From the
Oncoprotein 18 (Op18) is a conserved cytosolic protein that is a
target for both cell cycle and cell surface receptor-regulated
phosphorylation events. The four residues Ser
Progression through the cell cycle and cell division is
regulated by cyclin-dependent kinases (CDKs)
Oncoprotein 18 (Op18) is a conserved
cytosolic protein that has been identified in several cellular systems
and studied under different names such as p19, 19K, p18, prosolin,
stathmin, and
Op18
(7, 8, 9, 10, 11) . This
protein has evoked interest due to its up-regulated expression in
various neoplasms, prompting the designation
Op18
(12, 13) , and its complex pattern of
phosphorylation. Many investigators have proposed a regulatory role of
Op18 based on the following: (i) phosphorylation of Op18 in response to
diverse extracellular signals, (ii) developmental control and
differentiation-specific regulation of Op18 expression levels, and
(iii) profound up-regulation of Op18 in various
malignancies
(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
Previous studies have identified all four sites of Op18 that are
phosphorylated in intact cells, namely Ser
To address the function
of Op18 and the potential importance of its cell cycle-regulated
phosphorylation, we have recently expressed a CDK target site-deficient
mutant of Op18 and searched for defects on the level of cell cycle
regulation
(30) . The result demonstrated that Ala substitution
of the two CDK sites of Op18 results in rapid accumulation of cells in
the G2/M phase of the cell cycle. The block in G2 was transient, and
prolonged incubation resulted in a large fraction of the transfected
cells entering S-phase in the absence of mitosis, i.e. endoreduplication. In addition, a fraction of the transfected
cells was blocked in mitosis. These cells appeared to be blocked in
early M-phase, since they lacked the mitotic spindle, and also
exhibited a serious defect during mitotic chromosome segregation.
Analyses of the mechanism behind the phenotype of the mutants
containing Ala substitutions of Ser
In the present study we
investigated the stoichiometry of phosphorylation of the four targets
of Op18 and the cell cycle phenotypes of mutants containing Ala
substitutions of the phosphorylated residues. The data demonstrate that
a cascade of phosphorylation on all four Ser residues results in
multisite-phosphorylated Op18 and that Op18 may play a more central
role in G2/M transition than previously anticipated.
Op18 has been studied by several groups due to its rapid
phosphorylation in response to a stimulation of a variety of receptor
systems
(7, 8, 9, 14) and dramatic
up-regulation in various
neoplasms
(12, 13, 18, 19, 20, 21) .
The potential function of Op18 during receptor signaling is still
unknown, but our present and previous study present genetic evidence
for an essential function of this protein during cell division (30).
Hence, our data have revealed that mutation of the phosphorylated Ser
residues, which are all subject to cell cycle-regulated fluctuations,
results in dominant Op18 mutants that block cell division. As argued in
a previous study, the CDK site-deficient Op18 mutant is likely to
mediate its phenotype by interfering with the function of the
endogenous gene product, i.e. Op18-S25A,S38A is a dominant
negative mutant
(30) . This conclusion was based on the
observation that antisense mRNA-mediated suppression of Op18 expression
results in a G2/M block similar to Op18-S25A,S38A-expressing cells and
that the phenotype of the S25A,S38A mutant was resistant to an
extensive COOH-terminal deletion. Moreover, it was also shown that the
Op18 mutant did not act by sequestering of the endogenous gene product,
which suggests that the mutant acts by sequestering a putative
interacting protein. The minor, but still significant, phenotype
observed by overexpression of wild type Op18 is also in line with a
sequestering mechanism as previously discussed
(30) , since
overexpression of the wild type may result in a fraction of Op18 that
is nonphosphorylated on critical sites.
We have observed cell
cycle-regulated phosphorylation on all four residues of Op18 that are
phosphorylated in intact cells and identified CDKs as one of the kinase
systems involved
(23, 29) . The evidence that Ser
The
present report extends our previous studies of cell cycle-regulated
phosphorylation of Op18
(29, 30) . First, site-mapping
analysis of all phosphoisomers present in mitotic cells demonstrates
that both of the CDK target sites, Ser
A possible concern in
interpreting the results from overexpression of kinase target site
mutants of Op18 is that such mutants may interfere with specific kinase
systems, e.g. by acting as a pseudo-substrate site. In the
case of the S25A,S38A mutant, in vitro phosphorylation assays
have shown that Ala substitutions of Ser
As outlined above, multisite phosphorylation of Op18 during mitosis
involves CDKs and a distinct but as yet unidentified protein kinase
system. Initially it was surprising that mutations of the target sites
for these protein kinases result in an identical phenotype on all
levels tested. However, analysis of Op18 phosphoisomers by native PAGE
revealed that Ser
Deficiency of the two CDK target sites of Op18 may
interfere with phosphorylation of Ser
Most previous studies on
the Op18 protein have been concerned with phosphorylation in response
to extracellular signals. As outlined in the introduction section, both
Ser
We thank Dr. Victoria Shingler for critical reading of
the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,
Ser
, Ser
, and Ser
are all
subject to cell cycle-regulated phosphorylation. Ser
and
Ser
are targets for cyclin dependent kinases (CDKs), while
Ser
and Ser
are phosphorylated by an
unidentified protein kinase. We have recently shown that induced
expression of a CDK target site-deficient mutant, Op18-S25A,S38A,
blocks human cell lines during G2/M transition. In the present report
we show that mitosis is associated with complete phosphorylation of the
two Op18 CDK target sites Ser
and Ser
and
that Ser
and Ser
are also phosphorylated to a
high stoichiometry. To evaluate the function of multisite
phosphorylation of Op18, we expressed and analyzed the cell cycle
phenotype of different kinase target site-deficient mutants. The data
showed that induced expression of the S16A,S63A, S25A,S38A, and
S16A,S25A,S38A,S63A mutants all resulted in an indistinguishable
phenotype, i.e. immediate G2/M block and subsequent
endoreduplication, a given fraction of G2 versus M-phase
blocked cells, and a characteristic nuclear morphology of M-blocked
cells. This result was unexpected; however, a likely explanation was
provided by analysis of Op18 phosphoisomers, which revealed that
mutations of the CDK sites interfere with phosphorylation of Ser
and Ser
. The simplest interpretation of our results
is that phosphorylation of Ser
and Ser
is
essential during G2/M transition and that the phenotype of the
S25A,S38A mutant is mediated by the observed block of
Ser
/Ser
phosphorylation.
