted immunoprecipitation reactions. Whole cell extracts were prepared from unlabeled cells, and 0.5 mg of protein in the extract was used in each of seven two-step immunoprecipitation reactions. The first immunoprecipitation reactions were carried out with either nonimmune, anti-alpha, or anti-alpha` antibodies (1st IP). Cleared extracts from the first immunoprecipitation reactions were used in the second immunoprecipitation reactions with either nonimmune (NRS), anti-alpha, or anti-alpha` antibodies (2nd IP). Protein A bead immune complexes from the second immunoprecipitation reactions were the source of enzyme in the activity assay using two synthetic peptide substrates (see ``Materials and Methods''). Panel A, activity assay performed with RRRDDDSDDD peptide. Panel B, assay performed with RRREEETEEE peptide using a second aliquot of protein A beads. Activity was calculated as pmol of phosphate incorporated/min/mg of whole cell extract. Activities associated with immunoprecipitates arise from single isoforms or isoforms in combination and are indicated by symbols in A and B.





To compare isoform specific activities, the relative abundance of each isoform was calculated from the beta subunit signal associated with isoforms immunoprecipitated from HeLa cells radiolabeled for 12 h with [S]methionine. The beta subunit signal (PSL units) for each isoform was calculated as a percentage of the total beta signal obtained with the three isoforms and compared with the isoform relative activity values shown in Table 1. Comparison of columns 2 and 3 in Table 1indicates that the isoform activity contributions closely reflected isoform abundance and suggests that under our activity assay conditions there were no significant differences in the specific activities of isoforms.

Immunodepletion Analysis of CKII from Subcellular Fractions

To evaluate the possibility that the different forms of CKII might have different subcellular localization, immunodepletion analysis was performed on lysates from cytosolic and nuclear fractions. Subcellular fractionation was performed on both [S]methionine-labeled and unlabeled cells. For immunoprecipitations with labeled extracts, cells were labeled for 2 h and chased with complete medium for 10-14 h prior to subcellular fractionation and protein assay. For nuclear and cytosolic fractions, 200 µg of protein in each extract was used in each two-step immunoprecipitation reaction. The immunoprecipitation result with labeled extracts (Fig. 4, C and D) indicates that all three holoenzyme isoforms were present in both the nucleus and cytosol and that the three forms of the enzyme were more abundant in the nucleus. An immunodepletion assay was performed on subcellular fractions from unlabeled cells, and 100 µg of protein from each extract was used in two-step immunoprecipitation reactions followed by activity assay with the DSD peptide. Fig. 4, A and B, shows that activity (in cpm) was associated with CKII isoforms for each fraction, and there was approximately 3-fold more activity associated with nuclear isoform immunoprecipitates as compared with their cytosolic counterparts. The greater activity of nuclear immunoprecipitates reflects a higher specific activity of nuclear as compared with cytosolic extract and is explained by the greater relative abundance of nuclear as compared with cytosolic isoforms.


Figure 4: Immunodepletion analysis of CKII from subcellular fractions. Subcellular fractions were prepared from unlabeled and [S]methionine-labeled cells and analyzed by immunodepletion assay. For labeled extracts, 0.2 mg of protein was used in each of four two-step immunoprecipitation reactions for both cytosolic and nuclear fractions. Antibodies used to clear extracts are indicated (1st IP), and products from second immunoprecipitation reactions with cleared extracts (2nd IP) were subjected to electrophoresis and autoradiography (panels C and D). For unlabeled extracts, 0.1 mg of protein was used in each of seven two-step immunoprecipitation reactions for both cytosolic and nuclear fractions. Antibodies used for first immunoprecipitation reactions are indicated (1st IP). Activity assay was performed with synthetic peptide substrate RRRDDDSDDD using protein A bead immune complexes from the second immunoprecipitation reaction as the source of enzyme (2nd IP). Relative activities associated with immunoprecipitates are plotted (cpm) and arise from single isoforms or isoforms in combination; they are indicated by symbols, for cytosolic (panel A), and nuclear (panel B) fractions. Autoradiograms of SDS-PAGE for S-labeled immunoprecipitates that correspond (connecting lines) to activity assay immunoprecipitates are indicated for cytosolic (panel C, lanes 1-4) and nuclear (panel D, lanes 1-4) fractions. Molecular mass standards are indicated (kDa). The positions of alpha, alpha`, and beta subunits are shown (arrows) for cytosolic and nuclear fractions (panels C and D). For direct comparison, autoradiograms in panels C and D were processed in parallel.



Pulse-Chase Analysis of CKII Subunits and Isoforms

In an effort to determine if there were differences in the rates of turnover between CKII isoforms, pulse-chase labeling analysis was performed to determine turnover of the subunits and individual isoforms. Cells were labeled with [S]methionine for 12 h and chased for intervals up to 36 h. Fig. 5shows the result of an experiment in which three subunits for all isoforms were immunoprecipitated simultaneously from extracts normalized by protein assay by the addition of both alpha and alpha` antibodies. The signals were quantitated and are plotted in Fig. 5B. The calculated turnover numbers for each of the subunits were quite similar at 22, 24, and 27 h for alpha`, alpha, and beta, respectively. To determine if there were any isoform-specific differences in turnover, pulse-chase analysis was coupled with two-step immunoprecipitation, and the decay of the signal was measured for subunits associated with different isoforms. In Fig. 6, B and D, comparison of turnover between alpha(2)beta(2) and alpha`(2)beta(2) did not reveal obvious differences for the half-life between subunits for an isoform, or for a subunit between isoforms; the four subunits that compose the two isoforms shown have half-lives in the range of 24-29 h. The immunodepletion method does not allow us to immunoprecipitate the alphaalpha`beta(2) heterodimeric isoform directly, but evaluation of anti-alpha and anti-alpha` one-step immunoprecipitations enabled calculation of the turnover for the alpha and alpha` subunits associated with the alphaalpha`beta(2) isoform, respectively. The half-life of alpha was determined to be 25 h, and that of alpha` was 31 h (data not shown). These catalytic subunits fell approximately within the range determined for all subunits associated with homodimeric isoforms. Based on pulse-chase analysis, there are no significant differences in the rates of turnover for the three identified isoforms.


