©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABF1 Ser-720 Is a Predominant Phosphorylation Site for Casein Kinase II of Saccharomyces cerevisiae(*)

Todd Upton , Steven Wiltshire , Stephen Francesconi , Shlomo Eisenberg (§)

From the (1)Department of Microbiology, University of Connecticut Medical School, Farmington, Connecticut 06030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

ABF1 is a multifunctional phosphoprotein that binds specifically to yeast origins of replication and to transcriptional regulatory sites of a variety of genes. We isolated a protein kinase from extracts of Saccharomyces cerevisiae on the basis of its ability to specifically phosphorylate the ABF1 protein. Physical and biochemical properties of this kinase identify it as casein kinase II (CKII). The purified kinase has a high affinity for the ABF1 substrate as indicated by a relatively low K value. Furthermore, when incubated with ABF1 and anti-ABF1 antibodies, the kinase forms an immunocomplex active in the phosphorylation of ABF1.

Biochemical and genetic mapping localized a major site for phosphorylation at Ser-720 near the C terminus of the ABF1 protein. This serine is embedded within a domain enriched for acidic amino acid residues. A Ser-720 to Ala mutation abolishes phosphorylation by CKII in vitro. The same mutation also abolishes phosphorylation of this site in vivo, suggesting that CKII phosphorylates Ser-720 in vivo as well.

Although three CKII enzymes, yeast, sea star, and recombinant human, utilize casein as a substrate with similar efficiencies, only the yeast enzyme efficiently phosphorylates the ABF1 protein. This suggests that ABF1 is a specific substrate of the yeast CKII and that this specificity may reside within one of the regulatory subunits of the enzyme. Thus, phosphorylation of ABF1 by yeast CKII may prove to be a useful system for studying targeting mechanisms of CKII to a physiological substrate.


INTRODUCTION

ABF1/OBF1/BAF1 is a multifunctional phosphoprotein with a role in transcription and replication. It was independently isolated in several laboratories as a DNA-binding protein that interacts specifically with a DNA element present in ARSs from Saccharomyces cerevisiae (1-6). A number of studies suggested that ABF1 has a role in DNA replication. Purified ABF1 bound specifically to a wide spectrum of ARSs, albeit with different affinities, including repetitive telomeric ARSs, and single copy origins such as ARS1, HMRE, and ARS121(4) . In addition, the ABF1 DNA binding site present in the ARS121 origin was shown to function as a DNA replication enhancer(7) . Studies with the ARS1 origin of replication made similar observations(8) . Furthermore, recent analysis from our laboratory indicated that ABF1 is necessary for the in vitro assembly of a multiprotein complex that interacts specifically with the ARS121 origin of replication(9) . Finally, other studies demonstrated in vivo that DNA replication is impaired in yeast bearing an abf1 temperature-sensitive mutation (10) and that the ABF1 protein is bound to the ARS1 and 2µ origins of replication(11) .

Studies from other laboratories implicated ABF1 in transcription. A DNA binding motif, RTCRNACG, which is recognized by ABF1(12) , was identified as a transcriptional regulatory site of a large number of genes some of which are important for cell growth. These genes include the silencer region of HMRE(12) ; the genes adjacent to a ty2 element(13) ; genes encoding ribosomal proteins (14, 15); the genes involved in mitochondrial biogenesis(16) ; the YPT1 (Ras-like GTP-binding protein) and TUB2 (-tubulin) genes(17) ; and the DED1(6) , CAR1 (18), and COX6(19) genes.

The mechanism controlling these multiple roles of ABF1 is not known. However, the observation that ABF1 is multiply phosphorylated(19, 20) , primarily at serine residues(20) , raised the possibility that phosphorylation may be involved in regulating its various functions. This prompted us to search for the kinases that may be involved in this phosphorylation. Here we report the isolation and characterization of one such kinase, which we identified as the yeast CKII.()This kinase phosphorylates in vitro a unique site in the ABF1 protein that is also phosphorylated in vivo. The characteristics of the CKII-ABF1 interaction suggest that ABF1 is a physiological substrate for the yeast CKII and that this phosphorylation may be important for ABF1 function.


EXPERIMENTAL PROCEDURES

Yeast Strains and Escherichia coli Strains

S. cerevisiae strain BJ926 (/a prb1-1122/prb1-1122 prc1-407/prc1-407 pep4-3/pep4-3 can1/can1 gal2/gal2 his1/+ +/trp) was obtained from Dr. S. Berger of MIT; TD5 (a his 4-712 ura 3-52) was from Dr. Phil Farabaugh of the University of Maryland. E. coli HB101 (Fhsd 20 recA13 ara14 proA2 lacY1 galK2 straA xyl-5 mtl1 supE44) was used for transformation and plasmid propagation. E. coli strain BL21-DE3 (Fhsd5 (r- m-) gal sup( D69 lac P::Tgene 1), obtained from Dr. A. Das of this department, was used to overexpress and purify wild type and mutant ABF1.

Plasmids

Plasmid pETABF1, obtained from Heather G. Estes of this laboratory, contained the ABF1 protein with its initiation codon cloned into the NdeI site of the pET11a expression vector (obtained from Dr. A. Das of this department), downstream to the T7 promoter. Plasmid pETABF1 s/a-720 contained the ABF1 gene in which Ser-720 was mutated to alanine by a single base substitution, using polymerase chain reaction. The mutagenesis was confirmed by DNA sequencing. Plasmid pMHOBF1 was described previously(20) .

