(Received for publication, June 6, 1995; and in revised form, August 8, 1995)
From the
The and
subunits of casein kinase II are
dramatically phosphorylated in cells that are arrested in mitosis
(Litchfield, D. W., Lüscher, B., Lozeman, F. J.,
Eisenman, R. N., and Krebs, E. G.(1992) J. Biol. Chem. 267,
13943-13951). Comparative phosphopeptide mapping experiments
indicated that the mitotic phosphorylation sites on the
subunit
of casein kinase II can be phosphorylated in vitro by
p34
. In the present study, we have demonstrated
that a glutathione S-transferase fusion protein encoding the
C-terminal 126 amino acids of the
subunit is phosphorylated by
p34
at the same sites as intact casein kinase
II, indicating that the mitotic phosphorylation sites are localized
within the C-terminal domain of
. Four residues within this
domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal
consensus sequence for p34
phosphorylation.
Synthetic peptides corresponding to regions of
that contain each
of these residues are phosphorylated by p34
at
these sites. Furthermore, alterations in the phosphorylation of the
glutathione S-transferase proteins encoding the C-terminal
domain of
are observed when any of the four residues are mutated
to alanine. When all four residues are mutated to alanine, the fusion
protein is no longer phosphorylated by p34
at
any of the sites that are phosphorylated in mitotic cells. These
results indicate that Thr-344, Thr-360, Ser-362, and Ser-370 are the
sites on the
subunit of casein kinase II that are phosphorylated
in mitotic cells.
Biochemical and genetic studies have demonstrated that the
p34 protein kinase is an indispensable
regulator of events leading to the division of eukaryotic cells (for
reviews see (1, 2, 3, 4) ). To
ensure that the division of cells is very precisely regulated, the
activity of this protein serine/threonine kinase is exquisitely
controlled through its interactions with regulatory cyclins and through
phosphorylation of p34
itself. p34
is defined as a cyclin-dependent kinase since it is inactive
unless it is associated with a regulatory cyclin subunit. Furthermore,
p34
is inhibited by phosphorylation of Tyr-15
and/or Thr-14 (5, 6) but requires phosphorylation of
Thr-161 to be activated (7, 8) . CAK (the
p34
activating kinase) is responsible for
phosphorylation of Thr-161(9, 10, 11) , while
the phosphorylation state of Thr-14 and/or Tyr-15 is at least in part
controlled by the relative activities of the Wee1 protein
kinase (12, 13) and cdc25 protein
phosphatase(14, 15) .
Concomitant with the
activation of p34 at the G
-M
transition of eukaryotic cells is a massive burst of protein
phosphorylation. Many of the events that are associated with entry into
mitosis including nuclear envelope breakdown, transcriptional
termination, nucleolar disassembly, cytoskeletal reorganization, and
chromosome condensation appear to be associated with protein
phosphorylation. While it is evident that p34
directly phosphorylates a number of proteins at the
G
-M transition, there are also indications that
p34
could indirectly regulate phosphorylation
events through its phosphorylation of other protein
kinases(16, 17, 18, 19, 20, 21, 22, 23) .
One protein serine/threonine kinase that could be regulated by
p34 is casein kinase II (CKII), (
)which has been shown to be dramatically phosphorylated in
mitotic cells(19, 20, 21) . CKII is a
tetrameric enzyme composed of two catalytic (
and/or
`-subunits) and two additional subunits (
subunits) (for
reviews, see (24, 25, 26) ). Our previous
studies demonstrated that p34
phosphorylates
the
subunit of CKII at Ser-209, a site that is maximally
phosphorylated in mitotic cells(20) . Interestingly, the
subunit (but not the
`-subunit) of CKII is also dramatically
phosphorylated in mitotic avian and mammalian cells(21) . This
result suggests that there may be differences in the functional or
regulatory properties of the isozymic forms of the catalytic subunit of
CKII. Our analyses demonstrated that the mitotic phosphorylation sites
on
can be phosphorylated in vitro by
p34
. To facilitate efforts to examine the
functions of CKII during mitosis and how phosphorylation may affect
these functions, the objective of the present study was directed toward
identification of the sites on the
subunit of CKII that are
phosphorylated by p34
so that
non-phosphorylatable forms of CKII could be prepared by mutagenesis.