Figure 5: Determination of turnover for CKII alpha, alpha`, and beta subunits by pulse-chase analysis. Cells were labeled for 12 h with [S]methionine, washed, and chased with complete medium for intervals up to 36 h. Whole cell extracts were prepared from plates harvested at 0, 8, 16, 24, and 36 h. Protein in the extracts was quantitated, and an equal amount of protein was used in each immunoprecipitation reaction using anti-alpha and anti-alpha` antibodies in combination. Panel A, autoradiogram of immunoprecipitates after electrophoresis. Molecular mass markers are indicated (kDa); and alpha, alpha`, and beta subunit polypeptides are indicated by arrows.Panel B, PhosphorImager analysis of gel from panel A. Signals (PSL units) were plotted as a function of time and best fit lines drawn. Times that correspond to half-maximum signal (0 h) represent turnover numbers and are 24, 22, and 27 h for alpha, alpha`, and beta subunits, respectively.




Figure 6: Determination of turnover for CKII isoforms by pulse-chase analysis. Cells were labeled with [S]methionine for 12 h, washed, and chased with complete media for intervals up to 36 h. Whole cell extracts were prepared from plates harvested at 0, 8, 16, 24, and 36 h. Samples were quantitated by protein assay, and equal amounts of protein in the extracts were used in two-step immunoprecipitation reactions. Extracts were divided into two sets of immunoprecipitation reactions, and each set was cleared with either alpha or alpha` antibody. Immunoprecipitation of cleared sets of extracts with alternate catalytic subunit antibodies generated the alpha(2)beta(2) and alpha`(2)beta(2) isoforms, which were subjected to SDS-PAGE. Panel A, autoradiogram of alpha(2)beta(2) immunoprecipitates. Subunits alpha and beta are indicated by arrows. Panel B, autoradiogram of alpha`(2)beta(2) immunoprecipitates. Subunits alpha` and beta are indicated by arrows. PhosphorImager analysis was performed on gels from panels A and B, and signals for each isoform subunit were plotted as a function of time. Panel C, best fit plot of subunit signals associated with alpha(2)beta(2) isoform. Turnover numbers were calculated as times that correspond to half-maximum signal (0 h) and are 27 and 29 h for alpha and beta subunits, respectively. Panel D, best fit plot of subunit signals associated with the alpha`(2)beta(2) isoform. Turnover numbers are 29 and 24 h for alpha` and beta subunits, respectively.



Analysis of Complex Formation between Catalytic and Regulatory Subunits

We determined in our pulse-chase investigation that a long labeling period of 12 h was required for steady-state incorporation of [S]methionine into coimmunoprecipitated beta subunit, and this time interval was subsequently used for pulse-chase analysis (above). We set out to elucidate a mechanism for the lag in the appearance of coimmunoprecipitated beta subunit signal. Continuous labeling experiments were carried out for 16 h with dishes harvested at 2-h intervals, and extracts were prepared. The extracts were adjusted to the same protein concentration, and all three subunits immunoprecipitated by the addition of alpha and alpha` antibodies. Fig. 7, A and C, shows that the beta subunit signal reached steady state in the interval of 12-16 h, whereas signals for both the alpha and alpha` subunits reached steady state at 6 h. Furthermore, the signal increase from 2 to 12 h was 4-fold for subunit beta and only 1.7-fold for the alpha and alpha` subunits in the 2-6-h interval. To evaluate further these differences in signal incorporation, we tested if free beta subunit could be immunoprecipitated from extracts cleared with anticatalytic subunit antibody (Fig. 7A). To guard against the possibility that some beta subunit associated with complex remaining in the extract might be immunoprecipitated, supernatants were cleared a total of three times with excess alpha and alpha` antibodies. The autoradiogram in Fig. 7B shows the presence of free beta subunit at early intervals, which decreased during the time course, and this signal change is plotted in Fig. 7C. Taken together, these results suggest that signal lag for the beta subunit is a result of its slow assimilation into holoenzyme complex. To quantify the kinetics of complex formation, signals associated with free beta subunit and three complex polypeptides (Fig. 7C) were plotted either as a ratio of free or associated beta subunit signal/catalytic subunit signal, and the highest value obtained was used for normalization to 1.0 for each of the four ratios. Fig. 7D indicates that there is a reciprocal relationship between free and incorporated beta subunit, such that concomitant with the decrease in free beta subunit was an increase in complexed beta subunit. The alpha and alpha` versus beta subunit plots exhibit similar trends for both free and complexed beta subunit.


Figure 7: Analysis of complex formation between catalytic and regulatory subunits. Lag in beta subunit signal appearance relative to catalytic subunit signal was evaluated by continuous labeling analysis. Cells were labeled for 16 h with time points harvested at 2-h intervals. Whole cell extracts were prepared, samples quantitated by protein assay, and equal amounts of protein in the extracts immunoprecipitated with alpha and alpha` antibodies in combination. Panel A, autoradiogram of SDS-PAGE of immunoprecipitates containing alpha, alpha`, and beta subunits (arrows) for all isoforms. Panel C, PhosphorImager analysis of gel in panel A. The signal (PSL units) was plotted as a function of time for each subunit immunoprecipitated with anticatalytic subunit antibodies. Panel B, autoradiogram of SDS-PAGE of free beta subunit immunoprecipitated from denatured supernatants of immunoprecipitation reactions in panel A (see ``Materials and Methods''). The signal associated with the beta subunit is plotted in panel C. For direct comparison, autoradiograms in panels A and B were processed in parallel. Panel D, signals from panel C are replotted as a ratio of beta/catalytic subunit and normalized for each of four ratios: free beta/alpha or alpha` and complex-associated beta/alpha or alpha`.