Growth of Yeast and E. coli Cells

Yeast cells were grown in a fermentor (New Brunswick Scientific Co. Inc. 30L Micros BD. Auto) in a YPD (1% yeast extracts, 2% Bacto-peptone, and 2% D-glucose) medium at 30 °C to an optical density of 5.0 at 600 nm. E. coli BL21-DE3 harboring either pETABF1 or pETABF1 s/a-720 were grown in a Luria broth medium containing ampicillin at 30 °C to an optical density of 0.5 at 600 nm. The cells were then supplemented with 1 mM isopropyl-1-thio--D-galactopyranoside and allowed to grow for 4 h at 30 °C. Both yeast and E. coli cells were harvested by centrifugation and resuspended by adding an equal volume of a buffer containing 50 mM Tris-hydrochloride, pH 7.5, 10% sucrose. Cells were frozen in liquid nitrogen and stored at -100 °C.

Buffers

Buffer A contained 100 mM Tris acetate, pH 7.5, 50 mM potassium acetate, pH 7.5, 10 mM magnesium sulfate, 2 mM EDTA, 20% glycerol. Buffer B contained 20 mM HEPES, pH 7.5, 200 mM potassium glutamate, 1 mM EDTA, 5 mM EGTA, 10% glycerol. Buffer C contained 50 mM Tris-hydrochloride, pH 7.5, 250 mM NaCl, 1 mM EDTA. Buffer D contained 50 mM imidazole-hydrochloride, pH 6.9, 1 mM EDTA, 20% glycerol. Prior to use all buffers were supplemented with 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM NaF, 1.43 µg/ml pepstatin A, 3.33 µg/ml leupeptin, and 2 µg/ml aprotinin, final concentration.

Purification of Casein Kinase II

All procedures were carried out at 0-4 °C. Frozen BJ926 cells (150 g wet weight) were thawed, resuspended in buffer A (0.5 g/ml), and lysed as described(4) . After removal of cell debris by centrifugation, the supernatant was supplemented with NaCl to 1.25 M final concentration. Following a stirring on ice for about 30 min, the lysate was subjected to centrifugation in a 45 Ti rotor at 40K rpm for 1 h. Solid ammonium sulfate (0.4 g/ml) was added to the supernatant, and the suspension was stirred overnight. The protein precipitate was pelleted by centrifugation in a 45 Ti rotor at 40,000 rpm for 40 min and then resuspended in buffer B containing ammonium sulfate (60%). The protein pellet was again collected by centrifugation, and the above procedure was successively repeated using buffer B containing 55, 50, and 45% ammonium sulfate. The final pellet was resuspended in buffer C, dialyzed to a conductivity equivalent to buffer C, and applied to a DEAE-cellulose column (7.2 by 5 cm). Ammonium sulfate (0.4 g/ml) was added to the flow-through fraction, and a protein pellet was obtained as described above. The ammonium sulfate precipitate was resuspended in buffer D plus 50 mM KCl, dialyzed to a conductivity equivalent to buffer D plus 50 mM KCl, and then applied to a Bio-Rex 70 column (Bio-Rad, 7.2 by 5 cm). The column was washed with buffer D plus 50 mM KCl (5 bed volumes). Proteins that bound to the resin were eluted with buffer D plus 500 mM KCl (10 bed volumes). Active fractions were pooled and dialyzed to a conductivity equivalent to buffer D plus 50 mM KCl. A precipitate formed during dialysis was removed by centrifugation and the supernatant was applied to a Mono S column (HR 10/10) for fast protein liquid chromatography. The column was washed with buffer D plus 50 mM KCl (5 bed volumes) followed by a linear gradient of 50-250 mM KCl. Active fractions were pooled, dialyzed in buffer D plus 50 mM KCl, and loaded onto a Mono Q column (HR 10/10). The column was successively washed with buffer D plus 50 and 250 mM KCL (5 bed volumes each). Bound proteins were eluted by a linear salt gradient of 250-500 mM KCl. Active fractions were pooled and supplemented with potassium phosphate to 5 mM, final concentration. This Mono Q fraction was then applied to a hydroxylapatite column (0.9 by 0.5 cm) equilibrated with buffer D containing 50 mM KCl, 5 mM potassium phosphate, and 10% glycerol. The column was washed successively with buffer D plus 50 mM KCl containing 5 mM, 100 mM, and 150 mM potassium phosphate (5 bed volumes each). Kinase activity was eluted in the 150 mM potassium phosphate eluate. Final purification was achieved by sedimentation through a glycerol gradient as described in the legend to Fig. 1.


Figure 1: Purification of a kinase that phosphorylates the ABF1 protein. The ABF1 kinase was purified as described under ``Experimental Procedures.'' Aliquots of the following pools of active fractions (the lysate (20 µg), ammonium sulfate precipitate (20 µg), Bio-Rex 70 (20 µg), Mono S (20 µg), Mono Q (20 µg), and hydroxylapatite (10 µg)) were subjected to electrophoresis on a polyacrylamide-SDS gel and the proteins were stained by Coomassie Blue (A). An aliquot (40 µg) of the hydroxylapatite fraction was further purified by sedimentation on a 20-40% glycerol gradient, performed as described before (4). Sedimentation was in a SW 50.1 rotor at 50 krpm, 0 °C for 36 h. At the end of the centrifugation period, 0.2-ml fractions were collected from the bottom of the gradient. Aliquots of the indicated fractions were assayed for kinase activity as described under ``Experimental Procedures.'' Radioactively labeled ABF1 was identified by electrophoresis on a polyacrylamide-SDS gel and visualized by autoradiography (B). The remainder of each fraction was then subjected to electrophoresis on a polyacrylamide-SDS gel and the proteins were stained by Coomassie Blue (C). Numbers on the left designate the position of migration of the following protein standards: myosin (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and trypsin inhibitor (21 kDa). Numbers on the right are the estimated molecular weights of the corresponding protein bands. The arrows at the top designate the position of sedimentation in a parallel gradient of the following protein standards: catalase (cat.) (240 kDa); aldolase (ald.) (158 kDa), and bovine serum albumin (66 kDa).



