Ishida et al. 1
Phosphorylation at serine-10, a major phosphorylation
site of p27Kip1, increases its protein stability
Noriko Ishida, Masatoshi Kitagawa, Shigetsugu Hatakeyama, and
Kei-ichi Nakayama
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation,
Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582,
Japan, and CREST, Japan Science and Technology Corporation (JST),
Kawaguchi 332-0012, Japan
Running title: Control of p27Kip1 stability by Ser10 phosphorylation
Address correspondence to: Kei-ichi Nakayama, Department of Molecular and
Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
Tel.: +81-92-642-6815. Fax: +81-92-642-6819.
E-mail: [email protected]
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on May 30, 2000 as Manuscript M001144200 by guest on A
pril 3, 2018http://w
ww
.jbc.org/D
ownloaded from
Ishida et al. 2
SUMMARY
The association of the p27Kip1 protein with cyclin and cyclin-dependent kinase
complexes inhibits their kinase activities and contributes to the control of cell
proliferation. The p27Kip1 protein has now been shown to be phosphorylated in
vivo, and this phosphorylation reduces the electrophoretic mobility of the protein.
Substitution of Ser10 with Ala (S10A) markedly reduced the extent of p27Kip1
phosphorylation and prevented the shift in electrophoretic mobility.
Phosphopeptide mapping and phosphoamino acid analysis revealed that
phosphorylation at Ser10 accounted for ~70% of the total phosphorylation of p27Kip1,
and the extent of phosphorylation at this site was ~25- and 75-fold greater than
that at Ser178 and Thr187, respectively. The phosphorylation of p27Kip1 was markedly
reduced when the positions of Ser10 and Pro11 were reversed, suggesting that a
proline-directed kinase is responsible for the phosphorylation of Ser10. The extent
of Ser10 phosphorylation was markedly increased in cells in the G0-G1 phase of the
cell cycle compared with that apparent for cells in S or M phase. The p27Kip1
protein phosphorylated at Ser10 was significantly more stable than the
unphosphorylated form. Furthermore, a mutant p27Kip1 in which Ser10 was
replaced with glutamic acid in order to mimic the effect of Ser10 phosphorylation
exhibited a marked increase in stability both in vivo and in vitro compared with the
wild-type or S10A mutant proteins. These results suggest that Ser10 is the major
site of phosphorylation of p27Kip1, and that phosphorylation at this site, like that at
Thr187, contributes to regulation of p27Kip1 stability.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 3
INTRODUCTION
Progression of the cell cycle in all eukaryotic cells depends on the activity of a
series of kinase complexes composed of cyclins and cyclin-dependent kinases
(CDKs). The activity of cyclin-CDK complexes is regulated by various
mechanisms, including association of the kinase subunit with the regulatory cyclin
subunit, phosphorylation-dephosphorylation of the kinase subunit, and
association of the complex with a group of CDK inhibitors (CKIs) (1,2). The
interaction of CKIs with cyclin-CDK complexes is triggered by a variety of
antimitogenic signals and results in inhibition of the catalytic activity of the
complexes and consequent restraint of cell cycle progression. CKIs are classified
into two families on the basis of their amino acid sequence similarity and putative
targets (3,4). The Cip or Kip family comprises p21Cip1 (also known as Waf1, Sdi1,
and CAP20), p27Kip1, and p57Kip2, each of which possesses a conserved domain,
termed the CDK binding-inhibitory domain, at its NH2-terminus. The Ink4 family
consists of p16Ink4A, p15 Ink4B, p18 Ink4C, and p19Ink4D, and its members each contain
four tandem repeats of an ankyrin motif. Whereas members of the Ink4 family
inhibit the activity of CDK4 or CDK6 specifically, members of the Cip-Kip family
show a broad spectrum of inhibitory effects on cyclin-CDK complexes.
The p27Kip1 protein plays a pivotal role in the control of cell proliferation
(5,6). Transition from G1 phase to S phase of the cell cycle is promoted by G1
cyclin-CDK complexes, and p27Kip1 inhibits the activities of these complexes
directly by binding to them. In normal cells, the amount of p27Kip1 is high during
G0-G1 phase, but it rapidly decreases on reentry into S phase triggered by specific
mitogenic factors (7,8). Forced expression of p27Kip1 results in cell cycle arrest in
G1 phase (5,6), and, conversely, inhibition of p27Kip1 expression by antisense
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 4
oligonucleotides increases the number of cells in S phase (9). Moreover, mice
with a homozygous deletion of the p27Kip1 gene are larger than normal mice and
exhibit multiple organ hyperplasia and a predisposition to spontaneous and
radiation- or chemical-induced tumors (10-13).
The concentration of p27Kip1 is thought to be regulated predominantly by
posttranslational mechanisms (14,15). We recently showed that p27Kip1 is
degraded by both the ubiquitin-proteasome pathway and ubiquitin-independent
proteolytic cleavage (16). Regulation of ubiquitin-mediated proteolysis is often
achieved by phosphorylation of the target protein, which renders it more
susceptible to degradation (17-21). Such may also be the case with p27Kip1, given
that its down-regulation is promoted by its phosphorylation on Thr187 by the cyclin
E-CDK2 complex (22-24). Recent data have also suggested that Fbl1 (also known
as Skp2), an F-box protein that is thought to function as the receptor component
of an SCF ubiquitin ligase complex, binds to p27Kip1 only when Thr187 is
phosphorylated; such binding then results in the ubiquitination and degradation of
p27Kip1 (25-27).
Various kinases, such as mitogen-activated protein kinases (MAPKs) and
CDKs, may trigger the degradation of p27Kip1 in response to different upstream
signaling pathways. For example, activation of members of the MAPK family is
mediated through Ras (28), whereas rapid activation of cyclin E-CDK2 results
from the induction of Myc (29,30). Kaposi's sarcoma herpesvirus also destabilizes
p27Kip1 through phosphorylation of Thr187 by the complex of the virus cyclin (K-
cyclin) and CDK6 (31,32). These observations indicate that phosphorylation of
p27Kip1 controls its stability. However, because most studies have focused on the
role of phosphorylation of Thr187 in p27Kip1 stability, little is known about the
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 5
potential roles of other phosphorylation sites of this protein.
We now show that p27Kip1 is phosphorylated on many sites, including
Thr187, in vivo, with the predominant phosphorylation site being Ser10.
Phosphorylation of Ser10 is regulated in a cell cycle-dependent manner and may
function to stabilize p27Kip1. Given that the level of phosphorylation of Ser10 is
substantially greater than that apparent at other phosphorylation sites,
phosphorylation-dephosphorylation of p27Kip1 at Ser10 may be critical for regulation
of cell cycle progression from the resting state to proliferation.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 6
EXPERIMENTAL PROCEDURES
Cell Culture and Synchronization—293T, COS-7, and HeLa cells were cultured at
37°C and under an atmosphere of 5% CO2 in Dulbecco’s modified Eagle's
medium (DMEM) (Life Technologies, Rockville, MD) supplemented with 10% (v/v)
fetal bovine serum (FBS) (Life Technologies). NIH 3T3 cells were cultured in
DMEM supplemented with 10% (v/v) calf serum (Life Technologies). For analysis
of synchronized cells, HeLa or NIH 3T3 cells were arrested at G0-G1 phase by
subjecting them to contact inhibition during culture to confluence and to serum
deprivation with medium supplemented with 0.1% FBS or calf serum, respectively.
Cells were arrested in S phase by exposure to aphidicolin (1 µg/ml) as described
by Fang et al (33). For analysis of cells in M phase, HeLa cells were arrested in
aphidicolin-containing medium for 16 h, washed with phosphate-buffered saline,
and then incubated in aphidicolin-free medium for 3 h. They were subsequently
incubated with nocodazole (100 ng/ml) for 12 to 15 h to induce arrest at M phase,
after which culture dishes were shaken and floating cells were harvested for
recovery of only those cells in M phase.