Determination of Rate of beta Subunit Synthesis

The previous experiment suggested that the behavior of coimmunoprecipitated beta subunit signal is caused by a slow rate of incorporation into holoenzyme. However, it is possible that a slower rate of synthesis of beta subunit polypeptide may account for its slow rate of incorporation into complex. To test this possibility, a time course was conducted which was similar to that described above, and immunoprecipitation was performed on denatured extracts using a combination of alpha, alpha`, and beta subunit antibodies. Fig. 8A shows that substantially more beta subunit was observed at early time points when directly immunoprecipitated than was seen by coimmunoprecipitation (Fig. 7A). Comparison of the signal plots for each of the three subunits in Fig. 8B shows no obvious differences in the time required to reach maximal label incorporation, which was about 12 h for all three subunits. Two types of ratios for subunit/subunit signal were determined and are plotted in Fig. 8C. Evaluation of beta/alpha or alpha`, and alpha/alpha` plots shows little deviation from unity, which indicates that each subunit was synthesized at a similar rate. The result from this experiment strengthens our conclusion that the lag in the appearance of complexed beta subunit was caused by slow incorporation of previously synthesized polypeptide.


Figure 8: Determination of rate of beta subunit synthesis. The rate of beta subunit synthesis was evaluated by direct immunoprecipitation from extracts prepared from a 16-h continuous labeling time course with dishes harvested at 2-h intervals. Denaturing immunoprecipitation was performed as described under ``Materials and Methods'' on equal amounts of protein in the extracts. Reactions included, in addition to beta subunit antibody, alpha and alpha` subunit antibodies as controls. Panel A, autoradiogram of SDS-PAGE of immunoprecipitated alpha, alpha`. and beta subunits. Positions are indicated (arrows), and signals associated with each subunit are plotted in panel B. Panel C, signals from panel B are replotted as a ratio of subunit/subunit and normalized for each of three ratios: beta/alpha, beta/alpha`, and alpha/alpha`.



Analysis of Isoform Complex Formation

The analysis was extended to complex formation with individual isoforms. Immunoprecipitation of isoforms from whole cell extracts was accomplished by two-step immunodepletion using a time course similar to that above. Comparison of beta subunit signals for the alpha(2)beta(2) and alpha`(2)beta(2) isoforms in Fig. 9, A and B, respectively, reveals less overall increase and a shorter time to reach maximal strength for alpha`(2)beta(2) than for alpha(2)beta(2). These differences are shown in the signal incorporation versus time plot in Fig. 9C. When beta/alpha and beta/alpha` signal ratios are compared (Fig. 9D), the alpha(2)beta(2) isoform exhibited slow incorporation of the beta subunit similar to that shown for the combination of three isoforms (Fig. 7). However, the alpha`(2)beta(2) isoform did not exhibit an increase in the beta subunit signal during the time course. Taken together, these data suggest that there was a marked difference in the rates of complex formation between the regulatory and catalytic subunits for different forms of CKII. The alpha(2)beta(2) isoform took 12-16 h (Fig. 9C), and alpha`(2)beta(2) 2-4 h (Fig. 9D), to assemble tetrameric complexes.


Figure 9: Analysis of isoform complex formation. Cells were labeled continuously for 16 h, dishes were harvested at 2-h intervals, cell extracts prepared, and protein quantitated and normalized as standard. The alpha(2)beta(2) and alpha`(2)beta(2) isoforms were immunoprecipitated in two-step immunoprecipitation reactions and subjected to electrophoresis. Analysis of beta subunit appearance was determined for isoforms by autoradiography and imaging analysis. Panel A, autoradiogram of alpha(2)beta(2) immunoprecipitates. alpha and beta subunits are indicated by arrows. Panel B, autoradiogram of alpha`(2)beta(2) immunoprecipitates. alpha` and beta subunits are indicated by arrows. Panel C contains signal plots of four subunits for the two isoforms from panels A and B. Panel D, signals from panel C are replotted as a ratio of beta/catalytic subunit and normalized for two ratios: beta/alpha and beta/alpha`.



To determine the rate of complex formation for the alphaalpha`beta(2) isoform, anti-alpha and anti-alpha` immunoprecipitates generated from the first immunoprecipitation step in this experiment were evaluated, and beta subunit signal incorporation was found to be slow for both mixed isoform immunoprecipitates (data not shown). From this, we conclude that both the alpha(2)beta(2) and alphaalpha`(2)beta(2) isoforms exhibited similar slow complex formation times. We believe that this result does not depend on the label incorporation protocol because substitution of the continuous label procedure by a pulse-chase method that used short pulses of 2 h followed by 14-h chases yielded similar results for the three CKII isoforms. Complex formation for the major abundance isoforms, alpha(2)beta(2) and alphaalpha`beta(2), took 10-14 h of chase to reach completion (data not shown).