(
)
in
all eukaryotes (for review, see Ref. 1). The enzymatic activity of
these kinases requires association with a family of regulatory subunits
called cyclins (for review, see Ref. 2). In higher eukaryotes, the G1
and S-phase functions are controlled by the CDK2, CDK4, and CDK5
kinases, while the prototypic member of the CDK family, p34-cdc2,
controls entry into mitosis (for review, see Refs. 3 and 4). In
addition to CDKs, other protein kinases have also been implicated in
cell cycle control, such as mitogen-activated protein kinases, MPM-2
epitope kinases, calcium/calmodulin kinase II, or NimA (for review, see
Refs. 4 and 5). However, the specific role of these protein kinases
during the cell cycle is not yet as well understood as the role of
CDKs. Cell cycle control by protein kinases clearly depends on
phosphorylation of specific substrates, but only a limited number of
cell cycle-regulated physiological substrates have been definitively
identified (for review, see Ref. 6). Moreover, even fewer protein
substrates have been identified whose phosphorylation is known to be
important during cell division.
,
Ser
, Ser
, and
Ser
(22, 23, 24, 25, 26, 27, 28) .
During our search for the function of Op18, we have identified three
protein kinase systems that specifically phosphorylate these residues
(23, 24, 27, 29). Analysis of T-cell antigen receptor-induced
phosphorylation of Op18 revealed two distinct protein kinase families
that phosphorylate Ser
and Ser
. Our
site-mapping studies performed in vivo and in vitro have shown Ser
is a major target for members of the
mitogen-activated protein kinase family and that Ser
is a
major target for the Ca
/calmodulin-dependent
kinase-Gr(23, 24, 27). We have also observed cell
cycle-regulated fluctuations of Op18 phosphorylation on all four Ser
residues that are phosphorylated in intact cells. Site-mapping studies
performed in vivo and in vitro identified CDKs as the
kinase system involved in cell cycle-regulated phosphorylation of
Ser
and Ser
, but the cell cycle-regulated
kinase system(s) involved in Ser
and Ser
phosphorylation remains to be identified
(23, 29) .
The analysis of Op18 phosphorylation outlined above suggests that Op18
resides at a junction where receptor and cell cycle-regulated kinase
families interact with the same substrate.
and Ser
suggested an essential CDK-regulated role for Op18 during cell
division and that the mutant interfered with the function of the
endogenous gene product
(30) .
Reagents
Rabbit anti-Op18 was raised against
Escherichia coli produced Op18. Anti-Op18 specific for the
COOH-terminal part (amino acid residue 34-149,
anti-Op18:34-149) was affinity purified on recombinant Op18 as
described in detail elsewhere (19). The PC10 monoclonal
anti-proliferating cell nuclear antigen was purchased from Santa Cruz
Biotechnology Inc. Protein A and Protein A bound to Sepharose was
purchased from Pharmacia Biotech Inc.
DNA Transfection, Suppression of the Human
Metallothionein IIa (hMTIIa) Promotor, and Cell Culture
Condition
Construction of mutant Op18 cDNA, where the codons for
Ser (S16A), Ser
(S25A), Ser
(S38A), and Ser
(S63A) are changed to Ala, have
previously been described
(23, 24, 27) . Using
standard DNA manipulation techniques (31), we have combined these
single point mutations to generate constructs expressing di-, tri-, and
tetra- combinations of the amino acid substitutions. Construction of
Op18 cDNA with the sequence encoding amino acids 4-55 deleted,
designated Op18-
4-55, has also been described
(30) .