Utilizing synthetic peptides and glutathione S-transferase
(GST) fusion proteins containing the C-terminal domain of the human
CKII
subunit, p34
phosphorylation sites
were identified as Thr-344, Thr-360, Ser-362, and Ser-370. Furthermore,
following mutation of each of these residues to alanine residues,
fusion proteins containing the C-terminal domain of CKII
were no
longer phosphorylated by p34
at any of the
sites that are phosphorylated in mitotic cells.
Fusion
proteins were expressed in Escherichia coli JM109 and purified
using glutathione-agarose as described previously(29) . Fusion
proteins were eluted from glutathione-agarose using buffer (20 mM Tris-Cl pH 8.0, 100 mM NaCl, 1 mM EDTA)
containing 5 mM reduced glutathione. Protein determinations
were by the method of Bradford (38) using -globulin as
standard.
To determine the stoichiometry of phosphorylation of fusion
proteins, the following procedure was followed. Known amounts (2, 4, 6
µg) of each fusion protein were subjected to SDS-polyacrylamide gel
electrophoresis and visualized by staining with Coomassie Blue.
Typically, each fusion protein displayed one major band corresponding
to undegraded GST-fusion protein since it reacted on immunoblots with
antibodies directed against the C terminus of CKII
(anti-
) as well as bands representing
degradation products that were not reactive with
anti-
antibodies. Densitometry of the
Coomassie Blue-stained gel was performed to determine the proportion of
intact GST fusion protein. The phosphate incorporation into the
undegraded GST fusion proteins were subsequently determined by analysis
on a Phosphorimager (Molecular Dynamics). Stoichiometry of
phosphorylation was calculated by the following formula: pmol of
phosphate incorporated into intact fusion protein/pmol of intact fusion
protein.
Previous work had shown that sites within the C-terminal
domain of the subunit of CKII were phosphorylated in cells
arrested in mitosis with nocodazole and that immunopurified
p34
was capable of phosphorylating the same sites in
vitro(21) . To provide additional evidence that the
p34
phosphorylation sites are localized to the
C-terminal domain of CKII
, a GST fusion protein encoding the
C-terminal 126 amino acids of CKII
(GST-
C126) was tested as
a substrate for purified p34
. As shown in Fig. 1,
GST-
C126 is effectively phosphorylated by p34
. By
comparison, neither GST nor a GST fusion protein encoding the
C-terminal 51 amino acids of CKII-
` (GST-
`C51) is
phosphorylated. In addition, no phosphorylation of GST-
C126 was
observed when p34
was omitted from the reaction mixture.
Figure 1:
Phosphorylation of GST Fusion Proteins. A, schematic representation of GST and the GST-C126 and
GST-
`C51 proteins. GST-
C126 is composed of GST fused to the
126 C-terminal amino acids of the
subunit of CKII (indicated by solid black rectangle). GST-
`C51 encodes GST and the 51
C-terminal residues of CKII
` (indicated by striped
rectangle). B, equal amounts (4 µg) of purified GST,
GST-
C126, and GST-
`C51 proteins were incubated with purified
p34
(lanes 1, 3, and 6) and without purified p34
(lanes
2, 4, and 5, respectively) for 90 min using the in vitro phosphorylation conditions described under
``Experimental Procedures.'' The proteins were recovered from
the phosphorylation reactions using glutathione-agarose before analysis
on a 12% SDS-polyacrylamide gel and were visualized by
autoradiography.