Analysis of Minor Abundance Isoform Complex Formation

Since the previous time course did not show complex formation occurring for alpha`(2)beta(2), we were interested in evaluating the possibility that an increase in the beta/alpha` signal ratio could be observed for this isoform during a shorter time course. We conducted a pulse-chase experiment that used a short [S] methionine label incubation period of 30 min followed by a chase period with plates harvested at intervals up to 4 h. Two-step immunoprecipitation was performed, and in addition to evaluating signal ratios for the alpha(2)beta(2) and alpha`(2)beta(2) isoforms, signal plots that corresponded to beta/catalytic subunit signals for anti-alpha and anti-alpha` first step immunoprecipitations were also evaluated for comparison. Fig. 10shows that the beta/alpha` signal ratio for alpha`(2)beta(2) exhibited a steep 2-fold increase from 0 to 1 h and eventually reached a maximum at 3 h. In contrast, beta/catalytic signal ratios for all other immunoprecipitations exhibited shallow linear increases from 0 to 4 h and did not plateau. This result indicates that complex formation for alpha`(2)beta(2) can be observed and reaches completion with rapid kinetics of 2-4 h. In direct contrast, the major abundance isoforms required longer time courses to reach completion. We have also observed a similar increase in the beta/alpha` ratio for alpha`(2)beta(2) using a short continuous label time course (data not shown).


Figure 10: Analysis of minor isoform complex formation. Cells were pulsed labeled with [S]methionine for 30 min, washed, and chased with complete medium for intervals up to 4 h. Extracts were prepared from plates harvested at 0 h, 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h. Protein in the extracts was quantitated and equal amounts of protein in the extracts subjected to two-step immunoprecipitation and electrophoresis. In addition to imaging analysis of subunits associated with the alpha`(2)beta(2) isoform, included as controls are alpha(2)beta(2) isoform, and anti-alpha and anti-alpha` mixed isoform immunoprecipitates. Subunit signals associated with each type of immunoprecipitation were quantitated (PSL units), plotted as a ratio of beta/catalytic subunit signal, and normalized. Closed circles indicate the plot of the minor abundance alpha`(2)beta(2) isoform; open circles indicate the alpha(2)beta(2) isoform plot. beta/alpha and alpha` plots for anti-alpha immunoprecipitations are shown by open squares and closed triangles, respectively, and for anti-alpha` immunoprecipitations, by closed squares and open triangles, respectively.



Complex Formation of Isoforms in Subcellular Fractions

Nuclear translocation of subunits could be a post-translational mechanism to explain slow complex formation for the major abundance isoforms alpha(2)beta(2) and alphaalpha`beta(2). Consistent with nuclear localization of CKII, the alpha and alpha` subunits share the basic KPVKKKKIKR region, which is a putative nuclear targeting motif (Dang and Lee, 1989; Lozeman et al., 1990). We conducted experiments to measure catalytic subunit translocation and to relate translocation to complex formation with the regulatory subunit. Cells were pulse labeled with [S]methionine for 2 h and chased with complete medium for intervals up to 14 h, after which subcellular fractions were prepared, immunodepletion was performed, and CKII immunoprecipitates from various time points were analyzed for degree of complex formation. Examination of anti-alpha immunoprecipitation reactions from nuclear fractions shows that the beta subunit associated with the combination of the alpha(2)beta(2) and alphaalpha`beta(2) isoforms exhibited an increase in signal intensity during the chase period and reached a peak value at 6 h (Fig. 11, A and E). Similarly, anti-alpha` immunoprecipitations revealed an increase in beta subunit signal with the combination of alpha`(2)beta(2) and alphaalpha`(2)beta(2) isoforms (Fig. 11, B and F). In contrast, the beta subunit signal associated with the alpha`(2)beta(2) isoform immunoprecipitated from the nuclear fraction exhibited no increase during the time course (Fig. 11, C and F). Evaluation of mixed isoform immunoprecipitates allowed determination of a beta subunit signal change for the major abundance isoforms alpha(2)beta(2) and alphaalpha`beta(2), whereas separate immunoprecipitation of the minor abundance isoform alpha`(2)beta(2) was required to assay for beta subunit signal. Evaluation of alpha(2)beta(2) isoform immunoprecipitates also revealed a lag in the appearance of beta subunit signal (data not shown). In Fig. 11G, comparison of beta/alpha and beta/alpha` signal ratio plots for the mixed isoform immunoprecipitations and for the alpha`(2)beta(2) immunoprecipitation reveals a marked difference in the rate of complex formation. This suggests that each major abundance isoform at early time points was not yet fully assembled, whereas complex formation for the minor abundance isoform was essentially complete at 0 h.


Figure 11: Analysis of nuclear isoform complex formation. Complex formation of nuclear isoforms was determined by pulse-chase analysis of nuclear fractions. Cells were labeled for 2 h, washed, chased with complete medium up to 14 h, and dishes were harvested every 2 h. Subcellular fractionation was coupled to two-step immunoprecipitation as described under ``Materials and Methods,'' and isoform immunoprecipitates from nuclear lysates were evaluated by autoradiography and imaging analysis by SDS-PAGE. Panel A, autoradiogram of anti-alpha immunoprecipitates that contain alpha(2)beta(2) and alphaalpha`beta(2) isoforms. Signals for subunits are plotted in panel E. Complex-associated beta subunit is shown by closed circles, and alpha and alpha` subunits are indicated by open circles and crosses, respectively. Panel C, autoradiogram of alpha`(2)beta(2) isoform immunoprecipitated from supernatants of anti-alpha immunoprecipitates (Panel A). Subunit signals are plotted in panel F. The beta subunit signal is shown by closed circles and the alpha` subunit by crosses. Panel D, autoradiogram of free beta subunit immunoprecipitated from supernatants of alpha`(2)beta(2) immunoprecipitates, and E (closed squares), beta signal plot. For direct comparison, autoradiograms in panels A and D were processed in parallel. Panel B, autoradiogram of anti-alpha` immunoprecipitations that contain alphaalpha`beta(2) and alpha`(2)beta(2) isoforms. Signal plots are in F. Open circles indicate complex associated beta subunit, and alpha and alpha` subunits are shown by closed symbols. Panel G, signals from panels E and F are replotted as a ratio of beta/catalytic subunit and normalized for each of five ratios: beta/alpha (crosses) or alpha` (closed circles) for anti-alpha mixed isoform immunoprecipitates; beta/alpha (open squares) or alpha` (closed triangles) for anti-alpha` mixed isoform immunoprecipitates; and beta/alpha` for alpha`(2)beta(2) immunoprecipitates (open circles).