Op18 cDNA derivatives were excised from pBluescript SK(+)
(Strategene) as BamHI to HindIII fragments and cloned
into the corresponding sites in the poly-linker of the episomal
Epstein-Barr virus (EBV) expression vector pMEP4
(Invitrogen)
(32) . The resulting plasmids allow expression of
Op18 derivatives under control of the Cd
-induced
hMTIIa promotor. The pMEP4 shuttle vector contains the EBV origin of
replication and the EBNA-1 gene to allow high copy episomal
replication, and the hph gene, which confers hygromycin B
resistance in mammalian cells. pCMV-EBNA (Invitrogen) contains the
EBNA-1 gene under the control of the cytomegalovirus promotor,
which directs transient expression of EBNA-1. To maximize the
frequency of hygromycin B-resistant transfectants of the EBV-negative
K562 erythroleukemia cell line, pCMV-EBNA was co-transfected with
pMEP-Op18 derivatives. A detailed description of the transfection
procedure has been presented elsewhere
(30) . In brief, K562
cells were transfected by electroporation in the presence of 25 µg
of pMEP4-Op18 derivatives, 10 µg of pCMV-EBNA, and 25 µg of
pBluescript SK(+) as carrier DNA. Cells were thereafter recultured
in a medium containing EDTA (50 µM), which has been
specifically designed to support cell growth under conditions that
minimize expression from the hMTIIa promotor
(30) . Hygromycin
(0.5 mg/ml; Boehringer Mannheim) was used to select for transfected
cells as described
(30) , and about 50-70% of all
pMEP4-transfected cells surviving electroporation were resistant to the
drug, while essentially all mock-transfected cells were killed within 3
or 4 days. Flow cytometric analysis of Op18 expression of transfected
cells revealed that Cd
(0.1 µM) induced
a 5-20-fold increased ectopic expression of Op18 within 12 h. To
avoid selection for fast growing clones, cells were used between 5 and
10 days post-electroporation. Flow Cytometric Analysis and [
H]Thymidine
Incorporation-Single parameter DNA stainings were performed
with propidium iodide as described
(30) . The BrdU labeling and
detection kit I (Boehringer Mannheim) was used to for the detection of
BrdU incorporation into cellular DNA according to the
manufacturer's instructions. In brief, cells (0.5-1
10
) were cultured for 24 h in the presence of BrdU (1
µM) and thereafter fixed in 70% ethanol buffered with 50
mM glycine buffer, pH 2 (-20 °C). Cells were
thereafter washed and incubated with a monoclonal anti-BrdU, which was
revealed by fluorescein-conjugated sheep anti-mouse immunoglobulin.
Prior to analysis, cells were resuspended in phosphate-buffered saline
containing 10 µg/ml propidium iodide, 0.1% Triton X-100, and 10
µg/ml RNase. DNA and BrdU dual parameter analysis was performed
using a FACScan (Becton Dickinson). Evaluation of flow cytometry data
was performed using the Consort 30 or FACScan software. Cellular
proliferation was analyzed by culturing cells (0.2 ml/culture,
triplicate cultures) in flat bottom microtiter plates in the presence
of [
H]thymidine (1 µCi/well) for 2 h.
Cultures were thereafter precipitated on glass filter and incorporated
[
H]thymidine determined by liquid scintillation.
Western Blot, SDS-Polyacrylamide Gel Electrophoresis
(SDS-PAGE), and Immunoprecipitation
Preparation of cellular
proteins by cell lysis in Triton X-100 and separation of proteins by
10-20% gradient SDS-PAGE has been described
(30) .
Affinity-purified anti-Op18, specific for the COOH-terminal part
(anti-Op18:34-149), was used for Western blot analysis and
immunoprecipitation as described
(30) . I-Protein A
was used to reveal bound antibodies in Western blot analysis and
PhosphorImager analysis of radioactive bands was used for
quantification. As a control for equal loading, the relevant parts of
filters were routinely probed with a monoclonal anti-proliferating cell
nuclear antigen (PC10) or anti-triose-phosphate isomerase.
Separation of Op18 Phosphoisomers by Native PAGE and
Tryptic
To separate
Op18 phosphoisomers, we employed a native PAGE system that separates
Op18 according to the charge differences introduced by phosphorylation
(23). Unlabeled Op18 was purified prior to analysis by a small scale
purification protocol
(23) , and P-Phosphopeptide Analysis
P-labeled Op18 was
purified by immunoprecipitation. Cells were
P-labeled by
incubating HeLa cells for the indicated time in phosphate-free
Dulbecco's minimum Eagle's medium (10
cells/ml,
1 ml) containing
P
(100 µCi/ml).
Thereafter, cells were solubilized in Triton X-100 containing lysis
buffer, and Op18 was immunoprecipitated using anti-Op18:34-149 as
described
(23) . Bound Op18 was eluted by heating the beads to 70
°C (5 min) in 40 mM Tris-glycine, pH 8.8, 10% glycerol,
and 1% 2-mercaptoethanol. Eluted proteins were separated by native PAGE
and electrotransferred to nitrocellulose filters. Radioactive bands
were localized by autoradiography and excised. Immobilized proteins
were digested with tosylphenylalanyl chloromethyl ketone-treated
trypsin (Worthington, 4
7.5 µg of trypsin was added at 2-h
intervals) and processed as described
(33) . Phosphopeptide
mapping was performed as described previously
(23, 34) .
PhosphorImager analysis of radioactive spots was used for
quantification of phosphopeptides.
Site Specificity and Stoichiometry of Op18
Phosphorylation during Mitosis
A previous study has shown that
mitosis is associated with multisite phosphorylation of Op18 on up to
four distinct Ser residues
(29) . Op18 phosphoisomers can be
resolved on a native PAGE system that separates proteins according to
the charge differences introduced by each phosphate
group
(23, 29) . This method reveals that about 90% of
all Op18 molecules are nonphosphorylated in interphase cells and that
the remaining 10% are phosphorylated on one site
(Fig. 1A). In contrast, cells blocked in mitosis by
nocodazole treatment contain Op18 that is phosphorylated on at least
two sites, with most phosphoisomers phosphorylated on three or four
sites. We have previously reported a similar pattern of phosphoisomers
in mitotic cells recovered after ``mitotic shake off,'' which
suggests that multiphosphorylation of Op18 is indeed associated with
mitosis, even without nocodazole treatment
(29) .