To determine whether GST-C126 and intact CKII
are
phosphorylated at the same sites, we performed comparative
phosphopeptide mapping. Thermolytic digestion of phosphorylated
GST-
C126 and purified CKII
resulted in the production of
four major phosphopeptides that comigrated when phosphopeptides from
each of the two samples were mixed (Fig. 2). It is therefore
apparent that all of the sites that are phosphorylated on CKII
by
p34
are present in the C-terminal 126 amino acids of the
protein. Furthermore, since comparative phosphopeptide mapping
experiments had previously demonstrated that phosphopeptides a, b, and
c obtained following in vitro phosphorylation of purified CKII
comigrate with phosphopeptides obtained following
P
labeling of mitotic Jurkat cells(21) , these results indicate
that GST-
C126 is phosphorylated at sites that are phosphorylated
in mitotic cells. We previously noted that purified CKII is
phosphorylated at a site, represented by phosphopeptide d (Fig. 2), that was not detected from samples of CKII obtained
from intact cells(21) . It is apparent that this
phosphopeptide, which is also present following phosphorylation of
GST-
C126, does not represent an in vivo phosphorylation
site.
Figure 2:
Comparative phosphopeptide maps of
GST-C126 and of the
subunit of CKII phosphorylated in
vitro by purified p34
. GST-
C126 and
purified bovine CKII were phosphorylated in vitro using
purified p34
and were then immunoprecipitated
from kinase reactions using anti-
antiserum. Immunoprecipitates were analyzed by autoradiography after
separation of proteins on a 12% SDS-polyacrylamide gel. The
phosphorylated fusion protein and the
subunit of CKII were
recovered from homogenized gel slices excised from the
SDS-polyacrylamide gel. The samples were exhaustively digested with
thermolysin and then separated by electrophoresis at pH 1.9 (horizontal dimension with anode to the left), followed by ascending chromatography as described under
``Experimental Procedures.'' The MIX phosphopeptide map was
obtained by mixing aliquots (equal cpm) of each phosphorylated sample
(GST-
C126 and the
subunit of CKII) prior to two-dimensional
separation. The positions of the origins are marked by arrows and the letter O. Individual phosphopeptides are
identified with letters as in (21) .
Inspection of the amino acid sequence of the C-terminal domain
of CKII revealed the presence of four residues, Thr-344, Thr-360,
Ser-362, and Ser-370, that conform to the minimal consensus sequence
for p34
phosphorylation(39) . Synthetic peptides
corresponding to portions of CKII
containing each of the putative
phosphorylation sites were synthesized and phosphorylated in vitro with purified p34
(see Table 1). It is
interesting to note that replacement of Ser-362 with alanine (compare
peptide 3 with peptide 2) abolishes serine phosphorylation, suggesting
that p34
does not likely phosphorylate Ser-356 or
Ser-357 within this peptide. In a similar vein, peptide 1 is
exclusively threonine phosphorylated, demonstrating that p34
does not likely phosphorylate Ser-343, Ser-348, or Ser-349 within
the peptide.
To identify the p34 phosphorylation
sites on CKII
, a mutagenesis strategy was employed. We prepared
fusion proteins in which one or more of the putative p34
phosphorylation sites had been mutated to non-phosphorylatable
alanine residues (see Table 2for summary). Each of these fusion
proteins was tested as an in vitro substrate for p34
and analyzed by SDS-polyacrylamide gel electrophoresis followed
by autoradiography (see Fig. 3). Each of the fusion proteins
displays at least one phosphorylated band with noticeably reduced
electrophoretic mobility, except for the fusion protein in which all
four putative phosphorylation sites had been mutated (Fig. 3K). This fusion protein yields a single
phosphorylated band of unaltered electrophoretic mobility. In addition
to differences in the locations or shift in electrophoretic mobility of
each protein, differences in the extent of phosphorylation were
observed for each fusion protein. Interestingly, fusion proteins
containing both residues Thr-344 and Ser-370 (lanes A, B, C and I; TTSS, TTAS, TASS, and TAAS,
respectively) exhibited the most significant shifts in electrophoretic
mobility and also produced some of the most intense bands. Taken
together, these results suggest that Thr-344 and Ser-370 are important
for the optimal phosphorylation of the C-terminal domain of CKII
by p34
. Phosphorylation of the wild-type GST-
C126
fusion protein achieved an approximate stoichiometry of 3 mol of
phosphate/mol of protein (Fig. 4), while the AAAA mutant was
phosphorylated to a stoichiometry of approximately 0.3 mol of
phosphate/mol of protein.