Since this result was similar to that obtained with immunoprecipitates from whole cell lysates, we tested if free beta subunit could be immunoprecipitated from the nuclear fraction. Supernatants from alpha`(2)beta(2) isoform immunoprecipitates that had been cleared three times with alpha antibody and once with alpha` antibody were used for immunoprecipitation with beta subunit antibody. The experiment shown in Fig. 11D indicates the presence of free beta subunit in the nuclear fraction. The loss of signal during the time course mirrored that seen with continuously labeled whole cell lysate (Fig. 7B), suggesting incorporation of beta into complexes and also suggesting that the major abundance nuclear isoforms are assembled in the nucleus. Our choice of a pulse-chase as opposed to a continuous label protocol allowed us to follow incorporation of only previously synthesized free beta subunit into complex. The presence of free nuclear beta subunit was unexpected. In contrast to the catalytic subunits, beta subunit lacked a putative nuclear localization motif (Jakobi et al., 1989) and therefore would not be expected to translocate as free polypeptide.

Cytosolic fractions obtained from the pulse-chase experiment were analyzed in a manner identical to that for the nuclear fraction. Immunoprecipitation of alpha(2)beta(2) and alphaalpha`beta(2) isoforms with alpha antibody is shown in Fig. 12A, alpha(2)beta(2) isoform immunoprecipitates are shown in Fig. 12B. Quantitative analysis of change in beta subunit signal for the major abundance isoforms did not reveal the same magnitude or duration of increase as exhibited for the nuclear fraction (Fig. 12, E and F). However, beta/alpha and beta/alpha` signal ratio plots shown in Fig. 12G indicate some increase in beta subunit association. In contrast, evaluation of alpha`(2)beta(2) immunoprecipitates does not reveal an increase in beta subunit signal (Fig. 12, C and E) or in complex formation (Fig. 12G). Immunoprecipitation of the beta subunit from supernatants of alpha`(2)beta(2) immunoprecipitates shows the presence of free subunit in this fraction, which also exhibited a signal decrease similar to that of free nuclear beta subunit (Fig. 12D). Consistent with the higher abundance of nuclear holoenzyme isoforms, twice as much free beta subunit was immunoprecipitated per mg of protein in the extract from the nuclear fraction than from the cytosolic fraction at 0 h (Fig. 11E and 12E). The observation that free subunit can be immunoprecipitated from subcellular fractions does not depend on the labeling procedure. We have immunoprecipitated free catalytic and regulatory subunit from nuclear and cytosolic fractions using a continuous label protocol (data not shown). Our ability to immunoprecipitate free subunit is not limited to the two fractions reported here. We have observed complex formation for the major isoforms from free regulatory and catalytic subunit in extracts obtained from the 100,000 times g pellet which resulted after centrifugation of the soluble cytosolic fraction. (^2)This fraction may contain CKII from organelles and/or the particulate membrane fraction.


Figure 12: Analysis of cytosolic isoform complex formation. Complex formation of isoforms in the cytosolic fraction was analyzed by pulse-chase analysis. Cytosolic extracts from the subcellular fractionation experiment that yielded the nuclear fractions in Fig. 11were used in two-step immunoprecipitation reactions. Immunoprecipitates were evaluated by autoradiography and imaging analysis of SDS-PAGE gels. Panel A, autoradiogram of anti-alpha immunoprecipitations that contain alpha(2)beta(2) and alphaalpha`beta(2) isoforms. Signals for subunits are plotted in panel E. Closed circles indicate complex-associated beta subunit; alpha and alpha` are shown by open circles and crosses, respectively. Panel C, autoradiogram of alpha`(2)beta(2) isoform immunoprecipitated from supernatants of anti-alpha immunoprecipitates (panel A). Subunit signals are plotted in panel F. The beta subunit signal is shown by closed circles and the alpha` subunit by crosses. Panel D, autoradiogram of free beta subunit immunoprecipitated from supernatants of alpha`(2)beta(2) immunoprecipitates. Panel E, closed squares indicate the beta signal plot. For direct comparison, autoradiograms from panels A and D were processed in parallel. Panel B, autoradiogram of alpha(2)beta(2) isoform immunoprecipitated from supernatants of anti-alpha` immunoprecipitates (not shown). Subunit signals are plotted in panel F. Open circles indicate complex-associated beta subunit, and the alpha subunit signal is indicated by closed squares. Panel G, signals from panels E and F are replotted as a ratio of beta/catalytic subunit and normalized for each of four ratios: beta/alpha (closed circles) or alpha` (open circles) for anti-alpha mixed isoform immunoprecipitates; beta/alpha for alpha(2)beta(2) immunoprecipitates (closed squares); and beta/alpha` for alpha`(2)beta(2) immunoprecipitates (open triangles).




DISCUSSION

In this report we describe an immunodepletion assay that allowed us to determine the forms of CKII holoenzyme present in HeLa cells. Furthermore, this assay facilitated biochemical analysis of isoforms, and our in vivo metabolic labeling investigations revealed differences in the rate of complex formation between regulatory and catalytic subunits for different isoforms.