Figure 1:
Site-mapping
analysis of Op18 phosphoisomers in mitotic HeLa cells. A,
exponentially growing HeLa cells were either left untreated
(Interphase) or treated with nocodazole for 16 h
(M-Block). Op18 was thereafter purified from either adherent
(Interphase) or non-adherent (M-Block) cells and
phosphoisomers of Op18 resolved by a native PAGE system. After transfer
to nitrocellulose filter, Op18 was revealed as described under
``Materials and Methods.'' The arrows indicate
nonphosphorylated Op18 (non-P) as well as migration of Op18
with various numbers of phosphate groups (1-P, 2-P,
3-P, and 4-P). B-F, interphase or
M-blocked HeLa cells were labeled with
[P]orthophosphate for 16 h, Op18-purified,
resolved by a native PAGE, and transferred to a nitrocellulose filter.
The indicated Op18 phosphoisomers were digested with trypsin, and
phosphopeptides were resolved by two-dimensional separation on thin
layer cellulose plates. The electrophoretic dimension (pH 8.9, cathode
right), the chromatography dimension, and the location of sample
application (+) are shown. Each plate was loaded with
approximately 200 cpm and exposed for 14 days. The previously
identified Ser
(S16), Ser
(S25), Ser
/Ser
(S16/25), Ser
(S38), and
Ser
(S63) phosphopeptides are shown in panel
C. S16`, S38`, and S16/S25` represent
partially cleaved phosphopeptides. Results shown are representative for
two independent experiments.
Op18 is
phosphorylated during mitosis by specific CDKs, most likely by p34-cdc2
and p33-cdk2 on Ser and Ser
, and on
Ser
and Ser
by an as yet unidentified protein
kinase
(29) . The stoichiometry of Op18 phosphorylation by these
two kinase systems during mitosis was determined by site-mapping of
distinct phosphoisomers. Accordingly,
P-labeled Op18
phosphoisomers from interphase and M-blocked HeLa cells
(Fig. 1A) were separated by native PAGE and subjected to
tryptic phosphopeptide mapping. Previous studies have identified all
potential tryptic phosphopeptides of Op18, as well as a
diphosphorylated peptide containing both Ser
and
Ser
(Fig. 1C)
(23, 24, 27) .
Since Ser
phosphorylation interferes with trypsin cleavage
between amino acids 14 and 15, the Ser
and Ser
peptides migrate differently in the electrophoretic dimension,
which allows independent identification of Ser
and
Ser
phosphorylation (Fig. 1C)
(24) .
Fig. 1B shows that monophosphorylated Op18 derived from
interphase cells contains phosphopeptides corresponding to all four
previously mapped Ser residues. PhosphorImager analysis of the plate
reveals that Ser
contains 21%, Ser
contains
6%, Ser
contains 65%, and Ser
contains about
8% of the total radioactivity. M-blocked cells do not contain
monophosphorylated Op18, but interestingly, the diphosphorylated Op18
phosphoisomers contain only the Ser
and Ser
peptides (Fig. 1D). As expected, the Ser
and Ser
peptides derived from diphosphorylated Op18
contain a similar amount of radioactivity on their two CDK target
sites. Analysis of the tri- and tetraphosphorylated Op18 species
reveals phosphorylation of all four previously identified Ser residues.
As shown in Fig. 1E, the Ser
peptide is
absent in triphosphorylated Op18, demonstrating that Ser
is phosphorylated to completion in triphosphorylated Op18, since
the Ser
peptide is replaced by the
Ser
/Ser
peptide. Finally, as can be predicted
for tetraphosphorylated Op18, the S25 peptide is absent, since all Op18
is phosphorylated on both Ser
and Ser
, which
results in the Ser
/Ser
peptide. Hence, site
mapping of all phosphoisomers of Op18 shows that the two CDK target
sites, Ser
and Ser
, are phosphorylated to
completion during mitosis. Moreover, quantification of Ser
and Ser
phosphopeptides shows that both are
phosphorylated to a high stoichiometry, but analysis of
triphosphorylated Op18 reveals that the Ser
contains more
radioactivity than Ser
(taking into account that half of
the radioactivity of the Ser
/Ser
peptide is
on Ser
; Ser
contains 14% and Ser
contains 23% of the total radioactivity).
Analysis of the Phenotype of Kinase Target Site-deficient
Mutants of Op18
As shown above and in a previous study,
phosphorylations of four distinct sites of Op18 are subject to dramatic
cell cycle-dependent fluctuations
(29) . To address the potential
function of these phosphorylation events, we constructed and expressed
Op18 mutants containing Ala substitutions of each of the phosphorylated
Ser residues. Previous experiments have shown that mutations of
Ser and Ser
block cell growth, and this
prompted us to optimize a system allowing regulated expression of cDNA
inserts cloned into the episomal EBV-based vector pMEP4
(30) .
The hMTIIa promotor of this vector can be suppressed by nontoxic levels
of EDTA, while high level expression can be induced by Cd
(see ``Materials and Methods'' and Fig. 2).