Figure 3:
Phosphorylation of the GST-C126
fusion proteins by p34
. Wild-type and mutant
GST-
C126 fusion proteins were phosphorylated in vitro for
90 min using purified p34
as described under
``Experimental Procedures.'' Equivalent amounts of each
fusion protein were subsequently subjected to SDS-polyacrylamide gel
electrophoresis on a 12% gel, and phosphoproteins were visualized by
autoradiography. Lanes A-L contain the following GST
fusion proteins, respectively: TTSS (i.e. wild-type
GST-
C126), TTAS, TASS, ATSS, TTSA, ATSA, AASS, TTAA, TAAS, AASA,
AAAA, AAAS. Fusion protein designation is according to Table 2and represents the identity of the amino acid residues
present at positions 344, 360, 362, and 370, respectively (numbering
according to the deduced sequence of human CKII
; see (28) ).
Figure 4:
Time course of phosphorylation of
wild-type GST-C126 (TTSS) and four-site GST-
C126 mutant
(AAAA). Equal amounts (4 µg) of each fusion protein were
phosphorylated in vitro using purified
p34
. Aliquots of each fusion protein assay
mixture were taken at the various time points indicated, and phosphate
incorporation was determined as described under ``Experimental
Procedures.''
Alterations in the intensity of
phosphorylation and in the extent of the electrophoretic mobility shift
of individual GST fusion proteins (GST-C126 and corresponding
mutants) suggested that some of the p34
sites had been
eliminated by mutation. To directly examine the phosphorylation pattern
of the individual mutants, two-dimensional phosphopeptide mapping
procedures were utilized (Fig. 5). In all cases, the most highly
phosphorylated form (i.e. the uppermost phosphorylated band
observed in Fig. 3) was subjected to analysis. A number of
observations are apparent from examination of these phosphopeptide
maps. As evidenced by examination of maps D, F, G, J, K, and L (ATSS,
ATSA, AASS, AASA, AAAA, and AAAS, respectively), mutation of Thr-344 to
alanine results in loss of phosphopeptide a, indicating that this
phosphopeptide contains Thr-344. Phosphoamino acid analysis
demonstrated that phosphopeptide a contains exclusively
phosphothreonine (data not shown) supporting this interpretation.
Phosphopeptide b is absent on all phosphopeptide maps obtained from
fusion proteins that are mutated at both Ser-362 and Ser-370 (panels H and K; TTAA and AAAA, respectively),
suggesting that phosphopeptide b is derived from similar, if not
identical, peptides that are phosphorylated at either Ser-362 or
Ser-370. Phosphoamino acid analysis of phosphopeptide b shows that this
peptide is composed exclusively of phosphoserine (data not shown).
Figure 5:
Thermolytic phosphopeptide mapping of
GST-C126 fusion proteins. Fusion proteins containing different
forms of the C-terminal domain of the
subunit of CKII (as in Fig. 3) were phosphorylated in vitro using purified
p34
followed by immunoprecipitation using
anti-
antibodies as described under
``Experimental Procedures.'' Following separation by
SDS-polyacrylamide gel electrophoresis and visualization by
autoradiography, the most heavily phosphorylated form (i.e. the band with the least electrophoretic mobility as in Fig. 3) of each fusion protein was recovered from the
SDS-polyacrylamide gel, oxidized with performic acid, and exhaustively
digested with thermolysin. Thermolytic peptides were then separated in
two dimensions as described in the legend to Fig. 2.
Phosphopeptide maps were visualized by autoradiography. Capital
letters over each map designate the identity of the amino
acid residues present at positions 344, 360, 362, and 370, respectively
(see Table 2). Individual phosphopeptides are indicated by small letters on each map as in Fig. 3.