Based on the presence of the alpha and alpha` catalytic subunits in HeLa cells (Pyerin et al., 1987; Yu et al., 1991), there are three possible tetrameric forms of the enzyme of subunit stoichiometry: alpha(2)beta(2), alphaalpha`beta(2), and alpha`(2)beta(2). In support of this we have demonstrated the existence of three holoenzyme isoforms in HeLa cells, and because our assay shows only coimmunoprecipitation of subunits, tetrameric stoichiometry for isoforms is inferred. Purified CKII from HeLa cells has been characterized previously by Pyerin et al.(1987). Plasma membrane protein extract contained CKII alpha, alpha`, and beta subunits by SDS-PAGE and yielded a molecular mass estimate of 120 kDa by sedimentation velocity analysis, which is consistent with the existence of tetrameric complexes. Pyerin et al.(1987) proposed that their preparation contained two isoforms of subunit stoichiometry, alphaalpha`beta(2) and alpha`(2)beta(2). Comparison of these data and the results presented in this paper is problematic because different types of extracts were used. The results presented in this report ( Fig. 2and Fig. 4and Table 1) show that the alpha`(2)beta(2) isoform is the least abundant of the three holoenzyme isoforms.

Analysis of specific activity and subunit composition of holoenzyme forms of CKII to date has been limited to the alpha(2)beta(2) isoform, which by reconstitution of recombinant alpha and beta subunit polypeptides produced in Escherichia coli, has been demonstrated to have a tetrameric subunit composition; kinetic parameters for this isoform have been determined (Hu and Rubin, 1990; Grankowski et al., 1991). Reconstitution of recombinant alpha` and beta subunits resulted in enhancement of catalytic subunit activity^2 (Bodenbach et al., 1994). However, neither K(m) or V(max) values nor subunit stoichiometry has been reported for this isoform. Determination of subunit stoichiometry and specific activity may prove to be difficult for the alphaalpha`beta(2) isoform because in vitro reconstitution from purified alpha, alpha`, and beta polypeptides is predicted to generate additional isoforms. Filhol et al. (1994) separated bovine adrenocortical alpha(2)beta(2) and alphaalpha`beta(2) isoforms by phosphocellulose chromatography, but it was not known if the alphaalpha`beta(2) preparation contained the alpha`(2)beta(2) isoform.

Our CKII subcellular localization analysis revealed that the three holoenzyme isoforms are present in both the cytosol and nucleus. However, activity analysis showed approximately 3-fold greater activity in the nuclear fraction. Evaluation of [S]methionine-labeled immunoprecipitates indicates that this is due to greater polypeptide abundance (Fig. 4). These data confirm the result of Krek et al.(1992), obtained by immunolocalization of transfected CKII alpha and beta subunits in HeLa cells. They also agree with the predominant nuclear localization described for other tissue types (for review, see Pinna, 1990) but are inconsistent with our earlier localization study that concluded that CKII is mainly cytosolic (Yu et al., 1991). The data obtained previously were based on reactivity of antibodies made to the amino terminus of the alpha subunit and to an internal epitope of the beta subunit, both of which by immunofluorescence, exhibited nearly exclusive cytoplasmic staining. The behavior of these antibodies in immunoprecipitation reactions suggests that under native conditions, epitopes may not be accessible: (i) antibody to beta subunit will not immunoprecipitate free or complex associated beta without heat denaturation of extract; and (ii) antibody to the amino terminus of alpha does not quantitatively immunoprecipitate. (^3)Although it is not possible to relate immunoprecipitation and immunofluorescence assays directly, incomplete epitope recognition by these antibodies may help explain the lack of nuclear staining obtained by immunofluorescence. In contrast, evaluation of the carboxyl-terminal anti-alpha and anti-alpha` antibodies by immunofluorescence revealed strong nuclear and weak cytoplasmic staining (data not shown). This is consistent with immunoprecipitation of a greater amount of polypeptide from nuclear extracts by both antibodies.

Our analysis of CKII complex formation in subcellular fractions indicated that alpha(2)beta(2) and alphaalpha`beta(2) are assembled in the nucleus from free catalytic and regulatory subunit polypeptides. In contrast to the catalytic subunits, which contain putative nuclear localization motifs, the beta subunit lacks a similar signal sequence, and therefore would not be expected to be present in the nuclear fraction in uncomplexed form. It is possible that beta subunit translocation is chaperone-mediated, but we cannot currently evaluate this possibility because our anti-beta subunit immunoprecipitation protocol is predicted to abolish all associations between proteins. The location of complex formation for the corresponding cytosolic alpha(2)beta(2) and alphaalpha`beta(2) isoforms is less certain. These could be assembled in the cytosol but at a faster rate than their nuclear counterparts, or alternatively, complex formation could occur in the nucleus followed by export of holoenzyme to the cytosol. Our ability to immunoprecipitate free regulatory and catalytic subunit from the cytosolic fraction is consistent with either of these two mechanisms of assembly. Our hypothesis for assembly of the alpha(2)beta(2) and alphaalpha`beta(2) isoforms is that complex formation from free polypeptides occurs in situ where these forms of CKII are found in a cell, and this includes both nuclei and the cytosol. We propose that the most likely model for the apparent rapid assembly of the minor abundance alpha`(2)beta(2) isoform is that in contrast to the major abundance isoforms, the alpha`(2)beta(2) form of CKII is assembled at a single site (probably the cytosol) and exported as a holoenzyme to other cellular locations. Our data are also consistent with a more rapid rate of complex formation occurring in situ where this isoform is found. Our analysis of holoenzyme complex formation from free subunit polypeptides raises the question of whether tetramers are assembled from monomers or whether dimer formation of free catalytic and regulatory subunits is a prerequisite for assembly of tetrameric complexes.