Construction of Plasmids and Site-Directed Mutagenesis—Complementary
DNAs encoding all p27Kip1 derivatives were prepared from the human p27Kip1 cDNA,
kindly provided by M. Nakanishi. The p27Kip1 mutants were generated by replacing
Ser10, Ser178, or Thr187 with Ala (S10A, S178A, and T187A, respectively), or
replacing Ser10 with Glu (S10E) with the use of a QuickChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). Proteins tagged at their NH2-termini
with the Flag epitope were generated with the use of the polymerase chain
reaction as performed with the high-fidelity thermostable DNA polymerase KOD
(Toyobo, Tokyo, Japan). The sequences of all mutant cDNAs were confirmed in
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 7
their entirety. The cDNAs encoding the various p27Kip1 proteins, with or without the
Flag epitope tag, were then subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) for
transfection experiments, or into pGEX6P (Amersham Pharmacia Biotech, Little
Chalfont, UK) for production in bacteria of glutathione S-transferase (GST) fusion
proteins.
Transfection, Immunoprecipitation, and Immunoblot Analysis—Transfection,
immunoprecipitation, and immunoblot analysis were performed as previously
described (20,21,34). Immunoblots were probed with antibodies (1 µg/ml) to the
Flag epitope (M5; Sigma, St. Louis, MO), to p27Kip1 (Transduction Laboratories,
Lexington, KY), to phosphorylated MAPK (Promega, Madison, WI), or to α-tubulin
(TU01; Zymed).
Alkaline Phosphatase Treatment of p27Kip1—Immunoprecipitates containing
p27Kip1 were washed thoroughly three times with ice-cold lysis buffer and once
with lysis buffer without phosphatase inhibitors. They were then incubated for 5 h
at 37°C in a final volume of 30 µl containing 40 U of calf intestinal alkaline
phosphatase (CIAP) (Takara), 50 mM Tris-HCl (pH 9.0), and 1 mM MgCl2. The
reaction mixture was then subjected to SDS-polyacrylamide gel electrophoresis
(PAGE) and immunoblot analysis with antibodies to (anti-) p27Kip1.
[32P]Pi Labeling of p27Kip1—Transfected 293T cells were incubated for 2 h in
phosphate-free DMEM supplemented with 10% dialyzed FBS and then
metabolically labeled for 4 h at 37°C with [32P]Pi (Amersham Pharmacia Biotech)
at a concentration of 1 mCi/ml in the same medium. After extensive washing of the
cells in isotope-free medium, they were then lysed and subjected to
immunoprecipitation with anti-Flag or anti-p27Kip1. The immunoprecipitates were
fractionated by SDS-PAGE and subjected to autoradiography and quantitative
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 8
analysis with a BAS-2000 image analyzer (Fuji Film, Kanagawa, Japan).
Phosphorylation of p27Kip1 in Vitro—GST-p27Kip1 fusion proteins were
expressed in Escherichia coli XL1-blue and affinity-purified with glutathione-
Sepharose CL-4B (Amersham Pharmacia Biotech), after which the GST moiety
was cleaved from the fusion proteins with the use of PreScission protease
(Amersham Pharmacia Biotech). The recombinant wild-type p27Kip1 protein (0.2
µg) was then incubated for 30 min at 30°C in a final volume of 20 µl containing
purified MAPK p42 (100 U) (ERK2; New England Biolabs, Beverly, MA), 50 µM (1
µCi) [γ-32P]ATP (Amersham Pharmacia Biotech), 20 mM Tris-HCl (pH 7.3), 10 mM
MgCl2, 4.5 mM 2-mercaptoethanol, and 1 mM EGTA.
CDK Inhibition Assay by p27Kip1 in Vitro—The recombinant wild-type p27Kip1
protein and its S10A and S10E mutants (0, 0.01, 0.05, and 0.25 µg) were
incubated for 15 min at 30°C in a final volume of 20 µl containing purified
baculovirus-produced cyclin E-CDK2 complex or cyclin D2-CDK4 complex, 25 µM
(0.5 µCi) [γ-32P]ATP (Amersham Pharmacia Biotech), 20 mM Tris-HCl (pH 7.3), 10
mM MgCl2, 4.5 mM 2-mercaptoethanol, and 1 mM EGTA. The reaction mixture
was then subjected to SDS-PAGE, autoradiography, and quantitative analysis
with a BAS-2000 image analyzer.
Phosphopeptide Mapping and Phosphoamino Acid Analysis—32P-Labeled
proteins were prepared for phosphopeptide mapping as described (23). Dried
samples were treated with 10 µg of trypsin (Boehringer Mannheim) for at least 8 h
at 37°C. The reaction mixtures were then lyophilized twice in 0.4-ml volumes of
water and finally resuspended in 10 µl of pH 1.9 buffer (20 ml of formic acid and
156 ml of glacial acetic acid per 1794 ml of water) prior to application to thin-layer
chromatography (TLC) plates. Electrophoresis and ascending chromatography
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 9
were performed as described (35) with minor modifications; phospho-
chromatography buffer (750 ml of n-butanol, 500 ml of pyridine, and 150 ml of
glacial acetic acid per 600 ml of water) was used. Plates were air-dried and then
subjected to quantitative analysis with a BAS-2000 image analyzer.
Phosphoamino acid analysis of tryptic phosphopeptides derived from p27Kip1 was
performed as described (35), with the exception that Multiphor II (Amersham
Pharmacia Biotech) was used.
Two-Dimensional Gel Electrophoresis and Immunoblot Analysis—Two-
dimensional gel electrophoresis (2D-PAGE) with separation in the first dimension
by nonequilibrium pH gradient electrophoresis (NEPHGE) was performed as
described by O'Farrell et al (36). Cell lysate containing 0.15 to 0.5 mg of total
protein was applied to a NEPHGE tube (130 by 3 mm, inside diameter) gel [4%
(w/v) acrylamide, 9.2 M urea, 2% (v/v) Ampholytes (Bio-Lyte, pH 3-10; Bio-Rad),
2% (v/v) Nonidet P-40] and electrophoresis was performed for 5 to 8 h at 400 V.
The separated proteins were then resolved in the second dimension by standard
PAGE on a 10% gel, which was subsequently subjected to immunoblot analysis
with anti-p27Kip1.
In Vitro Degradation Assay—NIH 3T3 cell extracts (S100) were prepared as
described (16). Human recombinant p27Kip1 proteins (0.1 µg) or lysate (2 µg) of
transfected 293T cells were incubated at 37°C for the indicated times in 20 µl of a
degradation mixture containing 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 2 mM
dithiothreitol, 10 mM ATP, 1 mM phosphocreatine, phosphocreatine kinase (500
U/ml) with or without 2 µM Okadaic acid, and 10 µg of NIH 3T3 cell lysate proteins.