Purification of ABF1 Expressed in E. coli

Unless indicated otherwise, all procedures were carried out at 0-4 °C. Frozen cells were thawed and lysed to obtain a Fraction II (ammonium sulfate precipitate), essentially as described previously(21) . The ammonium sulfate pellet was resuspended in buffer C. This fraction was passed through a DEAE-cellulose column. Proteins in the flow through fraction of the DEAE-cellulose column were precipitated by ammonium sulfate, resuspended in buffer D plus 25 mM NaCl, and dialyzed to a conductivity of the same buffer. The dialyzed fraction was loaded onto a DEAE-cellulose column equilibrated with buffer D plus 25 mM NaCl. The column was washed successively with buffer D plus 25 mM, 100 mM, and 1 M NaCl (5 bed volumes each). Fractions containing ABF1 (1 M NaCl eluate) were dialyzed to a conductivity of buffer D plus 50 mM NaCl and loaded onto a Mono Q column for fast protein liquid chromatography. The column was washed successively with buffer D plus 50 mM and 125 mM NaCl (5 bed volumes each). ABF1 was eluted with a linear salt gradient of 125-300 mM NaCl. Fractions containing ABF1 were further purified by DNA affinity chromatography and assayed by DNA band shift in agarose gels, as described before(4) .

Kinase Assays

ABF1 protein that was expressed and purified from E. coli was used in all assays. Immunocomplexes active in the kinase reaction were obtained as follows: extracts (5-300 µg) were preincubated with purified ABF1 (0.6 µg) for 5 min on ice. Purified anti ABF1 antibodies (15 µg) were then added and the incubation continued for 1 h at 0 °C. This was followed by the addition of prewashed protein A-agarose beads (Genzyme Corp. Cambridge, MA.) and an additional incubation for 1 h with constant mixing. The agarose beads (30 µl) were harvested by centrifugation and washed two successive times with a buffer containing 50 mM Tris acetate, pH 7.5, 20 mM magnesium acetate, and 1 mM dithiothreitol. Then, a reaction mixture (25 µl) containing 50 mM Tris acetate, pH 7.5, 20 mM magnesium acetate, 1 mM dithiothreitol, 10 µM ATP, 100 µg/ml bovine serum albumin, and 1 µCi of [P]ATP (6000 Ci/mmol Amersham Corp.) was added. The reaction was allowed to proceed 10 min at room temperature and then was stopped by adding a solution (400 µl) containing 1 PBS and 1% Triton X-100. The beads were washed two to four successive times with the same solution and then boiled in a Tris-hydrochloride buffer (30 µl) containing sodium dodecyl sulfate (SDS) for electrophoresis in polyacrylamide gels. Polyacrylamide gel electrophoresis was carried out as before(4) . Radioactively labeled protein bands were identified and quantitated by electronic autoradiography using a Packard InstantImager. Kinase reactions in solution were performed similarly, except that purified ABF1 and CKII were added directly to the kinase reaction mixture.

Two-dimensional Phosphopeptide Mapping

Radioactively labeled ABF1 was subjected to electrophoresis on a polyacrylamide-SDS (6%) gel. After gel electrophoresis the protein was electrophoretically blotted to polyvinylidene difluoride membrane (Millipore) using a Hoeffer Transphor electrophoresis unit for 6-7 h at 4 °C in a buffer containing 20 mM Tris, 150 mM glycine, 20% methanol. The electroblotted protein was then cleaved on the polyvinylidene difluoride membrane by CNBr and trypsin essentially as described(22) . The two-dimensional analysis of phosphopeptides on thin layer cellulose plates was described before(23) . Electrophoresis was for 2.5 h and ascending chromatography was in butanol:pyridine:acetic acid:water (75:50:15:60) for 4-5 h.

Labeling of ABF1 with P in Vivo

Yeast strain TD5 harboring the plasmid pMHOBF1 was grown at 30 °C to an optical density of 0.25 at 600 nm in a phosphate-depleted medium (YP-phosphate) containing 2% raffinose. Cells were arrested in the S-phase with 80 mM hydroxyurea for 4 h. Then, the culture was harvested by centrifugation, washed, and resuspended in the same medium containing 80 mM hydroxyurea. The resuspended culture (500 ml) was supplemented with [P]orthophosphate (5 mCi, DuPont NEN) and galactose (2% final concentration) and incubated at 30 °C for 3.5- 4 h. The radioactively labeled ABF1 was purified essentially as described(20) , except that the DEAE-cellulose column was omitted. YP-phosphate was as described (24) except the pH was adjusted to 5.3.

Reagents

Casein kinase II from the sea star P. ochraceus and recombinant human were purchased from Upstate Biotechnology, Inc. and Boehringer Mannheim, respectively. Dephosphorylated casein from bovine milk, trypsin (sequencing grade), and CNBr were purchased from Sigma. Radioactively labeled isotopes [P]ATP and [-P]GTP (6000 Ci/mmol, each) were from Amersham Corp.


RESULTS

Purification of a Kinase That Phosphorylates Specifically the ABF1 Protein

Previous studies suggested that several kinases may be involved in the phosphorylation of the ABF1 protein(23) . In an attempt to identify these kinases, we developed an assay whereby purified ABF1 protein was incubated with yeast extracts and anti ABF1 antibodies in order to isolate immunocomplexes containing ABF1 and an active kinase. Indeed, when these complexes were isolated and incubated with [-P]ATP, the ABF1 protein was radioactively labeled. We used this immunocomplex assay (see ``Experimental Procedures'') to probe for the kinase in the crude lysate, ammonium sulfate precipitate and in the Bio-Rex 70 fraction shown in Fig. 1A. When probing the purer fractions, Mono S, Mono Q, and hydroxylapatite (Fig. 1A), it was possible to assay for the kinase activity in solution.