Biosynthesis of CKII has been evaluated in lymphoid cell lines (Lüscher and Litchfield, 1994), and the presence of excess beta subunit over that incorporated into complex was demonstrated in a pulse-chase experiment by comparison of beta subunit signals obtained with anti-beta and anti-alpha subunit immunoprecipitations. In HeLa cells, our data suggest that the beta subunit is not present in excess over that required to form complex. Quantitation of CKII beta by continuous labeling analysis indicated that free subunit is not synthesized in excess of what is required for complex formation (Fig. 7C). In addition, when a fixed population of beta subunit molecules was labeled by a pulse-chase labeling procedure, we did not observe in subcellular fractions more free beta subunit than that required for the assembly of complexes from the cytosolic and nuclear fractions combined (Fig. 11E and Fig. 12E). Also, our comparison of the rates of synthesis for the alpha, alpha`, and beta subunits indicated a high degree of similarity (Fig. 8). We do not know if differences between the cell lines used or in experimental protocols may account for differences between our results and those obtained by Lüscher and Litchfield(1994).

Our findings of three different forms of holoenzyme, coupled with different modes of complex formation between isoforms, support the concept that CKII, as an enzyme, exists in multifunctional forms, and different modes of regulation may exist for different isoforms. This research opens the path to evaluate the behavior of the enzyme in the context of cell cycle regulation.


FOOTNOTES

*
This research was supported by United States Public Health Service Grant CA13106 (to D. R. M.), by American Cancer Society Grant CB-72 (to D. R. M.), and National Institutes of Health Grants AG10208 and CA64593 (to D. R. M.). Funding was also provided by the Greenwall Foundation and the Sara Chait Memorial Foundation (to D. R. M.). 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.

§
Present address: Industrial Health Research Institute, Korea Industrial Safety Corporation, 34-4 Kusan-Dong, Buk-Ku, Inchon 403-120, Korea.

To whom correspondence should be addressed: Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, NY 11724. Tel.: 516-367-8426; Fax: 516-367-8873; or Osiris Therapeutics, Inc., 11100 Euclid Ave., Wearn Bldg., 4th floor, Cleveland, OH 44106. Tel.: 216-844-5909; Fax: 216-844-1581.

(^1)
The abbreviations used are: CKII, casein kinase II; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; ETE peptide, RRREEETEEE peptide; DSD peptide, RRRDDDSDDD peptide.

(^2)
N. Chester, unpublished observations.

(^3)
N. Chester and D. R. Marshak, unpublished observations.


ACKNOWLEDGEMENTS

We thank Georgia Binns and Maria Meneilly for peptide synthesis and purification, and James Duffy and Philip Renna for excellent photographic assistance. In addition, we also appreciate the critical review of the manuscript by Dr. Christian van den Bos, Dr. Young-Seuk Bae, and Dr. Grigori Enikolopov. We thank Joan Brooks at New England Biolabs for providing the two subunit alpha beta dicistronic E. coli expression vector and Drs. F. Lozeman and E. Krebs for the human alpha` subunit cDNA clone.