The region of CKII that contains the putative
p34
phosphorylation sites does not contain any charged
amino acids(28) . As a result, monophosphorylated peptides
would be nearly neutral at pH 1.9 (36) . Phosphopeptides
containing more than one phosphate would have sufficient negative
charge at pH 1.9 to migrate toward the positive electrode (to the left in Fig. 5). The minimal migration exhibited by
phosphopeptides a and b in the electrophoretic dimension is consistent
with the presence of only a single phosphate on each of these peptides.
Phosphopeptide c, observed only in A (TTSS), exhibits the
electrophoretic mobility of a peptide with a significant negative
charge. Furthermore, this peptide contains both phosphothreonine and
phosphoserine, suggesting that it is indeed a multiply phosphorylated
peptide (data not shown). When Ser-362 (map B, TTAS) or
Thr-360 (map C, TASS) were mutated to alanine residues,
phosphopeptide c was not observed. Instead, phosphopeptide e was
observed on map B (TTAS), and phosphopeptide f was observed on map C (TASS). Mixing experiments indicated that both
phosphopeptides e and f were less negatively charged than
phosphopeptide c (data not shown). These results indicate that
phosphopeptides e and f are phosphorylated at more than one site but
that they are phosphorylated to a lesser extent than phosphopeptide c.
Furthermore, phosphoamino acid analysis indicated that spot f was
phosphorylated exclusively on serine, whereas spot e contained a
mixture of phosphoserine and phosphothreonine. These results suggest
that the negatively charged phosphopeptide c is a triply phosphorylated
peptide that had been phosphorylated at Thr-360, Ser-362, and Ser-370.
Mutation of Thr-360 to alanine (map C, TASS) results in the
disappearance of phosphopeptide c and a concomitant appearance of
phosphopeptide f. Phosphopeptide f, which is exclusively serine
phosphorylated, has a lesser negative charge than phosphopeptide c but
still behaves as a multiply phosphorylated peptide, which is presumably
phosphorylated at Ser-362 and Ser-370. Similarly, mutation of Ser-362
to alanine (map B, TTAS) results in the loss of phosphopeptide
c with the gain of the less negatively charged phosphopeptide e, which
is most likely phosphorylated at both Thr-360 and Ser-370. The presence
of phosphoserine and phosphothreonine in phosphopeptide e supports this
conclusion. If phosphopeptides e and f are indeed diphosphorylated
peptides, it would naturally follow that phosphopeptide c is a
triphosphorylated peptide that is phosphorylated at Thr-360, Ser-362,
and Ser-370. The greater negative charge and diminished chromatographic
migration of phosphopeptide c in comparison to phosphopeptides e or f
is consistent with the presence of an additional phosphate on the
former peptide(36) .
Phosphopeptide d, which is only
observed on maps derived from CKII or GST fusion proteins that are
phosphorylated in vitro, is present on all phosphopeptide
maps, including the fusion protein in which all putative p34 phosphorylation sites, Thr-344, Thr-360, Ser-362, and Ser-370,
have been mutated to non-phosphorylatable alanine residues. In fusion
proteins with alanine instead of Thr-360 (maps C, G, I, J, K, L), it was noticed that
spot d (denoted as d` on maps) only contained phosphoserine
(data not shown). By comparison, when threonine was present at residue
360, phosphoserine and phosphothreonine were detected (data not shown)
at spot d (designated as spot d on the maps). This result
suggests that spot d, which is not observed following the
phosphorylation of CKII in cells(21) , was a mixture of
comigrating or identical mono-phosphorylated peptides arising either
from phosphorylation of Thr-360 or phosphorylation of an unidentified
serine. The result that the four-site mutant (i.e. AAAA) was
phosphorylated by p34
was somewhat unexpected since the
most likely p34
phosphorylation sites had been
eliminated through mutagenesis. It would therefore appear that the
additional phosphorylation site does not conform to the minimal
consensus for p34
phosphorylation since the C-terminal
region of CKII
does not contain any additional serine residues
that are followed by a proline with the exception of Ser-295.