The final step in the purification was sedimentation on a glycerol gradient. As demonstrated in Fig. 1, B and C, the kinase activity sedimented as a protein complex with a relative molecular mass of about 150 kDa. When fractions of the glycerol gradient were subjected to electrophoresis in polyacrylamide gels and stained by Coomassie Blue, the presence of four polypeptide chains (p42, p39, p37, and p32) cosedimenting with the kinase activity were revealed. The enrichment for these peptides during the course of the purification is evident in Fig. 1A. No other kinase able to phosphorylate the ABF1 protein was observed in the gradient.

The physical properties of the purified kinase shown in Fig. 1resemble the properties of the previously isolated yeast CKII(25) . To confirm that the purified kinase is CKII, we performed diagnostic tests for the CKII enzymes. As described in Fig. 2, A and B, the purified kinase was highly sensitive to heparin, it could use GTP instead of ATP in the phosphorylation reaction and it could autophosphorylate the and ` subunits. These results verified that the purified kinase is the known yeast CKII.


Figure 2: The purified ABF1 kinase is casein kinase II. A, purified kinase (6 ng, glycerol gradient fraction) was incubated in solution with ABF1 (100 ng) in a reaction mixture (25 µl) essentially as described under ``Experimental Procedures,'' except that increasing amounts of heparin were also added. Incorporation of P into the ABF1 protein was analyzed and estimated as described under ``Experimental Procedures.'' In B, lane 1 shows radioactively labeled ABF1 obtained by incubating ABF1 (100 ng) with the purified kinase (15 ng) in the presence of [-P]ATP; lane 2, the same reaction performed with [-P]GTP; lane 3, purified kinase (30 ng) was incubated with [-P]ATP for autophosphorylation (in the absence of ABF1) under similar conditions as in lanes 1 and 2, except that [-P]ATP of 100-fold higher specific activity was used. Prior to electrophoresis, purified kinase (6 µg) was mixed with the sample and loaded on the gel. The radioactively labeled bands ( and `) were visualized by autoradiography; lane 4 is lane 3 stained by Coomassie Blue. Symbols on the right refer to the names of the yeast CKII kinase subunits (25).



ABF1 Forms a Complex with CKII

A preference for the ABF1 protein as a substrate was demonstrated by incubating the kinase with increasing amounts of the ABF1 protein and casein, which is the standard substrate used in a CKII reaction (Fig. 3). To achieve the same amount of P label incorporated into both casein and ABF1, a 30-40-fold higher casein to ABF1 molar ratio was required, suggesting that CKII has a relatively high affinity for ABF1. The high affinity for ABF1 has also been indicated by a relatively low K value of 0.3 µM (determined by a Michaelis-Menten plot, data not shown), which is among the lowest K values for a CKII substrate(26) .


Figure 3: ABF1 is a preferred substrate for the yeast casein kinase II. Purified CKII (15 ng) was incubated in solution with increasing amounts of ABF1 and casein. The reactions were performed and the amount of P incorporated into ABF1, and casein was estimated as described under ``Experimental Procedures.'' A shows incorporation of P into the ABF1 protein, and B shows the incorporation of P into casein.



This high affinity of CKII for the ABF1 protein is consistent with our observation that in crude extracts the kinase can form an immunocomplex when incubated with ABF1 and anti ABF1 antibodies. Formation of an enzymatically active immunocomplex involving purified CKII, ABF1 and anti ABF1 antibodies is shown in Fig. 4. When either CKII or ABF1 were omitted from the immune reaction, an active immunocomplex was not formed. Likewise, when preimmune IGg substituted for anti-ABF1 antibodies, an active immunocomplex was not observed. These results suggested that the yeast CKII associates tightly with the ABF1 substrate.


Figure 4: ABF1 and CKII form a complex. Purified kinase (200 ng) was incubated with ABF1 (600 ng), anti-ABF1 antibodies (15 µg) and protein A-agarose beads. Then, the immunocomplexes bound to protein A-agarose beads were collected and incubated in a kinase reaction mixture containing [-P]ATP as described under ``Experimental Procedures.'' Incorporation of P into the ABF1 protein was analyzed on polyacrylamide gels as before. Lane 1, complete reaction containing ABF1, CKII, and anti-ABF1 antibodies; lane 2, preimmune IGg (15 µg) was used instead of anti ABF1 antibodies; lane 3, ABF1 was omitted; lane 4, ABF1 was initially omitted from the immune reaction and then added to the protein A-agarose beads together with the [-P]ATP; lane 5, CKII kinase reaction using ABF1 as a substrate performed in solution.



ABF1 Is a Specific Substrate of the Yeast CKII Kinase

The high affinity for the ABF1 protein and the formation of an active CKII-ABF1 immunocomplex suggested that ABF1 may be a specific substrate of the yeast CKII enzyme. To test this possibility we have examined the phosphorylation of ABF1 by CKII enzymes from other sources, the human recombinant and the sea star Pisaster ochraceus. As shown in Fig. 5, a-c, all three enzymes (the yeast CKII, human recombinant, and sea star P. ochraceus) phosphorylated casein at about equal efficiency. In contrast, under the same reaction conditions, the ABF1 protein was phosphorylated efficiently only by the yeast enzyme (Fig. 5d). Human recombinant CKII did not appear to phosphorylate the ABF1 protein and the sea star enzyme was at least 20-fold less efficient than the CKII from yeast (Fig. 5, e and f), suggesting that the ABF1 protein is a specific substrate of the yeast enzyme.