Increased expression of Op18 in Cd
-treated cells
transfected with either pMEP-Op18-wt or Ala-substituted derivatives
thereof are shown by Western blot analysis in Fig. 2, leftpanel. PhosphorImager analysis of the results reveals
that EDTA-suppressed transfected cells express about the same amount of
recombinant Op18 as the endogenous gene product, which is 1100 ng/mg
total cell proteins in the K562 erythroleukemia cell line. Moreover,
Cd
induction results in expression levels that are
more than 10-fold higher than the endogenous gene product. These
results also show that the introduced mutations do not alter the
expression levels of the proteins.
Figure 2:
Regulated ectopic expression of wild type
and mutated Op18. K562 cells were transfected with the indicated
pMEP4-Op18 constructs and ``stable'' transfectants were
selected by cultivation with hygromycin and EDTA for 6 days as
described under ``Materials and Methods.'' Cells were
thereafter cultured for 24 h with EDTA (50 µM) (rightpanels) or Cd (0.1 µM)
(leftpanels). Ectopic expression of Op18 was
analyzed by Western blot analysis using anti-Op18:34-149 and
I-Protein A. The positions of Op18 migrating at 19 kDa
and a triphosphorylated (Refs. 25 and 38) form of Op18 migrating at 23
kDa is indicated. An autoradiograph exposed for 24 h is shown. As a
control for equal loading the same filter was also probed with the PC10
monoclonal antibody (upperpanels). S25,38A,
S25A,S38A; S16,63A, S16A,S63A; S16,25,38,63A,
S16A,S25A,S38A,S63A.
Substitution of the CDK target
sites Ser and Ser
with Ala results in a
dominant Op18 mutant that blocks cell division
(30) . The
phenotype of these Op18 mutants, i.e. a G2/M block followed by
endoreduplication, was observed in both the K562 cell line and the
BL-42 Burkitt's lymphoma cell line. To delineate the potential
importance of Ser
and Ser
-specific
phosphorylation, we analyzed DNA synthesis and the cell cycle
distribution of K562 cells transfected with various Op18 derivatives.
The results of this analysis are shown in Fig. 3. Only minor
effects of the pMEP-Op18 constructs were observed in the presence of
EDTA, and as expected, the cell cycle profile of the vector
Co-transfected cells is not altered after addition of
Cd
. In agreement with our previous study, high level
expression of Op18-wt results in only minor alterations of the cell
cycle profile. However, addition of Cd
to cells
expressing either the S16A,S63A, S25A,S38A, or S16A,S25A,S38A,S63A Op18
mutants resulted in a drastic decrease in DNA synthesis and
accumulation of cells in the G2/M phase of the cell cycle within 24 h.
Most importantly, it appears that expression of the S16A,S63A mutant is
as efficient at causing a G2/M block as the previously characterized
CDK target site-deficient S25A,S38A mutant. Moreover, the similarities
between the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A Op18 mutants
are also evident at the 72-h time point, since a pronounced
endoreduplication response is observed with all these mutants
(Fig. 3, rightpanels).
Figure 3:
Mutation of the ``non-CDK''
phosphorylation sites Ser and Ser
results in
a transient G2 block followed by endoreduplication. K562 cells were
transfected with the indicated pMEP4-constructs, and
hygromycin-resistant cell lines were selected as in Fig. 2. Cells were
thereafter cultured in the presence of EDTA (50 µM), in
medium alone (None), or in Cd
(0.1
µM), as indicated. [
H]Thymidine
incorporation of cells cultured for 24 h (2
10
cells/well), using the indicated culture conditions, are
presented as percentage of incorporation in the presence of EDTA. It
should be noted that cells transfected with mutated Op18 routinely
incorporated 20-30% less [
H]thymidine per
cell in the presence of EDTA, as compared with cells transfected with
vector Co or Op18-wt. DNA profiles of cells cultivated for the
indicated time with EDTA or Cd
are also shown. The
results are representative for six inde-pendent experiments.
Op18-S25,38A, Op18-S25A,S38A; Op18-S16,63A,
Op18-S16A,S63A; Op18-S16,25,38,63A,
S16A,S25A,S38A,S63A.
To delineate the
importance of Serversus Ser
phosphorylation, the effect of single Ala substitutions at these
sites was investigated. The results in Fig. 4show that
expression of both wild type Op18 and the S16A mutant have only minor
effects on the cell cycle profile, while expression of the S63A mutant
caused a more pronounced inhibition of DNA synthesis and a substantial
G2/M block. However, although the S63A mutation alone exhibits a
pronounced phenotype, the result reveals that the phenotype of the
S16A,S63A mutant is even more dramatic at both the 24- and 72-h time
points. The differences between the S16A and S63A mutants outlined in
Fig. 4
were reproducible, and Western blot analysis revealed that
both mutants were expressed at equal levels (data not shown).
Figure 4:
The phenotype of Op18 mutants with single
site substitutions of Ser and Ser
.
[
H]Thymidine incorporation and DNA profile of
K562 cells expressing the indicated pMEP4 construct were assessed as in
Fig. 3. Op18-S16,63A,
Op18-S16A,S63A.