Elimination of this site by expressing a GST fusion protein encoding
residues 300-391 of CKII
did not abolish the ability of
p34
to phosphorylate the fusion protein (data not
shown), suggesting that Ser-295 is not the unknown phosphorylation
site. The phosphorylation of non-consensus residues by p34
has been previously noted(39, 40) . It is
important to emphasize that phosphopeptide d is not observed on
phosphopeptide maps derived from CKII
that had been isolated from
P-labeled human or chicken cells that had been arrested in
mitosis.
Overall, the results obtained by phosphorylation of mutant
fusion proteins and resultant phosphopeptide maps support the
conclusion that the preferred sites of phosphorylation by p34 in cells are Thr-344, Thr-360, Ser-362, and Ser-370 on the CKII
subunit (summarized in Table 3). In fact, mutation of each
of these residues to alanine results in elimination of all
phosphopeptides that are detected following the phosphorylation of CKII
in mitotic cells. Since p34
also phosphorylates
Ser-209 on CKII
in mitotic cells, the present results indicate
that p34
could phosphorylate the CKII holoenzyme to a
stoichiometry of up to 10 mol of phosphate/mol of
tetramer in mitotic cells. The high
stoichiometry of phosphorylation suggests that phosphorylation could
regulate functional properties of CKII and that it could in some way
participate in the burst of phosphorylation that accompanies the
activation of p34
at the G
-M
transition(1, 2, 3, 4, 41, 42, 43) .
At present, the role of phosphorylation in regulating the functions
of CKII in mitotic cells remains speculative. There have been a number
of studies in amphibian (44, 45, 46) or
starfish oocytes (47) and in mammalian cells (48) that
have suggested that the activity of CKII is regulated at different
stages in the cell cycle. However, a direct link between these changes
in the catalytic activity of CKII and the phosphorylation state of CKII
has not been demonstrated. In vitro studies by Mulner-Lorillon et al.(19) demonstrated that CKII isolated from Xenopus laevis can be phosphorylated and activated by
p34. By comparison, when we examined the activity of
CKII that had been isolated in its fully phosphorylated state by
immunoprecipitation from mitotic cells, the catalytic properties of the
enzyme were not significantly different from the unphosphorylated
enzyme(21) . The latter results suggest that phosphorylation of
CKII in mammalian or avian cells may not have direct effects on the
enzymatic properties of CKII. Despite the lack of evidence that
directly links the phosphorylation of CKII to changes in its catalytic
activity during mitosis, there are suggestions that the functions of
CKII could be altered during mitosis. For example, independent studies
using immunofluorescence have demonstrated that CKII is associated with
the mitotic spindle in dividing cells(49, 50) . The
factors that control the interaction of CKII with the mitotic spindle
remain uncharacterized. However, it should be noted that alterations in
the ability of CKII to phosphorylate substrate proteins could be
mediated by affects on its intracellular distribution or on its
interaction with specific substrates without obvious effects on its
enzymatic activity. In this regard, there are indications from the
studies of Cardenas et al.(51) that the
phosphorylation of topoisomerase II is increased in mitosis and that
the major mitotic phosphorylation sites are CKII sites. At the
restrictive temperature in yeast harboring a temperature-sensitive form
of CKII, topoisomerase II is hypophosphorylated, and the cells fail to
divide. In mammalian cells, the phosphorylation of topoisomerase II is
also elevated in mitosis(52) . With the exception of
topoisomerase II, very little is known regarding the stage in the cell
cycle when CKII phosphorylates its substrates. It will certainly be of
interest to examine the phosphorylation of other CKII substrates to
determine if any of these proteins are phosphorylated at the
G
-M transition. It is also noteworthy that the mitotic
phosphorylation sites that have been identified on the
subunit of
human CKII are highly conserved between mammalian species and are even
present in the
subunit of chicken CKII(28, 53) .
Interestingly, none of these sites are present of the
`-subunit of
CKII from either species. The latter observation suggests that the
regulation, and perhaps other functional properties, of the
and
`-isozymic forms of CKII could be distinct. Identification of the
mitotic phosphorylation sites on the
subunit of CKII will
undoubtedly facilitate efforts to define the role of CKII and its
phosphorylation during cell division.