The mixture was then subjected to SDS-PAGE on a 12% gel and immunoblot
analysis with anti-p27Kip1.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 10
Pulse-Chase Experiments—Transfected NIH 3T3 cells were metabolically
labeled with [35S]methionine and [35S]cysteine (L-[35S]in vitro Cell Labeling Mix;
Amersham Pharmacia Biotech) at a concentration of 80 µCi/ml for 1 h, and then
incubated in isotope-free medium for 0, 3, 6, or 12 h. Cell lysates were prepared
and subjected to immunoprecipitation with anti-p27Kip1, and the resulting
precipitates were subjected to SDS-PAGE on a 12% gel, autoradiography, and
quantitative analysis with a BAS-2000 image analyzer.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 11
RESULTS
Phosphorylation of p27Kip1 in Vivo—The p27Kip1 protein contains three serine or
threonine residues, at positions 10, 178, and 187 (Ser10, Ser178, and Thr187), that
are immediately upstream of proline residues (Fig. 1A and Table 1). We focused
on the potential roles of these sites in determining the stability of p27Kip1 because
members of a group of kinases (known as proline-directed kinases) that require a
proline immediately downstream of the target serine or threonine residue, and
which include MAPKs and CDKs, contribute to mitogenic signaling pathways. We
generated cDNAs that encode mutant human p27Kip1 proteins in which each of the
three residues Ser10, Ser178, and Thr187 was replaced individually (S10A, S178A,
and T187A) or together (S10A/S178A/T187A) with Ala (Fig. 1A). The
phosphorylation status of these three sites of p27Kip1 in vivo was investigated by
transiently expressing the Flag epitope-tagged wild-type and mutant proteins in
293T human embryonic kidney epithelial cells and metabolically labeling the cells
with [32P]Pi. The p27Kip1 proteins were then immunoprecipitated with anti-Flag, and
the extent of 32P incorporation was evaluated by autoradiography and image
analysis and normalized by the amount of p27Kip1 protein estimated by immunoblot
analysis of the immunoprecipitates with anti-p27Kip1 (Fig. 1, B and C). The amount
of 32P incorporated by the S10A mutant or by the S10A/S178A/T187A triple
mutant was ~30% of that incorporated by wild-type p27Kip1, whereas that
incorporated by the S178A or T187A mutants was virtually identical to that
incorporated by the wild-type protein. These results indicated that Ser10 is the
major phosphorylation site of p27Kip1 (accounting for ~70% of the total extent of
p27Kip1 phosphorylation).
Immunoblot analysis with anti-p27Kip1 of wild-type p27Kip1 expressed in
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 12
cultured cells revealed that these antibodies recognized two bands, suggesting
that the lower-mobility band might correspond to phosphorylated p27Kip1 (Fig. 1B
and Fig. 2A; the two bands are more evident in the latter as a result of a difference
in composition of the acrylamide gel). This electrophoretic mobility shift was
apparent for p27Kip1 expressed not only in 293T cells, but also in HeLa (human
cervical cancer), COS-7 (monkey kidney epithelial), and NIH 3T3 (mouse
fibroblast) cells (Fig. 2A). For all cells tested, mutation of Ser10 of p27Kip1 to Ala
resulted in the disappearance of the more slowly migrating band. To confirm that
the observed mobility shift was attributable to phosphorylation of p27Kip1, we
expressed wild-type p27Kip1 or the S10A mutant in 293T cells, immunoprecipitated
the recombinant protein, and treated it with CIAP. Treatment with CIAP resulted in
the disappearance of the lower-mobility form of wild-type p27Kip1, but it had
virtually no effect on the mobility of the S10A mutant (Fig. 2B). These results thus
suggested that phosphorylation at Ser10 was responsible for the observed shift in
the electrophoretic mobility of p27Kip1, and that the kinase or kinases that catalyze
this reaction are present in cells from various tissues and species.
Cell Cycle-dependent Phosphorylation of p27Kip1 on Ser10— To investigate
the biological role of phosphorylation of p27Kip1 on Ser10, we examined whether the
phosphorylation status of this residue is dependent on phase of the cell cycle.
Asynchronous NIH 3T3 cells were transfected with an expression plasmid
encoding Flag-tagged wild-type p27Kip1 or its S10A mutant, and cell lysates were
subjected to 2D-PAGE and immunoblot analysis with anti-p27Kip1 in order to
quantify the extent of phosphorylation at Ser10 (Fig. 3A). Wild-type p27Kip1 yielded
two immunoreactive spots, the upper of which, corresponding to the form of the
protein phosphorylated on Ser10, migrated in a more acidic position on NEPHGE
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 13
in the first dimension because of the negative charge of the phosphate group; this
spot was not detected with the S10A mutant. Endogenous p27Kip1 exhibited a
pattern similar to that of the recombinant wild-type protein, suggesting that
phosphorylation at Ser10 is not an artifact of overexpression.
Immunoblot analysis of synchronized HeLa or NIH 3T3 cells with anti-
p27Kip1 revealed that endogenous p27Kip1 was abundant in G0-G1 phase of the cell
cycle but was present in markedly smaller amounts during S and M phases (Fig.
3B), similar to results previously obtained with many other cell types (7-9). 2D-
PAGE and immunoblot analysis with anti-p27Kip1 of synchronized HeLa cells
revealed that ~80% of endogenous p27Kip1 was phosphorylated at Ser10 during
G0-G1 phase, whereas the amount of this form of the protein was reduced to
virtually zero (0.1%) during S phase. In M phase, although the abundance of
p27Kip1 was minimal, a small proportion (16.0%) of the total p27Kip1 protein was
phosphorylated at Ser10. Similar results were obtained with NIH 3T3 cells,
although the phosphorylation state of p27Kip1 in M phase could not be estimated
because of the "mitotic slippage" apparent in rodent cell lines (37). These
observations suggested that phosphorylation of p27Kip1 on Ser10 is cell cycle
dependent, and that phosphorylation at this site might contribute to regulation of
the stability of this protein.
Phosphopeptide Analysis of Phosphorylated p27Kip1—To characterize
further the phosphorylation status of p27Kip1, we performed two-dimensional
phosphopeptide mapping of wild-type and mutant p27Kip1 proteins labeled with 32P
in vivo. The expected length and sequence of tryptic peptides of p27Kip1 that
contain serine or threonine are shown in Table 1. Ser10 is contained in a peptide
composed of 10 amino acids, whereas Ser178 and Thr187 are both contained in the
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 14
same 20-residue peptide. We compared the phosphopeptide maps of wild-type
p27Kip1 (with or without the Flag tag) and its S10A, S178A, T187A, and
S10A/S178A/T187A mutants after their immunoprecipitation from transfected
293T cells (Fig. 4A). No differences were detected between the phosphopeptide
map of Flag-tagged wild-type p27Kip1 and that of the untagged protein. Six or
seven radioactive spots were reproducibly detected, four of which (spots 3 to 6)
appeared common to all maps. Two intensely labeled peptides (spots 1 and 2),
however, were detected only in the maps of wild-type p27Kip1 and those of its
S178A and T187A mutants, and not in those of the S10A or triple mutants. These
results suggested that the extent of phosphorylation of p27Kip1 at Ser10 in vivo was
markedly greater than the extent of phosphorylation at other sites, including Ser178
and Thr187. The observation that the phosphopeptide containing Ser10 yielded two
spots is likely attributable to treatment with performic acid during sample
preparation. We also showed that Ser178 and Thr187 were contained in spot 6 by
phosphopeptide analysis of recombinant p27Kip1 phosphorylated in vitro by cyclin
E-CDK2 (data not shown).
Phosphorylation of p27Kip1 at Thr187 by cyclin E-CDK2 is required for its
degradation by the ubiquitin-proteasome pathway (22-27). To estimate the
relative amount of 32P incorporated into p27Kip1 at Thr187, we compared the
autoradiographic intensity of the phosphopeptides derived from wild-type p27Kip1
and its mutants. The amount of radioactivity incorporated into the peptide
containing Ser10 was ~75 and 25 times that incorporated by Thr187 and Ser178,
respectively (Fig. 4B). This apparent high relative amount of Ser10 phosphorylation
relative to Thr187 phosphorylation is unlikely to reflect the fraction of p27Kip1 that
becomes phosphorylated at this site because the form phosphorylated on Thr187 is
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 15
thought be rapidly degraded.
Phosphoamino Acid Analysis of p27Kip1—To identify the phosphorylation
sites of p27Kip1 in vivo, and to confirm the phosphorylation at Ser10, Ser178, and
Thr187, we performed phosphoamino acid analysis of seven major
phosphopeptides of wild-type p27Kip1 phosphorylated in 293T cells. The results
revealed that peptides 1 and 2, which include Ser10, contained only phosphoserine,
whereas peptide 6, which includes Ser178 and Thr187, contained phosphoserine
and, to a lesser extent, phosphothreonine (Fig. 5). The analysis also revealed that
peptide 5 was phosphorylated on serine and to a lesser extent on threonine,
whereas peptide 3 was phosphorylated on threonine and to a lesser extent on
serine. Peptides 4 and 7 contained exclusively phosphoserine (the
phosphorylation of peptide 7 was not detected in Fig. 4A, probably due to
experimental variation among culture condition of the cells).