Figure 5: ABF1 is not an efficient substrate for the human recombinant and sea star casein kinase II. ABF1 (400 ng) and casein (10 µg) were incubated separately with the yeast CKII (YCKII), human recombinant CKII (hrCKII), and sea star CKII (SsCKII) enzymes. P-Labeled casein and ABF1 were visualized on polyacrylamide gels as in the legend to Fig. 1. a and d show incorporation of P into casein and ABF1 by increasing levels (0.1, 0.2, 0.4, and 1 ng) of yeast CKII; b and e show incorporation of P into casein and ABF1 by increasing levels of human recombinant CKII (0.3, 0.6, 1.2, and 3 ng); c and f show incorporation of P into casein and ABF1 by increasing levels of sea star CKII (0.35, 0.7, 1.4, and 3.5 ng). In all panels the increased amount of enzyme added in the four lanes is from left to right.



CKII Phosphorylates a Site in Vitro That Is Also Phosphorylated in Vivo

The specific phosphorylation of ABF1 by the yeast CKII suggested that ABF1 may be a physiological target for phosphorylation. To examine whether a similar phosphorylation occurs in vivo, the ABF1 protein was first labeled invivo using radioactively labeled inorganic phosphate, as described in Fig. 6. Then, P-labeled ABF1 was purified and digested with CNBr and trypsin for the analysis of phosphopeptides. Previous similar analysis have shown that ABF1 is phosphorylated in vivo at multiple sites(19, 20) . One of these was a predominant site phosphorylated at all stages of the cell cycle. The phosphorylation of another major site seemed to be enriched during the S-phase(23) . To visualize both phosphopeptides, ABF1 was isolated from P-labeled cells that were arrested in the S-phase with hydroxyurea. As shown in Fig. 6a, two major phosphopeptides (phosphopeptides 1 and 2) resulting from the digest of the in vivo labeled ABF1 were visible in the two-dimensional chromatogram. In addition, several fainter spots were seen. In contrast, when ABF1 was labeled with CKII in vitro only one major phosphopeptide was observed (Fig. 6b). This major phosphopeptide comigrated with phosphopeptide 1 of the in vivo ABF1 (Fig. 6c), indicating that CKII phosphorylates in vitro a site that is also phosphorylated in vivo.


Figure 6: Phosphopeptide analysis by two-dimensional thin layer chromatography of the in vivo and in vitro labeled ABF1 protein. ABF1 was labeled in vivo and in vitro with P. The radioactively labeled protein was isolated, digested with CNBr, and trypsin and the resulting phosphopeptides were analyzed by two-dimensional chromatography on thin layer cellulose (TLC) plates. Experimental details of these procedures are described under ``Experimental Procedures.'' a, two-dimensional chromatography of a digest of wild type (wt) ABF1 radioactively labeled in vivo; b, the same as a, except that the ABF1 protein was labeled in vitro with purified yeast CKII; c, two-dimensional chromatography of a mixture containing ABF1 radioactively labeled in vitro and in vivo; d, the same as in a, except that the labeled protein was the ABF1 Ser/Ala-720 mutant; e, the same as b, except that the labeled protein was ABF1 Ser/Ala-720 mutant. The panel in the top left is a schematic presentation of the two-dimensional chromatography. The numbers 1 and 2 designate the position of the two major phosphopeptides. The Ser/Ala-720 mutation eliminates phosphopeptide 1.



Mapping the CKII Phosphorylation Site

To elucidate the function of the phosphorylation of phosphopeptide 1 by CKII (Fig. 6), it is first necessary to map this site. Mapping of phosphopeptide 1 was performed by treating the in vitroP-labeled ABF1 with N-chlorosuccinimide, which cleaves the protein at tryptophan residues. Since ABF1 contains two tryptophan residues (Trp-36 and Trp-535), this treatment should produce three proteolytic fragments of a predicted size (Fig. 7). We found the P label comigrating with the protein fragment corresponding to the C-terminal portion of the ABF1 protein, identified in a Western blot using anti ABF1 antibodies (Fig. 7, lanes 1-3).


Figure 7: N-chlorosuccinimide treatment of P-labeled ABF1. ABF1 was labeled in solution and isolated on a polyacrylamide gel, as described under ``Experimental Procedures.'' A region of the gel corresponding to the P-labeled ABF1 was excised and crushed in a microcentrifuge tube. The crushed gel was immersed in a solution (1 ml) containing 0.1% SDS, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5, and 0.1 mM EDTA. The supernatant was removed following an overnight incubation at room temperature. The gel pieces were rinsed one time with HO (300 µl), and the two supernatants were combined for dialysis against HO. After concentrating the sample to 0.1-0.2 ml by ultrafiltration, an aliquot of the isolated P-labeled ABF1 was treated with N-chlorosuccinimide, essentially as described previously (52). This treatment cleaves the ABF1 protein at two tryptophan residues, producing three polypeptides. One of these encompasses amino acids 1-36 (N terminus). A second fragment contains amino acids 536-732 (C terminus), and the third fragment contains amino acids 37-535. These products were analyzed by electrophoresis in polyacrylamide gels as described above. Lane 1, untreated P-labeled ABF1; lane 2, ABF1 treated with N-chlorosuccinimide. Lanes 1 and 2 were visualized by autoradiography. Lane 3 is lane 2 analyzed by a Western blot, in addition to autoradiography, using anti-ABF1 antibodies. Numbers on the right designate the position of migration of protein markers described in the legend to Fig. 1. The arrow on the left points to the C-terminal polypeptide fragment containing the bulk of the radioactive label. The small fragment from the N terminus (amino acids 1-36) was run out and is not seen on the gel. In other similar experiments no radioactive label associated with this fragment could be detected.



