From the
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
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
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, RTCRN
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.
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
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)
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.
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,
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
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
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.
value. Furthermore,
when incubated with ABF1 and anti-ABF1 antibodies, the kinase forms an
immunocomplex active in the phosphorylation of ABF1.
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.
ACG, 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.
(
)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.
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 (F
hsd
20 recA13 ara14 proA2 lacY1 galK2 straA xyl-5 mtl1 supE44) was
used for transformation and plasmid propagation. E. coli strain BL21-DE3 (F
hsd5
(r
- m
-) gal
sup
(
D69 lac P::T
gene
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
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 in Vivo
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.
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.
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 H
O (300 µl), and the two supernatants
were combined for dialysis against H
O. 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.
)(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.
,
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) .
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 vitro
P-labeled ABF1, is identical. Furthermore, the
identity of this site was confirmed by demonstrating that Ser-720 is
the site of phosphorylation.
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) .
(
)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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.