Phosphorylation of p27Kip1 at Ser10 by a Proline-Directed Kinase—The Ser10
residue of p27Kip1 is located immediately upstream of a proline residue (Table 1)
and is therefore a potential target for proline-directed kinases such as MAPKs or
CDKs. Proline possesses a fixed, rigid conformation and serves to reduce the
flexibility of proteins at sites of its incorporation. We therefore constructed a p27Kip1
mutant (S10P/P11S, or PS) in which the positions of Ser10 and Pro11 were
reversed, in order to investigate whether the kinase responsible for
phosphorylation of Ser10 is a proline-directed kinase while minimizing any
introduced conformational change. Expression and metabolic labeling with 32P of
the PS mutant in 293T cells revealed that the extent of its phosphorylation was
about one-sixth of that of the wild-type protein (Fig. 6A). Phosphopeptide mapping
also revealed that the extent of phosphorylation of the peptides corresponding to
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 16
Ser10 (or Ser11 in the case of the mutant) was markedly greater for wild-type p27Kip1
than for the PS mutant (Fig. 6B). Of the proline-directed kinases important in cell
cycle control, MAPKs appeared more likely than did CDKs to be responsible for
phosphorylation of Ser10 of p27Kip1 because CDKs usually require a basic amino
acid immediately downstream of the Ser(Thr)-Pro sequence (38,39). Indeed,
p27Kip1 was phosphorylated by p42 MAPK (ERK2) in vitro, and the
phosphopeptide map of the protein so phosphorylated was similar to that of
p27Kip1 phosphorylated in vivo (Fig. 6C). In contrast, p27Kip1 was poorly
phosphorylated at Ser10 by recombinant cyclin E-CDK2 in vitro; rather, it was
preferentially phosphorylated on Thr187 by this kinase complex (data not shown).
These data suggested that a proline-directed kinase, possibly a member of the
MAPK family, phosphorylates p27Kip1 on Ser10.
We thus investigated the effect on p27Kip1 phosphorylation in vivo of
PD98059 (40), a specific inhibitor of MEK1 and MEK2, which phosphorylate and
thereby activate the MAPKs p44 (ERK1) and p42 (ERK2). Immunoblot analysis
with anti-p27Kip1 of 293T cells expressing wild-type p27Kip1 revealed that the lower-
mobility band of the p27Kip1 doublet, which corresponds to the form of the protein
phosphorylated on Ser10, was detected at similar intensities with cells cultured
with either dimethyl sulfoxide (DMSO) (vehicle control) or PD98059 (Fig. 6D). In
contrast, the phosphorylated forms of p42 and p44 MAPKs were detected in the
cells treated with DMSO but not in those treated with PD98059. These results
indicated that the MAPK isoforms p44 (ERK1) and p42 (ERK2) do not
phosphorylate p27Kip1 on Ser10 in vivo. It remains possible that other MAPKs, such
as ERK5, SAPK (or JNK), or p38 MAPK, may mediate the phosphorylation of
p27Kip1 on Ser10 in intact cells. Butyrolactone I (41), a potent inhibitor of CDK1,
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 17
CDK2, and CDK5, also did not affect the phosphorylation of p27Kip1 on Ser10 in
vivo (data not shown).
Effect of Mutation of Ser10 of p27Kip1 on CDK-Inhibitory Activity—We next
examined whether mutation of Ser10 of p27Kip1 affects the CDK-inhibitory activity of
the protein. A mutant p27Kip1 in which Ser10 was replaced with glutamic acid
(S10E), which mimics the negative charge of phosphate (42), was generated.
Bacterially expressed wild-type p27Kip1 and its S10A and S10E mutants were
subjected to an in vitro kinase assay either with cyclin E-CDK2 and its substrate
histone H1 (Fig. 7A) or with cyclin D2-CDK4 and its substrate Rb protein (Fig. 7B).
Each of the three p27Kip1 proteins inhibited the kinase activity of cyclin E-CDK2 or
cyclin D2-CDK4 to similar extents, suggesting that phosphorylation of p27Kip1 on
Ser10 does not affect the CDK-inhibitory function of the protein.
Effect of Phosphorylation of Ser10 on the Stability of p27Kip1 in Vitro and in
Vivo—Given that the p27Kip1 protein that accumulates in resting cells is highly
phosphorylated on Ser10 (Fig. 3), we compared the stability of phosphorylated and
unphosphorylated form of p27Kip1. Wild-type p27Kip1 and its S10A mutant were
expressed in 293T cells, and the lysates that contained both phosphorylated and
unphosphorylated forms of p27Kip1 protein were subjected to in vitro degradation
assay as described in Experimental Procedures. Phosphorylated p27Kip1 was
relatively stable compared with unphosphorylated form, whose kinetics of
degradation was similar to that of the S10A mutant (Fig. 8). The half-life of the
phosphorylated p27Kip1 was thus increased about twofold relative to that of the
unphosphorylated form or of the S10A mutant.
Furthermore, we examined the stability of wild-type p27Kip1 and its S10A
and S10E mutants in vitro and in vivo. We previously showed that p27Kip1 is
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 18
degraded in NIH 3T3 cell lysates in vitro and in vivo, by both ubiquitination-
dependent and -independent pathways, degradation by the latter pathway being
apparent by the generation of a 22-kDa intermediate (p27∆22k) (16). The stability
of the S10E mutant in this in vitro degradation assay was markedly increased
compared with those of the wild-type protein and the S10A mutant (Fig. 9).
However, the observation that both S10A and S10E mutants underwent
ubiquitination-independent cleavage suggests that phosphorylation of p27Kip1 on
Ser10 does not affect such cleavage. We also examined the stability of wild-type
p27Kip1 and its S10A and S10E mutants in intact transfected NIH 3T3 cells.
Consistent with the in vitro results, the stability of the S10E mutant was markedly
greater than that of either the wild-type protein or the S10A mutant (Fig. 10); the
half-life of the S10E mutant was thus increased more than twofold relative to that
of the wild-type protein. The S10A mutant appeared to be unstable compared with
the wild-type protein. The order of stability (S10E > wild-type > S10A) might be
explained by the possibility that the phosphorylated wild-type protein might be
rapidly dephosphorylated in cycling cells. Collectively, these data suggest that
phosphorylation of p27Kip1 on Ser10 contributes to regulation of the stability of this
protein.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 19
DISCUSSION
Regulation of the cell cycle at the G1-S boundary is thought to be important for the
control of cell proliferation. Kinase activity associated with two G1 cyclins, cyclins
D and E, is essential for this transition, predominantly because of the requirement
for phosphorylation of Rb and the consequent termination of its inhibition of cell
cycle progression (1,2). Among the mechanisms responsible for regulation of G1
cyclin-associated kinase activity, control of the abundance of p27Kip1 by external
mitogenic signals appears important (3,4). The amount of p27Kip1 is regulated
predominantly by posttranslational modification, which affects protein stability,
rather than by transcriptional control (14,15). The stability of p27Kip1 has thus been
shown to be affected by ubiquitin-dependent (14,25-27), ubiquitin-independent
(16), caspase-mediated (43,44), and Jab1-dependent (45) degradation.
The phosphorylation state of many proteins affects their stability, and
phosphorylation of p27Kip1 on Thr187 has been shown to be essential for binding of
Fbl1, an F-box protein component of an SCF ubiquitin ligase complex (25-27).