Another mapping experiment involved the isolation of the P-labeled phosphopeptide 1 followed by determination of its N-terminal amino acid sequence. The results of this analysis suggested that the phosphorylation has occurred in a tryptic peptide located at the very C terminus of the ABF1 protein (amino acids 712-730, data not shown). Since this tryptic peptide contains a single serine residue, Ser-720, this residue became the most likely target for the phosphorylation. To verify the involvement of Ser-720, we mutated this serine to an alanine and used the mutated protein as a substrate for CKII. The incorporation of P label into the mutant protein was consistently four to five times lower than the incorporation of radioactive label into the wild type ABF1 (data not shown), suggesting that Ser-720 is indeed the site of phosphorylation. To confirm this possibility, the mutant ABF1, which was labeled in vitro using [-P]ATP and CKII, was digested with CNBr and trypsin for phosphopeptide analysis, as described in Fig. 6e. In contrast to wild type ABF1 (Fig. 6b), phosphopeptide 1 was not present in the digest of the mutant protein (Fig. 6e), indicating that Ser-720 is the major phosphoacceptor when reacted with CKII and ATP. This is consistent with the calculated stoichiometry of ABF1 phosphorylation in the reaction of Fig. 6b, where we estimated that on the average less than one (0.3-0.4) phosphate is incorporated into an ABF1 molecule.

However, in the absence of Ser-720, other minor phosphopeptides were observed as indicated by the presence of several radioactively labeled tryptic peptides (Fig. 6e). ABF1 protein contains 13 sites that correspond to the CKII consensus sequence, S*/T*-(D/E/S(P))(27, 28) . The presence of radioactively labeled phosphopeptides in the mutant protein is presumably the result of incorporation into some of these sites. The amino acid sequence surrounding Ser-720 also conforms to the general features of a CKII consensus sequence(27, 28) . This sequence, DDEELSDENIQPE, is highly acidic, containing a cluster of acidic amino acids on the N-terminal side of the serine residue and acidic residues at positions 1 and 2 C-terminal to Ser-720. Although this sequence lacks an acidic residue at the Ser-720 position, which is the preferred position for the catalytic action of CKII(28) , it is an effective site for phosphorylation.

To examine whether the Ser/Ala-720 mutation also abolishes the phosphorylation of ABF1 in vivo, mutant ABF1 was radioactively labeled in vivo as described in Fig. 6a, and the labeled protein was analyzed as shown in Fig. 6d. Since phosphopeptide 1 of the mutant protein was not labeled (Fig. 6d), we conclude that Ser-720 is the major CKII phosphorylation site in vivo as well.


DISCUSSION

We have reported here the isolation of CKII from S. cerevisiae using an assay based on the phosphorylation of ABF1, a multifunctional protein with a role in DNA replication and transcription. The purified kinase, which is composed of four subunits with the following relative molecular masses, 42 kDa, 39 kDa, 37 kDa, and 32 kDa, was identified as CKII because of its physical and biochemical properties. The subunit structure of the purified enzyme is in close agreement with that previously published for the S. cerevisiae CKII(25) . In addition, the purified kinase is inhibited by heparin and can use GTP instead of ATP as the phosphate donor, which are diagnostic tests for CKII kinase(29, 30) .

CKII is a ubiquitous kinase found in a wide spectrum of eucaryotic organisms, including vertebrates and yeast(31, 32, 33) . The kinase localizes to the nucleus and cytoplasm(34) . A wide range of substrates of the CKII kinase have been identified, including proteins involved in transcription, translation, signal transduction, and cytoskeletal architecture, implying that this kinase is important for cell proliferation and cell growth (for review, see Refs. 31-33). Many of the nuclear CKII substrates are proteins that interact with DNA and are involved in DNA replication and transcription. These include the SV40 T antigen(35) , topoisomerase II(36, 37, 38) , DNA ligase(39) , and several transcriptional activators, c-myc, c-myb, and c-jun(40, 41, 42) .

CKII from most sources is a heterotetramer, , composed of two catalytic subunits and two regulatory subunits. In many organisms two isoforms ( and `) of the catalytic subunit, encoded by distinct genes, are expressed(30, 31, 32) . With the exception of CKII from S. cerevisiae, the subunit of CKII from various sources is encoded by a single gene(43) . In S. cerevisiae recent studies have shown that the regulatory subunits ( and `) are encoded by two distinct genes(44) .

Evidence supporting an important physiological function of CKII in different organisms has been described (for reviews, see Refs. 31-33). In S. cerevisiae this kinase is essential for cell viability, since simultaneous disruption of the two catalytic subunits is lethal(45) , whereas in Schizosaccharomyces pombe the disruption of the gene encoding the regulatory subunit causes a cold-sensitive phenotype and abnormalities in cell shape(46) . However, a deeper understanding of the physiological role of CKII in the cell will require further identification and analysis of CKII physiological targets. In S. cerevisiae not many physiological targets for phosphorylation by CKII are known. One likely target is topoisomerase II, which copurifies with CKII, suggesting the two tightly associate (47). It was also reported that phosphorylation of the C terminus of topoisomerase II is needed for activating the decatenation activity of the enzyme(48) . In addition, a recent report has indicated that the initiation factor 2 (eIF-2) may also be a physiological target for phosphorylation by CKII(49) .