Analysis of Cell Cycle Dynamics Suggests That both the
S16A,S63A and S25A,S38A Op18 Mutants Cause an Immediate G2/M
Block
The experiments outlined above show that expression of the
S16A,S63A mutant results in rapid accumulation of cells with G2/M
content of DNA, followed by endoreduplication, and this phenotype
appears similar to the one caused by the CDK target site-deficient
S25A,S38A mutant. To further analyze cell cycle dynamics and to search
for potential phenotypic differences between Op18 mutants we analyzed
incorporation of BrdU during the entire 20-h period of induced
expression of Ala-substituted Op18 derivatives. Subsequent dual
parameter flow-cytometric analysis of BrdU incorporation and DNA
content allowed identification of cycling and noncycling cells in
various stages of the cell cycle. The result in Fig. 5A shows that most cells expressing wild type Op18 incorporates BrdU
during the 20-h period and that the minor population of BrdU-negative
cells have a G1 content of DNA. In contrast, cells expressing all the
indicated Ser to Ala-substituted Op18 derivatives contain BrdU-negative
cell populations with both G1 and G2/M content of DNA. Most
importantly, the results clearly show that all cycling cells are
blocked in G2/M by expression of these Op18 mutants. It seems
reasonable to assume that the BrdU-negative cell populations with G2
content of DNA represent cells that were in the G2 phase at the time of
Cd addition and that these cells were immediately
blocked by the mutant Op18 protein. This interpretation agrees with the
rapid kinetics of Cd
-induced expression of the hMTIIa
promotor, which result in elevated Op18 levels within a few hours of
induction (data not shown). In conclusion, the result in
Fig. 5
shows that expression of all kinase target site-deficient
mutants tested results in a more or less immediate block in cell
division, and this block seems independent of expression of the mutants
during the preceding S-phase. Moreover, it appears that the phenotypes
of the S16A,S63A and the CDK target site-deficient S25A,S38A mutants
are identical. The observed phenotype of the S16A,S25A,S38A,S63A mutant
is in line with this interpretation, since mutation of all four
available phosphorylation sites results in a phenotype
indistinguishable from the other mutants.
Figure 5:
Analysis of BrdU incorporation in cells
expressing Op18 ``kinase site-deficient'' mutants reveals an
immediate block in cell division. K562 cells were transfected with the
indicated pMEP4 constructs, and hygromycin-resistant cell lines were
selected as in Fig. 2. Op18 expression was thereafter induced with
Cd in the presence of BrdU for 20 h. Cell fixation,
staining with anti-BrdU, and flow cytometric dual parameter analyses of
BrdU incorporation versus DNA content are described under
``Materials and Methods.'' The results are representative for
two independent experiments. S25,38A, S25A,S38A;
S16,63A, S16A,S63A; S16,25,38,63A,
S16A,S25A,S38A,S63A.
Previous morphological
studies of cells transfected with the Op18-S25A,S38A mutant and induced
with Cd for 24 h revealed that about two-thirds of
the cells appeared as normal healthy G2 cells
(30) . The
remaining cells appeared as mitotic cells, lacking the nuclear envelope
but without signs of spindle formation. Transmission electron
micrographs revealed various degrees of aberrant chromosome
aggregations, which appeared as compacted nuclei in light
microscopy
(30) . To compare the morphology of cells expressing
the S25A,S38A, S16A,S63A, and S16A,S25A,S38A,S63A Op18 mutants,
respectively, we analyzed May-Grunwald-Giemsa-stained cytocentrifuge
preparations of transfected cells. The results in show
that expression of all these Op18 mutants resulted in essentially the
same frequency of cells blocked in M-phase and of mitotic cells with a
compacted nucleus. Thus, morphological examination of G2/M-blocked
cells also suggest that the phenotypes of the S25A,S38A, S16A,S63A, and
S16A,S25A,S38A,S63A Op18 mutants are the same.
Deletion of an N-terminal Region of Op18 Abolishes the
Phenotype of the S63A Mutant
The phenotype of kinase target
sites mutants is most likely due to the absence of a phosphate group(s)
but may also, in some cases, be due to the creation of a
``pseudo-substrate site'' or some other alteration that
interferes with specific kinase systems. To address these questions we
expressed both full-length and N-terminal deleted Op18 derivatives with
or without the S63A mutation. Fig. 6A shows high level
expression of all these derivatives in Cd-induced
cells (lanesd and e), as compared with the
endogenous gene product and that the N-terminal deleted Op18 proteins
are expressed at almost the same levels as the full-length Op18 protein
(lanesb and c). Ala substitution of
Ser
, but not Ser
, is sufficient to induce a
substantial G2/M block (see above). To determine if this phenotype is
resistant to an N-terminal deletion, the cell cycle profile of cells
expressing Op18-
4-55 was compared with that of cells
expressing Op18-
4-55/S63A. The result in Fig. 6, B and C, shows that expression of Op18-
4-55/S63A
does not alter the DNA distribution compared with vector Co or
Op18-
4-55. For comparison, we also included Op18-wt- and
Op18-S63A-transfected cells in this experiment. As expected, the S63A
mutation caused a profound G2/M block as compared with the wild type
construct. Thus, the G2/M block caused by the S63A mutation requires
the N-terminal region of Op18, which suggests that a region distal to
the mutation is critical for the observed phenotype.