Thus, phosphorylation of Thr187 has been thought to be a central mechanism in
control of the stability of p27Kip1 by ubiquitin-mediated degradation. However, we
have now shown that the extent of phosphorylation of p27Kip1 on Thr187 represents
only ~1% of the total extent of phosphorylation of this protein in vivo. In contrast,
phosphorylation of Ser10 accounts for ~70% of the total extent of phosphorylation
of p27Kip1. Furthermore, the extent of phosphorylation at this site is increased in
resting cells, and Ser10 phosphorylation both affects protein stability and was
apparent in various types of cells from several species. These data suggest that
phosphorylation of Ser10 may represent another important mechanism by which
the stability of p27Kip1 is regulated. It is of note that the extent of phosphorylation of
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 20
Thr187 is almost certainly underestimated since this residue is phosphorylated
during a limited period of the cell cycle or if p27Kip1 phosphorylated at this site is
too unstable to be effectively detected by immunoblot analysis or labeling with 32P.
The observation that the abundance of p27Kip1 is increased in cells of Fbl1-
deficient mice (46) suggests that phosphorylation of Thr187 is indeed an important
determinant of this parameter. Although the degradation of p27Kip1 was slower in
Fbl1-deficient cells than in wild-type cells, the observation that a substantial extent
of p27Kip1 degradation was still apparent in these cells2 is consistent with the
existence of other pathways for p27Kip1 degradation.
The increased stability of the Ser10-phosphorylated form of p27Kip1 (Fig. 8)
and the S10E mutant, which mimics the Ser10-phosphorylated form of the protein
(Figs. 9 and 10), suggests that dephosphorylation of p27Kip1 at Ser10 might play an
important role in progression of the cell cycle from G0-G1 to S phase. However,
both the kinase and phosphatase responsible for the phosphorylation and
dephosphorylation at Ser10, respectively, as well as the mechanism by which
phosphorylation of Ser10 stabilizes p27Kip1, remain to be identified. It will also be
important to determine whether such regulation of p27Kip1 stability is linked to
external mitogenic signals. The stability of the protein IκBα is regulated by two
independent mechanisms: phosphorylation at sites near the NH2-terminus, which
is induced by external signals, and phosphorylation at sites near the COOH-
terminus, which controls the basal turnover rate (47). The signal-induced
phosphorylation of IκBα results in its targeting by the F-box protein Fbw1 (also
known as FWD1 or β-TrCP) and its consequent ubiquitination-dependent
degradation (20,48-50). The stability of p27Kip1 thus might also be subjected to
dual regulation by signal-induced phosphorylation at Thr187, which recruits the F-
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 21
box protein Fbl1 and results in ubiquitination-dependent degradation, and by
phosphorylation at Ser10.
The biochemical activity of p27Kip1 suggests that the protein functions as a
tumor suppressor. Indeed, mice lacking p27Kip1 are prone to spontaneous
tumorigenesis (10-12). Furthermore, mice that possess one normal allele of the
p27Kip1 gene develop tumors at a greatly increased frequency (compared with
wild-type animals) after exposure to chemical carcinogens or x-rays, without loss
of the functional p27Kip1 allele in the tumor cells (13). Although numerous clinical
studies have attempted to identify mutations within the p27Kip1 locus in individuals
with cancer, such mutations have proved to be extremely rare (51-59). Reduced
expression of p27Kip1 has nevertheless been correlated with poor prognosis in
cohorts of individuals with breast, colorectal, or stomach carcinoma (60-66).
Loda et al. (62) showed that tumors with low levels of p27Kip1 expression
exhibited relatively high rates of p27Kip1 degradation (and vice versa). It is
unlikely that this increased degradation of p27Kip1 was due to nonspecific
enhancement of general protein degradation, because degradation of neither
p21Cip1 nor cyclin A was affected in the same cancer patients. The mechanisms
that control the stability of p27Kip1 thus appear important in cancer development.
Characterization of these mechanisms should shed light on fundamental issues
such as how cell cycle regulation is linked to developmental control and how the
disturbance of cell cycle regulation results in carcinogenesis (and may lead to
the development of anticancer drugs with new modes of action).
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 22
ACKNOWLEDGEMENTS
We thank Dr. M. Nakanishi for the human p27Kip1 cDNA used in this study; S.
Hatakeyama, M. Matsumoto, N. Nishimura, and R. Yasukochi for technical
assistance; M. Kimura for secretarial assistance. This work was supported in
part by a grant from the Ministry of Education, Science, Sports and Culture of
Japan.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 23
REFERENCES
1. Morgan, D. O. (1995) Nature 374, 131-134
2. Sherr, C. J. (1996) Science 274, 1672-1677
3. Nakayama, K.-i., and Nakayama, K. (1998) BioEssays 20, 1020-1029
4. Sherr, J. C., and Roberts, J. M. (1999) Genes Dev. 13, 1501-1512
5. Polyak, K., Lee, M. H., Erdjument-Bromage, H., Koff, A., Roberts, J. M.,
Tempst, P., and Massague, J. (1994) Cell 78, 59-66
6. Toyoshima, H., and Hunter, T. (1994) Cell 78, 67-74
7. Nourse, J., Firpo, E., Flanagan, W. M., Coats, S., Polyak, K., Lee, M. H.,
Massague, J., Crabtree, G. R., and Roberts, J. M. (1994) Nature 372, 570-
573
8. Reynisdottir, I., Polyak, K., Iavarone, A., and Massague, J. (1995) Genes
Dev. 9, 1831-1845
9. Coats, S., Flanagan, W. M., Nourse, J., and Roberts, J. M. (1996) Science
272, 877-880
10. Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N.,
Horii, I., Loh, D. Y., and Nakayama, K.-i. (1996) Cell 85, 707-720
11. Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak,
K., Tsai, L. H., Broudy, V., Perlmutter, R. M., Kaushansky, K., and Roberts, J.
M. (1996) Cell 85, 733-744
12. Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V. C.,
Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A., and
Koff, A. (1996) Cell 85, 721-732
13. Fero, M. L., Randel, E., Gurley, K. E., Roberts, J. M., and Kemp, C. J. (1998)
Nature 396, 177-180
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 24
14. Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Del Sal, G.,
Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995) Science 269,
682-685
15. Hengst, L., and Reed, S. I. (1996) Science 271, 1861-1864
16. Shirane, M., Harumiya, Y., Ishida, N., Hirai, A., Miyamoto, C., Hatakeyama,
S., Nakayama, K.-i., and Kitagawa, M. (1999) J. Biol. Chem. 274, 13886-
13893
17. Feldman, R. M., Correll, C. C., Kaplan, K. B., and Deshaies, R. J. (1997) Cell
91, 221-230
18. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J., and Harper, J. W. (1997)
Cell 91, 209-219
19. Kominami, K., and Toda, T. (1997) Genes Dev. 11, 1548-1560
20. Hatakeyama, S., Kitagawa, M., Nakayama, K., Shirane, M., Matsumoto, M.,
Hattori, K., Higashi, H., Nakano, H., Okumura, K., Onoe, K., Good, R. A.,
and Nakayama, K.-i. (1999) Proc. Natl. Acad. Sci. USA 96, 3859-3863
21. Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M., Ishida, N.,
Hattori, K., Nakamichi, I., Kikuchi, A., Nakayama, K.-i., and Nakayama, K.
(1999) EMBO J. 18, 2401-2410
22. Vlach, J., Hennecke, S., and Amati, B. (1997) EMBO J. 16, 5334-5344
23. Sheaff, R. J., Groudine, M., Gordon, M., Roberts, J. M., and Clurman, B. E.
(1997) Genes Dev. 11, 1464-1478
24. Montagnoli, A., Fiore, F., Eytan, E., Carrano, A. C., Draetta, G. F., Hershko,
A., and Pagano, M. (1999) Genes Dev. 13, 1181-1189
25. Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H., and Zhang, H. (1999) Curr.