REFERENCES

  1. Ackerman, P., and Osheroff, N. (1989) J. Biol. Chem. 264, 11958-11965 [Abstract/Free Full Text]
  2. Baker, S. J., Kerppola, T. K., Luk, D., Vandenberg, M. T., Marshak, D. R., Curran, T., and Abate, C. (1992) Mol. Cell. Biol. 12, 4694-4705 [Abstract]
  3. Barany, G., and Merrifield, R. B. (1979) in Solid-phase Peptide Synthesis: Special Methods in Peptide Synthesis (Gross, E., and Meienhoffer, J. eds) part A, vol. 2, Academic Press, New York
  4. Berberich, S. J., and Cole, M. D. (1992) Genes & Dev. 6, 166-176
  5. Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J., and Pyerin, W. (1994) Eur. J. Biochem. 220, 263-273 [Abstract]
  6. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  7. Bresnick, E. H., Rories, C., and Hager, G. L. (1992) Nucleic Acids Res. 20, 865-870 [Abstract]
  8. Carroll, D., and Marshak, D. R. (1989) J. Biol. Chem. 264, 7345-7348 [Abstract/Free Full Text]
  9. Cochet, C., and Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406 [Abstract/Free Full Text]
  10. Dang, C. V., and Lee, W. M. F. (1989) J. Biol. Chem. 264, 18019-18023 [Abstract/Free Full Text]
  11. DeBenedette, M., and Snow, E. C. (1991) J. Immunol. 147, 2839-2845 [Abstract/Free Full Text]
  12. Edelman, A. M., Blumenthal, D. K., and Krebs, E. G (1987) Annu. Rev. Biochem. 56, 567-613 [CrossRef][Medline] [Order article via Infotrieve]
  13. Filhol, O., Cochet, C., and Chambaz, E. M. (1990a) Biochemistry 29, 9928-9936 [Medline] [Order article via Infotrieve]
  14. Filhol, O., Cochet, C., and Chambaz, E. M. (1990b) Biochem. Biophys. Res. Commun. 173, 862-871 [Medline] [Order article via Infotrieve]
  15. Filhol, O., Cochet, C., Wedegaertner, P., Gill, G. N., and Chambaz, E. M. (1991) Biochemistry 30, 11133-11140 [Medline] [Order article via Infotrieve]
  16. Filhol O., Baudier, J., Delphin, C., Loue-Mackenbach, P., Chambaz, E. M., and Cochet, C. (1992) J. Biol. Chem. 267, 20577-20583 [Abstract/Free Full Text]
  17. Filhol, O., Cochet, C., Loue-Mackenbach, P., and Chambaz, E. M. (1994) Biochem. Biophys. Res. Commun. 198, 660-665 [CrossRef][Medline] [Order article via Infotrieve]
  18. Glineur, C., Bailly, M., and Ghysdael, J. (1989) Oncogene 4, 1247-1254 [Medline] [Order article via Infotrieve]
  19. Grankowski, N., Boldyreff, B., and Issinger, O.-G. (1991) Eur. J. Biochem. 198, 25-30 [Abstract]
  20. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52 [Medline] [Order article via Infotrieve]
  21. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , pp. 83, 310, and 522, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY,
  22. Hathaway, G. M., and Traugh, J. A. (1982) Curr. Top. Cell Regul. 21, 101-127 [Medline] [Order article via Infotrieve]
  23. Heller-Harrison, R. A., and Czech, M. P. (1991) J. Biol. Chem. 266, 14435-14439 [Abstract/Free Full Text]
  24. Hu, E., and Rubin, C. S. (1990) J. Biol. Chem. 265, 20609-20615 [Abstract/Free Full Text]
  25. Inoue, A., Tei, Y., Qi, S.-L., Higashi, Y., Yukioka, M., and Morisawa, S. (1984) Biochem. Biophys Res. Commun. 123, 398-403 [Medline] [Order article via Infotrieve]
  26. Issinger, O.-G. (1993) Pharmacol. Ther. 59, 1-30 [CrossRef][Medline] [Order article via Infotrieve]
  27. Jakobi, R., and Traugh, J. A. (1992) J. Biol. Chem. 267, 23894-23902 [Abstract/Free Full Text]
  28. Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227-233 [Abstract]
  29. Klarlund, J. K., and Czech, M. P. (1988) J. Biol. Chem. 263, 15872-15875 [Abstract/Free Full Text]
  30. Krek, W., Maridor, G., and Nigg, E. A. (1992) J. Cell Biol. 116, 43-55 [Abstract]
  31. Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4021-4025 [Abstract]
  32. Kuenzel, E. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 737-741 [Abstract]
  33. Kuenzel, E. A., Mulligan, J. A., Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem. 262, 9136-9140 [Abstract/Free Full Text]
  34. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  35. Lee, C. Q., Yun, Y., Hoeffler, J. P., and Habener, J. F. (1990) EMBO J. 9, 4455-4465 [Abstract]
  36. Lin, A., Frost, J., Deng, T., Smeal, T., Al-Alawi, N., Kikkawa, U., Hunter, T., Brenner, D., and Karin, M. (1992) Cell 70, 777-789 [Medline] [Order article via Infotrieve]
  37. Litchfield, D. W., Lüscher, B., Lozeman, F. J., Eisenman, R. N., and Krebs, E. G. (1992) J. Biol. Chem. 267, 13943-13951 [Abstract/Free Full Text]
  38. Lorenz, P., Pepperkok, R., Ansorge, W., and Pyerin, W. (1993) J. Biol. Chem. 268, 2733-2739 [Abstract/Free Full Text]
  39. Lozeman, F. J., Litchfield, D. W., Piening, C., Takio, K., Walsh, K. A., and Krebs, E. G. (1990) Biochemistry 29, 8436-8447 [Medline] [Order article via Infotrieve]
  40. Lüscher, B., and Litchfield, D. W. (1994) Eur. J. Biochem. 220, 521-526 [Abstract]
  41. Lüscher, B., Kuenzel, E. A., and Krebs, E. G. (1989) EMBO J. 8, 1111-1119 [Abstract]
  42. Lüscher, B., Christenson, E., Litchfield, D. W., Krebs, E. G., and Eisenman, R. N. (1990) Nature 344, 517-522 [CrossRef][Medline] [Order article via Infotrieve]
  43. Manak, J. R., de Bisschop, N., Kris, R. M., and Prywes, R. (1990) Genes & Dev. 4, 955-967
  44. Marshak, D. R., and Carroll, D. (1991) Methods Enzymol. 200, 134-156 [Medline] [Order article via Infotrieve]
  45. Meek, D. W., Simon, S., Kikkawa, U., and Eckhart, W. (1990) EMBO J. 9, 3253-3260 [Abstract]
  46. Meggio, F., and Pinna, L. A. (1984) Eur. J. Biochem. 145, 593-599 [Abstract]
  47. Miyata, Y., and Yahara, I. (1992) J. Biol. Chem. 267, 7042-7047 [Abstract/Free Full Text]
  48. Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099 [Medline] [Order article via Infotrieve]
  49. Pepperkok, R., Lorenz, P., Jakobi, R., Ansorge, W., and Pyerin, W. (1991) Exp. Cell Res. 197, 245-253 [Medline] [Order article via Infotrieve]
  50. Pepperkok, R., Lorenz, P., Ansorge, W., and Pyerin, W. (1994) J. Biol. Chem. 269, 6986-6991 [Abstract/Free Full Text]
  51. Pinna, L. A. (1990) Biochim. Biophys. Acta 1054, 267-284 [Medline] [Order article via Infotrieve]
  52. Pyerin, W., Burow, E., Michaely, K., Kübler, D., and Kinzel, V. (1987) Biol. Chem. Hoppe-Seyler 368, 215-227 [Medline] [Order article via Infotrieve]
  53. Russo, G. L., Vandenberg, M. T., Yu, I. J., Bae, Y.-S., Franza, B. R., Jr., and Marshak, D. R. (1992) J. Biol. Chem. 267, 20317-20325 [Abstract/Free Full Text]
  54. Serrano, L., Díaz-Nido, J., Wandosell, F., and Avila, J. (1987) J. Cell Biol. 105, 1731-1739 [Abstract]
  55. Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem. 262, 3839-3843 [Abstract/Free Full Text]
  56. Stallcup, M. R., Ring, J., and Yamamoto, K. R. (1978) Biochemistry 17, 1515-1521 [Medline] [Order article via Infotrieve]
  57. Tuazon, P. T., and Traugh, J. A. (1991) Adv. Second Messenger Phosphoprotein Res. 23, 123-164 [Medline] [Order article via Infotrieve]
  58. Yu, I. J., Spector, D. L., Bae, Y.-S., and Marshak, D. R. (1991) J. Cell Biol. 114, 1217-1232 [Abstract]

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