Figure 6:
The phenotype of the S63A mutant requires
the N-terminal part of Op18. K562 cells were transfected with the
indicated pMEP4-Op18 constructs, and hygromycin-resistant cell lines
were selected as in Fig. 2. Op18 expression was thereafter induced with
Cd for 24 h. A, ectopic expression of Op18
was analyzed by Western blot analysis as in Fig. 2 (the minor band
migrating below full-length Op18 (lanesb and
c) is due to proteolytic cleavage). The positions of Op18
migrating at 19, 23, and 15 kDa (
4-55) are indicated. As a
control for equal loading, the same filter was also probed with the
PC10 monoclonal antibody (upperpanel).
Phosphorylation of the deleted Op18 proteins was analyzed by
P labeling of cells followed by immunoprecipitation. As
anticipated, while the Op18-
4-55 protein incorporated the
expected amount of [
P] label, the
Op18-
4-55/S63A protein was completely nonphosphorylated
(data not shown). DNA profiles of transfected cells cultivated for 24
(B) and 72 h (C) with Cd
are
shown.
The S25A,S38A Mutation Interferes with Phosphorylation of
Ser
Since
the G2/M transition is associated with multisite phosphorylation of
Op18 by two distinct kinase systems, it is possible that mutations of
some sites interfere with phosphorylation of others. To test this idea,
we resolved Op18 phosphoisomers by native PAGE from
Cd and Ser
-induced cells transfected with Op18-wt,
Op18-S25A,S38A, Op18-S16A,S63A, or Op18-S16A,S25A,S38A,S63A
(Fig. 7). The resulting blot of Op18 phosphoisomers reveal that
overexpression of the wild type protein results in a substantial
increase of the fraction of monophosphorylated Op18 as compared with
phosphorylation of the endogenous gene product (see Fig. 1A and Ref. 29). This finding is striking, but the low levels of
multisite-phosphorylated Op18 suggest that the phenomenon is not a
result of the minor alteration of the cell cycle profile caused by
overexpression of the wild type protein.
Figure 7:
Analysis of Op18 phosphoisomers in
Op18-transfected cells. K562 cells were transfected with the indicated
pMEP-Op18 constructs and hygromycin-resistant cell lines were selected
as in Fig. 2. Cells were thereafter cultured in the presence of
Cd (0.1 µM) for 24 h, and phosphoisomers
of Op18 were analyzed by separation on native PAGE as in Fig.
1A. Only the recombinant Op18 gene product is visualized on
the presented autoradiograph, since the transfected Op18 derivatives
are expressed at 10-20-fold higher levels than the endogenous
Op18 protein. S25,38A, S25A,S38A; S16,63A, S16A,S63A;
S16,25,38,63A, S16A,S25A,S38A,S63A; non-P,
nonphosphorylated.
Cells expressing the
S25A,S38A, S16A,S63A, or S16A,S25A,S38A,S63A Op18 mutants for 24 h all
have G2/M content of DNA. Analysis of the Op18 phosphoisomers derived
from these mutants reveals profound differences in the stoichiometry
and distribution of phosphoisomers. First, the S16A,S25A,S38A,S63A
protein is not phosphorylated at all, as could be anticipated. Second
and most importantly, while the S25A,S38A protein is mainly
nonphosphorylated, a major fraction of S16A,S63A protein is
phosphorylated on both of its remaining Ser and Ser
sites, as revealed by the predominant species of diphosphorylated
protein. Induced expression of these Op18 mutants blocks between 65 and
80% of all cells in G2, and the remaining cells are blocked in mitosis
( and Ref. 30). Hence, the presence of mitotic cells
readily explains diphosphorylation on the two non-mutated sites of the
S16A,S63A mutant. However, it was unexpected that the diphosphorylated
isomer is absent in Op-18-S25A,S38A-expressing cells, since
diphosphorylation of Ser
and Ser
is prominent
in nontransfected mitotic cells (Fig. 1). These results suggest
that mutation of the CDK sites interferes with phosphorylation of
Ser
and Ser
. It follows that the observed
phenotype of the S25A,S38A Op18 mutant may be caused by interference
with Ser
and Ser
phosphorylation, which would
explain the similarity of the phenotypes of the S16A,S63A and S25A,S38A
Op18 mutants.
and Ser
are physiological substrates for at least
some members of the CDK family includes the following: (i) S-phase
progression is associated with increased phosphorylation of these
residues, which reaches its peak during mitosis ( Fig. 1and Ref.
29); and (ii) both Ser
and Ser
contain CDK
consensus phosphorylation sites and are efficiently phosphorylated
in vitro by at least two of the members of the CDK family,
namely p34-cdc2 and p33-cdk2
(23, 29) . Hence, Ser
and Ser
are likely physiological targets for CDKs.
However, two lines of evidence exclude CDKs as the kinases
phosphorylating Ser
and Ser
; first, these
sites are not phosphorylated by the CDKs tested in vitro (e.g. p34-cdc2-, p33-cdk2-, and p13-suc1-precipitated
kinases)
(23, 29) . Second, the sequences surrounding
Ser
(KRASGQA) and Ser
(RRKSHEA) lack the Pro
residues and the other features that are essential for phosphorylation
by CDKs
(6, 35) . Thus, the kinase system(s) involved in
cell cycle-regulated Ser
and Ser
phosphorylation is still unknown, but there are many possible
candidates, e.g. MPM-2 epitope kinases, calcium/calmodulin
kinase II, or
NimA
(4, 5, 36, 37, 38) .