Biol. 9, 661-664
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 25
26. Carrano, A. C., Eytan, E., Hershko, A., and Pagano, M. (1999) Nature Cell
Biol. 1, 193-199
27. Sutterluty, H., Chatelain, E., Marti, A., Wirbelauer, C., Senften, M., Muller, U.,
and Krek, W. (1999) Nature Cell Biol. 1, 207-214
28. Kawada, M., Yamagoe, S., Murakami, Y., Suzuki, K., Mizuno, S., and
Uehara, Y. (1997) Oncogene 15, 629-637
29. Muller, D., Bouchard, C., Rudolph, B., Steiner, P., Stuckmann, I., Saffrich, R.,
Ansorge, W., Huttner, W., and Eilers, M. (1997) Oncogene 15, 2561-2576
30. Vlach, J., Hennecke, S., Alevizopoulos, K., Conti, D., and Amati, B. (1996)
EMBO J. 15, 6595-6604
31. Mann, D. J., Child, E. S., Swanton, C., Laman, H., and Jones, N. (1999)
EMBO J. 18, 654-663
32. Ellis, M., Chew, Y. P., Fallis, L., Freddersdorf, S., Boshoff, C., Weiss, R. A.,
Lu, X., and Mittnacht, S. (1999) EMBO J. 18, 644-653
33. Fang, G., Yu, H., and Kirschner, M. W. (1998) Genes Dev. 12, 1871-1883
34. Hattori, K., Hatakeyama, S., Shirane, M., Matsumoto, M., and Nakayama,
K.-i. (1999) J. Biol. Chem. 274, 29641-29647
35. Boyle, W. J., Van Der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201,
110-148
36. O'Farrel, Z. P., Goodman, M. H., and O'Farrel, H. P. (1977) Cell 12, 1133-
1142
37. Kung, L. A., Sherwood, W. S., and Schimke, T. R. (1990) Proc. Natl. Acad.
Sci. USA 87, 9553-9557
38. Kitagawa, M., Higashi, H., Jung, H. K., Suzuki-Takahashi, I., Ikeda, M.,
Tamai, K., Kato, J., Segawa, K., Yoshida, E., Nishimura, S., and Taya, Y.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 26
(1996) EMBO J. 15, 7060-7069
39. Kitagawa, M., Higashi, H., Takahashi, I. S., Okabe, T., Ogino, H., Taya, Y.,
Hishimura, S., and Okuyama, A. (1994) Oncogene 9, 2549-2557
40. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995)
Proc. Natl. Acad. Sci. USA 92, 7686-7689
41. Kitagawa, M., Okabe, T., Ogino, H., Matsumoto, H., Suzuki-Takahashi, I.,
Kokubo, T., Higashi, H., Saitoh, S., Taya, Y., Yasuda, H., Ohba, Y.,
Nishimura, S., and Okuyama, A. (1993) Oncogene 8, 2425-2432
42. Maciejewski, P. M., Peterson, F. C., Anderson, P. J., and Brooks, C. L.
(1995) J. Biol. Chem. 270, 27661-27665
43. Loubat, A., Rochet, N., Rezzonico, R., Far, D. F., Auberger, P., Rossi, B.,
and Ponzio, G. (1999) Oncogene 18, 3324-3333
44. Levkau, B., Koyama, H., Raines, E. W., Clurman, B. E., Herren, B., Orth, K.,
Roberts, J. M., and Ross, R. (1998) Mol. Cell 1, 553-563
45. Tomoda, K., Kubota, Y., and Kato, J. (1999) Nature 398, 160-165
46. Nakayama, K., Nagahama, H., Minamishima, Y. A., Matsumoto, M.,
Nakamichi, I., Kitagawa, K., Shirane, M., Tsunematsu, R., Tsukiyama, T.,
Ishida, N., Kitagawa, M., Nakayama, K.-i., and Hatakeyama, S. (2000)
EMBO J. 19, 2069-2081
47. Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16,
225-260
48. Yaron, A., Hatzubai, A., Davis, M., Lavon, I., Amit, S., Manning, A. M.,
Andersen, J. S., Mann, M., Mercurio, F., and Ben-Neriah, Y. (1998) Nature
396, 590-594
49. Winston, J. T., Strack, P., Beer-Romero, P., Chu, C. Y., Elledge, S. J., and
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 27
Harper, J. W. (1999) Genes Dev. 13, 270-283
50. Spencer, E., Jiang, J., and Chen, Z. J. (1999) Genes Dev. 13, 284-294
51. Ponce-Castaneda, M. V., Lee, M. H., Latres, E., Polyak, K., Lacombe, L.,
Montgomery, K., Mathew, S., Krauter, K., Sheinfeld, J., Massague, J., and
Cordon-Cardo, C. (1995) Cancer Res. 55, 1211-1214
52. Pietenpol, J. A., Bohlander, S. K., Sato, Y., Papadopoulos, N., Liu, B.,
Friedman, C., Trask, B. J., Roberts, J. M., Kinzler, K. W., Rowley, J. D., and
Vogelstein, B. (1995) Cancer Res. 55, 1206-1210
53. Kawamata, N., Seriu, T., Koeffler, H. P., and Bartram, C. R. (1996) Cancer
77, 570-575
54. Kawamata, N., Morosetti, R., Miller, C. W., Park, D., Spirin, K. S., Nakamaki,
T., Takeuchi, S., Hatta, Y., Simpson, J., Wilcyznski, S., Lee, Y. Y., Bartram,
C. R., and Koeffler, H. P. (1995) Cancer Res. 55, 2266-2269
55. Seriu, T., Erz, D., and Bartram, C. R. (1996) Leukemia 10, 345
56. Stegmaier, K., Takeuchi, S., Golub, T. R., Bohlander, S. K., Bartram, C. R.,
and Koeffler, H. P. (1996) Cancer Res. 56, 1413-1417
57. Ferrando, A. A., Balbin, M., Pendas, A. M., Vizoso, F., Velasco, G., and
Lopez-Otin, C. (1996) Hum. Genet. 97, 91-94
58. Spirin, K. S., Simpson, J. F., Takeuchi, S., Kawamata, N., Miller, C. W., and
Koeffler, H. P. (1996) Cancer Res. 56, 2400-2404
59. Morosetti, R., Kawamata, N., Gombart, A. F., Miller, C. W., Hatta, Y., Hirama,
T., Said, J. W., Tomonaga, M., and Koeffler, H. P. (1995) Blood 86, 1924-
1930
60. Porter, P. L., Malone, K. E., Heagerty, P. J., Alexander, G. M., Gatti, L. A.,
Firpo, E. J., Daling, J. R., and Roberts, J. M. (1997) Nature Med. 3, 222-225
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 28
61. Catzavelos, C., Bhattacharya, N., Ung, Y. C., Wilson, J. A., Roncari, L.,
Sandhu, C., Shaw, P., Yeger, H., Morava-Protzner, I., Kapusta, L., Franssen,
E., Pritchard, K. I., and Slingerland, J. M. (1997) Nature Med. 3, 227-230
62. Loda, M., Cukor, B., Tam, S. W., Lavin, P., Fiorentino, M., Draetta, G. F.,
Jessup, J. M., and Pagano, M. (1997) Nature Med. 3, 231-234
63. Mori, M., Mimori, K., Shiraishi, T., Tanaka, S., Ueo, H., Sugimachi, K., and
Akiyoshi, T. (1997) Nature Med. 3, 593
64. Fredersdorf, S., Burns, J., Milne, A. M., Packham, G., Fallis, L., Gillett, C. E.,
Royds, J. A., Peston, D., Hall, P. A., Hanby, A. M., Barnes, D. M., Shousha,
S., O'Hare, M. J., and Lu, X. (1997) Proc. Natl. Acad. Sci. USA 94, 6380-6385
65. Tan, P., Cady, B., Wanner, M., Worland, P., Cukor, B., Magi-Galluzzi, C.,
Lavin, P., Draetta, G., Pagano, M., and Loda, M. (1997) Cancer Res. 57,
1259-1263
66. Kawana, H., Tamaru, J., Tanaka, T., Hirai, A., Saito, Y., Kitagawa, M.,
Mikata, A., Harigaya, K., and Kuriyama, T. (1998) Am. J. Pathol. 153, 505-
513
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 29
FOOTNOTES
1Abbreviations: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; MAPK,
mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum; GST, glutathione S-transferase; CIAP, calf intestinal
alkaline phosphatase; PAGE, polyacrylamide gel electrophoresis; anti-,
antibodies to; TLC, thin-layer chromatography; 2D-PAGE, two-dimensional
PAGE; NEPHGE, nonequilibrium pH gradient electrophoresis; DMSO, dimethyl
sulfoxide.
