phosphorylation at serine-10, a major phosphorylation site of

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

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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.

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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


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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


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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.


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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


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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


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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


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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.


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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.


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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


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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


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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


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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


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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


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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,


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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


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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.


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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


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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-


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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).


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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.


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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.


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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


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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


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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


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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


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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


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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.


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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.


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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


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Fig. 2

A

B

293T HeLa COS-7 NIH 3T3

Flag-pp27Flag-p27

Wild-type S10A

CIAP - + - +Flag-pp27Flag-p27


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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


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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)


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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

(-)

(+)

*


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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 (+) (-)* **


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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

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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


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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


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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


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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


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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:

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phosphorylation at serine-10, a major phosphorylation site of

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