and
Ser
, are phosphorylated to completion. Second, a major
fraction of Op18 is tri- or tetraphosphorylated with Ser
phosphorylated to a somewhat higher stoichiometry than Ser
during mitosis. Third, phosphorylation of Ser
and
Ser
as well as the CDK sites is shown to be functionally
important for cell division. Our previous study of CDK site-deficient
mutants of Op18 revealed a clear cut dominant phenotype of single site
mutations of Ser
and Ser
and an even more
pronounced phenotype of a double mutant. This study shows that the S16A
mutant has only minor effects on the cell cycle profile as compared
with wild type Op18, while expression of the S63A mutant caused a
pronounced inhibition of DNA synthesis and a substantial G2/M block
(Fig. 4). Interestingly, the combination of the S16A and S63A
mutations resulted in a more dramatic phenotype that was
indistinguishable from that of the S25A,S38A mutant. Thus, these
genetic data suggest that all four kinase target sites of Op18 are
functionally important during cell division, although the S16A mutation
alone has an almost undetectable phenotype.
or Ser
do not interfere with CDK-mediated phosphorylation of the
alternative Op18 sites
(29) , nor have we detected inhibition of
CDK phosphorylation of histone H1 in vitro in the presence of
the Op18-S25A,S38A mutant protein (data not shown). In the present
study we analyzed the phenotype of N-terminal deleted Op18 derivatives,
with or without the S63A mutation (Fig. 6A). The result
showed that the phenotype of the S63A mutation requires the N-terminal
region of Op18. Since a region distal to the mutation is critical for
the phenotype, we assume that the phenotype is not caused by generation
of a putative inhibitory pseudo-substrate site. This interpretation is
in line with our observation that single site mutations of at least
three distal cell cycle-regulated phosphorylation sites
(Ser
, Ser
, and Ser
) are
sufficient to cause a profound phenotype on the level of cell cycle
regulation, i.e. G2/M block followed by endoreduplication.
and Ser
were only weakly
phosphorylated in cells expressing the S25A,S38A CDK sites mutant,
while cells expressing the S16A,S63A mutant contained a large fraction
of Op18 diphosphorylated on the Ser
and Ser
CDK sites (Fig. 7). Cells blocked at G2/M by either of
these mutants contain about 25% of M-blocked cells ().
Therefore, multisite phosphorylation would be predicted on the intact
sites of the mutated protein. While this was the case for cells
expressing the S16A,S63A mutant, it was not for the S25A,S38A mutant.
Hence, mutation of the CDK sites Ser
and Ser
interferes with phosphorylation of Ser
and
Ser
in transfected cells with the consequence that the
S25A,S38A as well as the S16A,S63A mutated protein lacks
diphosphorylation on Ser
and Ser
. It follows
that the expressed S25A,S38A protein is identical to the
S16A,S25A,S38A,S63A protein with respect to the complete absence of
multiphosphorylation, which may explain the identical phenotype. The
S16A,S63A protein is heavily phosphorylated on its intact CDK target
sites, but this phosphorylation does not seem to influence the observed
phenotype, which appears identical to that of the S25A,S38A and
S16A,S25A,S38A,S63A mutants. The simplest interpretation of these
observations is that phosphorylation of Ser
and Ser
is essential during a point at G2/M transition and that the
observed phenotype of the S25A,S38A CDK site-deficient mutant is caused
by interference with multiphosphorylation of Ser
and
Ser
.
and Ser
by at least two mechanisms. First, CDK dependent
prephosphorylation on Ser
and Ser
may be a
prerequisite for Ser
and Ser
phosphorylation
during G2/M transition. Second, phosphorylation on the CDK sites may by
involved in the activation of the unidentified Op18 Ser
and Ser
kinase. Discrimination between these two
alternatives has to await the future identification of the protein
kinase(s) responsible. However, the finding that cell division requires
phosphorylation of Op18 by at least two distinct protein kinase systems
suggests that Op18 is directly involved in some aspect of G2/M
transition. Such an involvement is consistent with the rapid block in
cell division caused by the kinase target site-deficient mutants and
the finding that this block does not require expression of the mutants
during the preceding S-phase (Fig. 5). Since both CDK target
sites are phosphorylated to completion in mitotic cells, it is tempting
to speculate that CDKs have an on/off function that allows
phosphorylation of Ser
and Ser
, while the
Ser
/Ser
kinase(s) may mediate the
phosphorylation event that is essential for G2/M transition. If this is
the case, the observation that the S25A,S38A, S16A,S63A, or
S16A,S25A,S38A,S63A Op18 mutants all result in an identical phenotype
would be predicted since all are defective in multisite phosphorylation
on Ser
and Ser
.
and Ser
are clearly substrates for
receptor-regulated kinase systems, but the role(s) of these
phosphorylation events is still obscure. The genetic evidence in this
and our previous study
(30) demonstrates the importance of a
complex multisite phosphorylation of Op18 during G2/M transition. A
distinct type of multisite phosphorylation is observed after triggering
of the T-cell antigen receptor, involving increased phosphorylation of
Ser
and Ser
but not of Ser
and
Ser
(24, 27) . Thus, it seems that a
plethora of protein kinases converge on Op18. An important extension of
the studies on Op18 will be to determine if phosphorylation of this
protein is of functional importance on other levels of cell regulation
besides G2/M transition.
Table:
M-phase frequency and nuclear morphology in
cells expressing various kinase site-deficient mutants of Op18
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.