2M. Kitagawa et al., in preparation.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 30
FIGURE LEGENDS
Fig. 1. Effects of mutation of Ser10, Ser178, and Thr187 of p27Kip1 to Ala on the
extent of protein phosphorylation in vivo. (A) Schematic representation of the
structure of human p27Kip1 (198 amino acids) showing the positions of residues
mutated in the present study. The cyclin binding domain, CDK binding domain,
and nuclear localization signal (NLS) are indicated. (B and C) Flag-tagged wild-
type (WT) p27Kip1 or S10A, S178A, T187A, or S10A/S178A/T187A (triple) mutants
of p27Kip1 were transiently expressed in 293T cells and metabolically labeled by
incubation of cells with [32P]Pi. Cell lysates (3 mg of protein) were then subjected
to immunoprecipitation (IP) with anti-Flag (α-Flag), and the resulting precipitates
were subjected to autoradiography (upper panel) or to immunoblot analysis (IB)
with anti-p27Kip1 (α-p27) (lower panel) (B). The extent of 32P incorporation into
wild-type and mutant p27Kip1 proteins was then quantified with a BAS-2000 image
analyzer and normalized by the abundance of p27Kip1 revealed by immunoblot
analysis (C). The normalized incorporation of 32P into the wild-type protein is
defined as 100%. Data are from an experiment that was repeated three times with
similar results.
Fig. 2. Electrophoretic mobility shift of p27Kip1 caused by phosphorylation of
Ser10. (A) 293T, HeLa, COS-7, or NIH 3T3 cells were transfected with empty
expression plasmid alone (mock) or plasmids encoding either Flag-tagged wild-
type p27Kip1 or its S10A mutant. Cell lysates (from 10 to 125 µg of protein) were
subjected to immunoblot analysis with anti-Flag. Bands corresponding to Flag-
tagged unphosphorylated and phosphorylated p27Kip1 are indicated by Flag-p27
and Flag-pp27, respectively. (B) Flag-tagged wild-type p27Kip1 and its S10A
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 31
mutant were immunoprecipitated from transfected 293T cells with anti-Flag, and
the resulting immunoprecipitates were incubated for 5 h at 37°C in the absence
(–) or presence (+) of CIAP. The samples were then subjected to immunoblot
analysis with anti-p27Kip1.
Fig. 3. Cell cycle-dependent phosphorylation of p27Kip1 on Ser10. (A) Flag-
tagged wild-type p27Kip1 (upper panel) or its S10A mutant (lower panel) was
expressed in NIH 3T3 cells, and cell lysates (200 µg of protein) were subjected to
2D-PAGE and immunoblot analysis with anti-p27Kip1. The directions of
electrophoresis (arrows) as well as the positions corresponding to transfected
(exo) and endogenous (endo) p27Kip1 are indicated. (B) Lysates (50 µg of protein)
of HeLa or NIH 3T3 cells synchronized in G0-G1, S, or M phases of the cell cycle
were subjected to immunoblot analysis with either anti-p27Kip1 (upper panel) or
anti-α-tubulin (lower panel). (C) Lysates of HeLa or NIH 3T3 cells synchronized in
G0-G1 (upper panels), S (middle panels), or M (lower panel) phase were subjected
to 2D-PAGE and immunoblot analysis with anti-p27Kip1. The amount of lysate
protein analyzed was varied from 150 to 500 µg in order to ensure that the
amounts of endogenous p27Kip1 were similar at the different phases of the cell
cycle. The blots of lysates from cells in S or M phases were overexposed. The
positions corresponding to unphosphorylated and phosphorylated p27Kip1 are
indicated, as are the amounts of each of these two forms of the protein expressed
as a percentage of total p27Kip1 (determined by image analysis with NIH Image
software).
Fig. 4. Two-dimensional tryptic phosphopeptide mapping of wild-type and
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 32
various mutant p27Kip1 proteins. (A) Wild-type (tagged or not with the Flag
epitope) or S10A, S178A, T187A, or S10A/S178A/T187A mutants of p27Kip1 were
expressed in 293T cells and metabolically labeled with [32P]Pi. The recombinant
proteins were immunoprecipitated with anti-Flag or anti-p27Kip1, and the resulting
immunoprecipitates were subjected to two-dimensional tryptic phosphopeptide
mapping. Major phosphopeptides are numbered 1 to 6. Phosphopeptides
containing Ser10 are indicated by open arrowheads, and those containing Ser178
and Thr187 are indicated by filled arrowheads. The origin of migration is indicated
by an asterisk, and the directions of separation by TLC and electrophoresis are
shown by arrows. (B) The relative incorporation of 32P by Ser10, Ser178, and Thr187
of p27Kip1 was estimated by image analysis of autoradiographs of phosphopeptide
maps. The extent of 32P incorporation by Ser10 was defined as 100%. Because
Ser178 and Thr187 are both present in the same tryptic peptide, the incorporation of
32P at each site was calculated from the difference in incorporation into spot 6
[filled arrowheads in (A)] between wild-type and either S178A or T187A,
respectively. Data are from an experiment that was repeated twice with similar
results.
Fig. 5. Phosphoamino acid analysis of p27Kip1. Tryptic phosphopeptides
derived from wild-type p27Kip1 expressed in 293T cells were subjected to
phosphoamino acid analysis. (Left panel) Two-dimensional phosphopeptide map.
Major phosphopeptides are numbered 1 to 7. (Right panels) The upper leftmost of
the smaller panels shows a schematic representation of the results of
phosphoamino acid analysis, with the positions of phosphoserine,
phosphothreonine, and phosphotyrosine indicated. Panels labeled 1 to 7
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 33
correspond to the results of phosphoamino acid analysis of the corresponding
phosphopeptides. Phosphoamino acids that were not detected are indicated by
dotted outlines. Spots 1 and 2 contain phosphorylated Ser10, and spot 6 contains
phosphorylated Ser178 and Thr187. The directions of phosphoamino acid separation
by electrophoresis at pH 1.9 and pH 3.5 are indicated by arrows.
Fig. 6. Role of a proline-directed kinase in the phosphorylation of p27Kip1 on
Ser10. (A) 293T cells transiently expressing Flag-tagged wild-type or the PS
mutant of p27Kip1 were metabolically labeled with [32P]Pi, lysed, and subjected to
immunoprecipitation with anti-Flag. The resulting precipitates were then analyzed
by SDS-PAGE and autoradiography. (B) Flag-tagged wild-type or the PS mutant
of p27Kip1 was immunoprecipitated from [32P]Pi-labeled transfected 293T cells with
anti-Flag and subjected to two-dimensional phosphopeptide mapping. The spots
corresponding to the phosphopeptides containing Ser10 (or Ser11 in the case of the
mutant) are indicated by open arrowheads. (C) The phosphopeptide map of wild-
type p27Kip1 phosphorylated in vivo (left panel) as in (B) was compared with that of
bacterially expressed wild-type p27Kip1 phosphorylated in vitro with purified p42
MAPK in the presence of [γ-32P]ATP (center panel). The identities of the spots in
the two maps were confirmed by mixing the two samples before mapping (right
panel). (D) 293T cells expressing recombinant wild-type p27Kip1 or its S10A mutant
were incubated for 5 h with 50 µM PD98059 (New England Biolabs Inc.) or 0.1 %
(v/v) DMSO (vehicle control), after which the cells were lysed and subjected to
immunoblot analysis with either anti-p27Kip1 (upper panel) or antibodies to
phosphorylated MAPK (lower panel). The positions corresponding to Flag-tagged
unphosphorylated and phosphorylated p27Kip1 as well as to phosphorylated p44
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 34
and p42 MAPKs are indicated.