Several lines of evidence suggest that ABF1 is also a physiological substrate of CKII in yeast. First, the enzyme displays a high affinity for the ABF1 protein, as indicated by the low estimated K value of 0.3 µM, which is on the lower end of K values for CKII(26) . Second, the enzyme appears to form a stable complex with the ABF1 protein which can be immunoprecipitated by the addition of anti ABF1 antibodies. The immunoprecipitate formed is active in phosphorylating ABF1. These results strongly suggest that the yeast CKII specifically recognizes and tightly interacts with the ABF1 protein. This notion is further supported by the intriguing result showing that two other kinases, human recombinant and sea star CKII, are ineffective in using ABF1 as a substrate. Third, the site phosphorylated in vitro is also phosphorylated in vivo. This was evident from the fingerprint analysis of tryptic peptides of ABF1, which has shown that the major labeled phosphopeptide of both, the in vivo and in vitroP-labeled ABF1, is identical. Furthermore, the identity of this site was confirmed by demonstrating that Ser-720 is the site of phosphorylation.

The mechanism for the recognition of ABF1 is not yet known. Although there are potentially thirteen CKII phosphorylation sites in the ABF1 protein, Ser-720 is a predominant site for phosphorylation in vitro and in vivo. It is possible that an ABF1-kinase complex is formed in which Ser-720 is positioned for a favorable interaction with one of the catalytic subunits of the kinase. The fact that this phosphorylation site does not conform precisely with the CKII consensus sequence is intriguing and may contribute to the stability of the CKII-ABF1 interaction. In absence of Ser-720, other types of interactions may take place, which could explain the phosphorylation of other sites in the mutant protein. The fact that ABF1 is not an efficient substrate for the other kinases (human recombinant and sea star CKII) suggest that the primary recognition of ABF1 occurs via an interaction with one of the regulatory subunits. Specifically, the ABF1 protein may interact with the ` subunit, since it appears to be unique to yeast and least conserved among the subunits analyzed to date(44) . Previous studies from other laboratories have also described formation of a tight complex between a CKII enzyme and its substrate. Co-immunoprecipitation of CKII with topoisomerase II and the tumor suppressor p53 has been demonstrated and it has been shown that these interactions are mediated by the subunit(50, 51) .

The biochemical analysis of ABF1 phosphorylation presented here strongly suggest that ABF1 is a physiological substrate for CKII in vivo. It is therefore likely that this phosphorylation is also biologically significant. We have recently constructed a yeast strain containing a Ser-720 to alanine mutation in the ABF1 gene. This mutated strain had no obvious growth defects and had no effect on the mitotic stability of plasmids containing the ARS121 origin of replication.()It seems therefore that this phosphorylation has no effect on the replication function of ABF1. However, the ABF1 protein has also been shown to affect the expression of a large variety of genes through either gene activation or repression (see Introduction). The phosphorylation of ABF1 may provide the mechanism by which the transcriptional activity of ABF1 is modulated at promoters of some genes. Consistent with this notion are the reported studies that correlated the extent of ABF1 phosphorylation and the level of transcription of the COX6 gene, when shifting the carbon source from glucose to ethanol(19) . It is possible that the function of Ser-720 phosphorylation by CKII is to modulate the transcriptional activity of ABF1 at promoters of certain genes. Identification of Ser-720 as a major phosphorylation site by CKII should now allow a direct examination of the effect of the Ser-720 to alanine mutation on the expression of specific genes.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM46760-03 (awarded to S. E.). 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.

§
To whom correspondence should be addressed.

The abbreviation used is: CKII, casein kinase II.

T. Upton and S. Eisenberg, unpublished observations.


REFERENCES
  1. Eisenberg, S., Civalier, C., and Tye, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A.85, 743-746 [Abstract]
  2. Diffley, J. F. X., and Stillman, B. (1988) Proc. Natl. Acad. Sci. U. S. A.85, 2120-2124 [Abstract]
  3. Sweder, K. S., Rhode, P. R., and Campbell, J. L. (1988) J. Biol. Chem.263, 17270-17277 [Abstract/Free Full Text]
  4. Francesconi, S. C., and Eisenberg. S. (1989) Mol. Cell. Biol.9, 2906-2913 [Medline] [Order article via Infotrieve]
  5. Halfter, H., Muller, U., Winnacker, E.-L., and Gallwitz, D. (1989) EMBO J.8, 3029-3037 [Abstract]
  6. Buchman, A. R., and Kornberg, R. D. (1990) Mol. Cell. Biol.10, 887-897 [Medline] [Order article via Infotrieve]
  7. Walker, S. S., Francesconi, S. C., and Eisenberg, S. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 4665-4669 [Abstract]
  8. Marahrens, Y., and Stillman, B. (1992) Science255, 817-823 [Medline] [Order article via Infotrieve]
  9. Estes, H. G., Robinson, B. S., and Eisenberg, S. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 11156-11160 [Abstract]
  10. Rhode, P. R, Elsasser, S., and Campbell, J. L. (1992) Mol. Cell. Biol.12, 1064-1077 [Abstract]
  11. Diffley, X. F. J., Cocker, H. J., Dowell, J. S., and Rowley, A. (1994) Cell78, 303-316 [Medline] [Order article via Infotrieve]
  12. Buchman, A. R., Kimmerly, W. J., Rine, J., and Kornberg, R. (1988) Mol. Cell. Biol.8, 210-225 [Medline] [Order article via Infotrieve]
  13. Goel, A., and Pearlman, R. E. (1988) Mol. Cell. Biol.8, 2572-2580 [Medline] [Order article via Infotrieve]
  14. Hamil, K. G., Nam, H. G., and Fried, H. M. (1988) Mol. Cell. Biol.8, 4328-4341 [Medline] [Order article via Infotrieve]
  15. Kraakman, L. S., Griffioen, G., Zerp, S., Gronveld, P., Thevelein, J. M., Mager, H. W., and Planta, J. R. (1993) Mol. & Gen. Genet.239, 196-204
  16. Dorsman, J. C., van Heeswijk, W. C., and Grivell, L. A. (1988) Nucleic Acids Res.16, 7287-7301 [Abstract]
  17. Halfter, H., Kavety, B., Vandekerckhove, J., Kiefer, F., and Gallwitz, D. (1989) EMBO J.8, 4265-4272 [Abstract]
  18. Kovari, L. Z., and Cooper, T. G. (1991) J. Bacteriol.173, 6332-6338 [Medline] [Order article via Infotrieve]
  19. Silve, S., Rhode, P. R., Coll, B., Campbell, J. L, and Poyton, R. O. (1992) Mol. Cell. Biol.12, 4197-4208 [Abstract]
  20. Francesconi, S. C., and Eisenberg, S. (1991) Proc. Natl. Acad. Sci. U. S. A.88, 4089-4093 [Abstract]
  21. Eisenberg, S., and Kornberg, A. (1979) J. Biol. Chem.254, 5328-5332 [Abstract]
  22. Stone, L. K., McNulty, E. D., LoPresti, L. M., Crawford, M. J., De Angelis, R., and Williams, R. K. (1992) Techniques in Protein Chemistry (Angeletti, R., ed) Vol. III, pp. 23-34 Academic Press, San Diego, CA
  23. Francesconi, S. C. (1992) Purification, Molecular Cloning, and Characterization of a DNA Replication Protein, OBF 1. Ph.D. thesis University of Connecticut, Farmington, CT
  24. Warner, J. R. (1973) Methods Enzymol.194, 423-428
  25. Padmanabha, R., and Glover, C. V. C. (1987) J. Biol. Chem.262, 1829-1835 [Abstract/Free Full Text]
  26. Tanasijevic. M. J. Myers, M. G., Jr., Thoma, R. S., Crimmins, D. L., White, M. F., and Sacks, D. B. (1993) J. Biol. Chem.268, 18157-18166 [Abstract/Free Full Text]
  27. Kuenzel, F. A., Mulligan, J. A., Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem.262, 9136-9140 [Abstract/Free Full Text]
  28. Kennelly, P. J., and Krebs, E. G. (1991) J. Biol. Chem.266, 15555-15558 [Free Full Text]
  29. Feige, J. J., Pirollet, F., Cochet, C., and Chambaz, E. M. (1980) FEBS Lett.121, 139-142 [CrossRef][Medline] [Order article via Infotrieve]
  30. Hathaway, G. M., and Traugh, J. A. (1982) Curr. Top. Cell. Reg.21, 101-127 [Medline] [Order article via Infotrieve]
  31. Issinger, O. G. (1993) Pharmacol. & Ther.59, 1-30 [CrossRef]
  32. Tuazon, P. T., and Traugh, J. A. (1991) Adv. Second Messenger Phosphoprotein Res.23, 124-164
  33. Pinna, L. A. (1990) Biochim. Biophys. Acta1054, 267-284 [Medline] [Order article via Infotrieve]
  34. Krek, W., Maridor, G., and Nigg, E. A. (1992) J. Cell Biol.116, 43-55 [Abstract]
  35. Grasser, F. A., Scheidtmann, K. H., Tuazon, P. T., Traugh, J. A., and Walter, G. (1988) Virology165, 13-22 [Medline] [Order article via Infotrieve]
  36. Sander, M., Nolan, J. M., and Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U. S. A.81, 6938-6942 [Abstract]
  37. Saijo, M., Enomoto, T., Hanaoka, F., and Ui, M. (1990) Biochemistry29, 583-590 [Medline] [Order article via Infotrieve]
  38. Cardenas, M. E., and Gasser, S. M. (1993) J. Cell Sci.104, 219-225 [Free Full Text]
  39. Prigent, C., Lasko, D. D., Kodama, K. Woodget, J. R., and Lindahl, T. (1992) EMBO J.11, 2925-2933 [Abstract]
  40. Luscher, B., Kuenzel, E. A., Krebs, E. G., and Eisenman, R. N. (1989) EMBO J.8, 1111-1119 [Abstract]
  41. Luscher, B., Christenson, E., Litchfield, D. W., Krebs, E. G., and Eisenman, R. N. (1990) Nature344, 517-522 [CrossRef][Medline] [Order article via Infotrieve]
  42. Lin, A., Frost, J., Deng, T., Smeal, T., Al-Alawi, N., Kikkawa, U., Hunter, T., Brenner, D., and Karin, M. (1992) Cell70, 777-789 [Medline] [Order article via Infotrieve]
  43. Takio, K., Kuenzel, E., A., Walsh, K. A., and Krebs, E. G. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 4851-4855 [Abstract]
  44. Reed, J. C., Bidwai, A. P., and Glover, C. V. C. (1994) J. Biol. Chem.269, 18192-18200 [Abstract/Free Full Text]
  45. 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]
  46. Roussou, I., and Draetta, G. (1994) Mol. Cell. Biol.14, 576-586 [Abstract]
  47. Cardenas, M. E., Walter, R., Hanna, D., and Gasser, S. M. (1993) J. Cell Sci.104, 533-543 [Abstract/Free Full Text]
  48. Cardenas, M. E., Dang, Q., Glover, C. V. C., and Gasser, S. M. (1992) EMBO J.11, 17885-1769
  49. Feng, L., Yoon, H., and Donahue, F. T. (1994) Mol. Cell. Biol.14 5139-5153 [Abstract]
  50. Bojanowski, K., Filhol, O., Cochet, C., Chambaz, E. M., and Larsen, A. K. (1993) J. Biol. Chem.268, 22920-22926 [Abstract/Free Full Text]
  51. 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]
  52. Schecter, Y., Patchornik, A., and Burtstein, Y. (1976) Biochemistry15, 5071-5075 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.