Fig. 7. Effect of mutation of Ser10 of p27Kip1 on the CDK-inhibitory activity of
the protein. Wild-type p27Kip1 and its S10A and S10E mutants expressed in and
purified from bacteria were incubated in the indicated amounts in in vitro kinase
assays either with histone H1 and recombinant cyclin E-CDK2 (E-K2) (A) or with
Rb protein and cyclin D2-CDK4 (D2-K4) (B). The reaction mixtures were then
subjected to SDS-PAGE and autoradiography. The positions corresponding to
histone H1 (HH1) and Rb are indicated.
Fig. 8. Effect of phosphorylation of Ser10 of the stability of p27Kip1. (A) Flag-
tagged wild-type p27Kip1 and its S10A mutants were expressed in 293T cells and
their lysates were subjected to an in vitro degradation assay for the indicated
times. Subsequently, the reaction mixtures were subjected to immunoblot analysis
with anti-p27Kip1. The positions corresponding to Flag-tagged unphosphorylated
and phosphorylated p27Kip1 are indicated as Flag-p27 and Flag-pp27, respectively.
(B) The intensities of the bands corresponding to phosphorylated wild-type p27Kip1
(open diamonds) and unphosphorylated wild-type p27Kip1 (filled squares) and
S10A mutant (filled circles) in the immunoblots shown in (A) were quantified and
expressed as a percentage of the corresponding value at time zero. Data are from
an experiment that was repeated two times with similar results.
Fig. 9. Effect of mutation of Ser10 on the stability of p27Kip1 in vitro. (A) Wild-
type p27Kip1 and its S10A and S10E mutants were expressed in and purified from
bacteria and then subjected for the indicated times to an in vitro degradation
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 35
assay with NIH 3T3 cell lysate. The reaction mixtures were analyzed by
immunoblotting with anti-p27Kip1. The positions corresponding to
unphosphorylated and phosphorylated p27Kip1 as well as to the p27∆22k cleavage
product are indicated. (B) The intensities of the bands corresponding to full-length
wild-type p27Kip1 (open diamonds) and its S10A (filled squares) and S10E (filled
circles) mutants in the immunoblots shown in (A) were quantified and expressed
as a percentage of the corresponding value at time zero. Data are from an
experiment that was repeated three times with similar results.
Fig. 10. Pulse-chase analysis of the stability of Ser10 mutants of p27Kip1 in
vivo. (A) NIH 3T3 cells transfected with vectors encoding wild-type p27Kip1 or its
S10A or S10E mutants were pulse-labeled with [35S]methionine and [35S]cysteine,
and then incubated in the absence of isotope for the indicated chase periods. Cell
lysates were then subjected to immunoprecipitation with anti-p27Kip1, and the
resulting precipitates were subjected to SDS-PAGE, autoradiography, and
scanning densitometry. (B) The intensities of the bands corresponding to wild-
type p27Kip1 (open diamonds) and its S10A (filled squares) and S10E (filled circles)
mutants in the autoradiograms shown in (A) were quantified and expressed as a
percentage of the corresponding value for the beginning of the chase period (time
0). Data are from an experiment that was repeated twice with similar results.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ishida et al. 36
TABLE LEGEND
Table 1. Tryptic peptides of p27Kip1 that contain serine or threonine. Serine
and threonine residues are shown in bold; those immediately upstream of a
proline residue are double-underlined.
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig.1
34.5%29.3%
110.1%100% 102.4%
0
20
40
60
80
100
WT S10A S178A T187A Triple
C
IP: α-Flag
IB: α-p27
B
A CDK binding NLS
198 a.a.p27
S10A S178AT187A
S10P/P11S
Cyclin binding
S10E
32P-p27
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 2
A
B
293T HeLa COS-7 NIH 3T3
Flag-pp27Flag-p27
Wild-type S10A
CIAP - + - +Flag-pp27Flag-p27
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 3
BA
C
p27
α-Tubulin
HeLa NIH 3T3
WT
S10A
Flag-pp27Flag-p27
pp27p27
Exo Endo
NEPHGESDS-PAGE
pp27 p27
G0-G1
S
M
NIH 3T3HeLa
pp27 p27
77.1% 22.9%
0.1% 99.9%
16.0% 84.0%
47.1% 52.9%
22.0% 78.0%
NEPHGESDS-PAGE
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 4
100%
3.7% 1.3%0
20
40
60
80
100
Ser10 Ser178 Thr187
A
B
S10A
S10A/S178A/T187AT187AS178A
*
TLC
Electrophoresis(+) (-)*
*
*
6 (S178,T187)
12
3 4
WT(no tag)
**
5
WT(Flag tag)
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig.5
WT p27
TLC
Electrophoresis (+) (-)
12
34
5
67
P-Ser P-Thr
P-Tyr
1P-Ser
2P-Ser
4P-Ser
5
P-ThrP-Ser
6
P-ThrP-Ser
7P-Ser
3
P-ThrP-Ser
pH 1.9(+) (-)
pH 3.5
(-)
(+)
*
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 6
A B
C
32P-p27
IP: α-Flag
D
α−p27 Flag-pp27
Flag-p27
α−pMAPK pp44 MAPKpp42 MAPK
p27 (WT) p27 (PS )
TLC
Electrophoresis (+) (-)**
In vivo In vitro In vivo + in vitro
TLC
Electrophoresis (+) (-)* **
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 7
WT
S10A
S10E
HH1
HH1
HH1
p27 (µg):
E-K2 (+) (-)A
Rb
Rb
Rb
D2-K4 (+) (-)
p27 (µg):
B
WT
S10A
S10E by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
0 1 2Time (h)
Flag-pp27Flag-p27
Flag-p27
WT
S10A
4
A
B
Fig. 8
100
80
60
40
20
02 4
Reaction time (h)
WT(pp27)
WT(p27)
S10A
0
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 9
B
AS10A WT S10E
Time (min): 0 5 10 20 40 0 5 10 20 40 0 5 10 20 40
pp27p27p27∆22k
20
40
60
100
010 20 30 40
Time (min)
S10AWTS10E
80
0
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 10
B
A
WT
S10A
S10E
100
80
60
40
20
06 12
Chase time (h)0
0 3 6 12
Chase time (h)
S10E
WT
S10A
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Table 1
MSNVRVSNGSPSLERPSACRNLFGPVDHEELTRDMEEASQRGSLPEFYYRVPAQESQDVSGSRPAAPLIGAPANSEDTHLVDPKTDPSDSQTGLAEQCAGIRPATDDSSTQNKTEENVSDGSPNAGSVEQTPKQT
Peptide Serine
27,10,1227
5683106,110,112125138,140160,161175,178,183
Threonine
42
128135,142157,162170,187198
Position
1-56-1526-3031-4351-5882-90101-113114-134135-152155-165170-189197-198
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Noriko Ishida, Masatoshi Kitagawa, Shigetsugu Hatakeyama and Kei-ichi Nakayamaits protein stability
, increasesKip1Phosphorylation at serine-10, a major phosphorylation site of p27
published online May 30, 2000J. Biol. Chem.
10.1074/jbc.M001144200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on April 3, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Top Related