Degradation of p27 Kip1 at the G 0 -G 1 transition mediated · The CKI p27 Kip1 plays a pivotal role in the control of cell proliferation. Transition from G 0 phase to S phase of

Hara et al. 1

Degradation of p27Kip1 at the G0-G1 transition mediated

by a Skp2-independent ubiquitination pathway

Taichi Hara,1,2 Takumi Kamura,1,2 Keiko Nakayama,2,3 Kiyotaka Oshikawa,2,3

Shigetsugu Hatakeyama,1,2 and Kei-Ichi Nakayama1,2,3,4

1Department of Molecular and Cellular Biology and 3Department of Molecular

Genetics, Medical Institute of Bioregulation, Kyushu University, 3-1-1

Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.

2CREST, Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi,

Saitama 332-0012, Japan.

4To whom correspondence should be addressed: Dr. 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]

Running title: Skp2-independent degradation of p27Kip1

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 26, 2001 as Manuscript M107274200 by guest on July 4, 2020

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ABSTRACT

Targeting of the cyclin-dependent kinase inhibitor p27Kip1 for proteolysis has been

thought to be mediated by Skp2, the F-box protein component of an SCF

ubiquitin ligase complex. Degradation of p27Kip1 at the G0-G1 transition of the cell

cycle has now been shown to proceed normally in Skp2–/– lymphocytes, whereas

p27Kip1 proteolysis during S-G2 phases is impaired in these Skp2-deficient cells.

Degradation of p27Kip1 at the G0-G1 transition was blocked by lactacystin, a

specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-

proteasome pathway. The first cell cycle of stimulated Skp2–/– lymphocytes

appeared normal, but the second cycle was markedly inhibited, presumably as a

result of p27Kip1 accumulation during S-G2 phases of the first cell cycle.

Polyubiquitination of p27Kip1 in the nucleus is dependent on Skp2 and

phosphorylation of p27Kip1 on threonine-187. However, polyubiquitination activity

was also detected in the cytoplasm of Skp2–/– cells, even with a threonine-187 →

alanine mutant of p27Kip1 as substrate. These results suggest that a

polyubiquitination activity in the cytoplasm contributes to the early phase of

p27Kip1 degradation in a Skp2-independent manner, thereby promoting cell cycle

progression from G0 to G1.

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INTRODUCTION

Progression of the cell cycle in eukaryotic cells depends on the activity of a series

of protein complexes composed of cyclins and cyclin-dependent kinases (CDKs).1

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 (3,4).

A key question with regard to regulation of the cell cycle concerns the

mechanism by which cells undergo the transition from the resting state (G0) to

proliferation. The CKI p27Kip1 plays a pivotal role in the control of cell proliferation.

Transition from G0 phase to S phase of the cell cycle is promoted by complexes of

G1 cyclins with CDKs, and p27Kip1 inhibits the activity of these complexes directly

by binding to them (5). In normal cells, the amount of p27Kip1 is high during G0

phase, but it rapidly decreases on reentry into G1-S phases triggered by specific

mitogenic factors (6,7). Forced expression of p27Kip1 results in cell cycle arrest in

G1 phase (8,9); conversely, inhibition of p27Kip1 expression by antisense


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oligonucleotides increases the number of cells in S phase (10). Moreover, we and

others have demonstrated that mice homozygous for deletion of the p27Kip1 gene

are larger than normal mice, and that they exhibit multiple organ hyperplasia as

well as a predisposition to both spontaneous and radiation- or chemical-induced

tumors (11-14).

The ubiquitin-proteasome pathway plays an important role in the

degradation of short-lived regulatory proteins, including those that participate in

the cell cycle, cellular signaling in response to stress and to extracellular signals,

morphogenesis, the secretory pathway, DNA repair, and organelle biogenesis (15).

The concentration of p27Kip1 is thought to be regulated predominantly by this

proteolytic pathway (16-18). The ubiquitin-mediated proteolysis of many proteins

is regulated by phosphorylation of the target, which increases its susceptibility to

degradation (19-22). The proteolysis of p27Kip1 may also be regulated by such a

mechanism, given that degradation of the protein is promoted by its

phosphorylation on Thr187 by the cyclin E–CDK2 complex (23-25). Recent data

have also suggested that Skp2, an F-box protein that is thought to function as the

receptor component of an SCF ubiquitin ligase complex, binds to p27Kip1 in

conjunction with Cks1 only when Thr187 of p27Kip1 is phosphorylated; such binding

then results in the ubiquitination and degradation of p27Kip1 (26-30). These

biochemical observations are supported by genetic evidence that p27Kip1


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accumulates to high levels in the cells of mice that lack either Skp2 or Cks1 (29-

31).

We now show that, although the degradation of p27Kip1 during S and G2

phases of the cell cycle is markedly impaired in Skp2–/– cells, the degradation of

this protein at the G0-G1 transition occurs normally in these cells. Our data

suggest the existence of a second pathway of p27Kip1 degradation that is also

mediated by the ubiquitin-proteasome system. This second pathway may be

critical for regulation of cell cycle progression from the resting state to proliferation,

and may be important in mechanisms of carcinogenesis.


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EXPERIMENTAL PROCEDURES

Immunoblot analysis

Single-cell suspensions of lymphocytes were prepared from the lymph nodes of

4-month-old Skp2+/+ or Skp2–/– mice and were cultured (1.0 × 107 cells in 5 ml) for

the indicated times in RPMI 1640 medium supplemented with 10% fetal bovine

serum, 10 nM phorbol 12,13-dibutyrate (PDBu) (Sigma), and 300 nM ionomycin

(Sigma). In some experiments, 10 µM lactacystin (Kyowa Medics), 10 µM

MG132 (Peptide Institute), or vehicle (dimethyl sulfoxide) was added to the

culture medium. The cells were subsequently harvested and lysed in 50 µl of

RIPA buffer containing 0.4 mM Na3VO4, 0.4 mM EDTA, 10 mM NaF, 10 mM

sodium pyrophosphate, and antipain, pepstatin, chymostatin, leupeptin, and

phenylmethylsulfonyl fluoride each at a concentration of 10 µg/ml. The lysates

were incubated on ice for 15 min and then centrifuged at 20,000 × g. After

determination of its protein concentration with the Bradford assay (Bio-Rad), the

resulting supernatant (30 µg of protein) was subjected to SDS–polyacrylamide

gel electrophoresis (PAGE). The separated proteins were transferred to a

Hybond P membrane (Amersham Pharmacia Biotech) and subjected to

immunoblot analysis with antibodies (1 µg/ml) to p27Kip1 (Transduction

Laboratories), to cyclin D2 (Santa Cruz Biotechnology), to cyclin E (Santa Cruz


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Biotechnology), to Skp2 (Zymed), or to glycogen synthase kinase–3β (GSK-3β)

(Transduction Laboratories). Immune complexes were detected with appropriate

horseradish peroxidase–conjugated secondary antibodies and either

SuperSignal West Pico or SuperSignal West Dura chemiluminescence reagents

(Pierce).

Cell cycle analysis by flow cytometry

Single-cell suspensions of lymphocytes were cultured as described above. Cells

were exposed to 10 µM bromodeoxyuridine (BrdU) (Sigma) for 30 min before

harvesting, and were then subjected to fixation overnight in 70% ethanol at

–20°C followed by denaturation for 30 min at room temperature in 2 M HCl

containing 0.5% Triton X-100. After neutralization with borax buffer (pH 8.5), the

cells were subjected to dual-color staining with fluorescein isothiocyanate

(FITC)–conjugated antibodies to BrdU (Becton Dickinson) and propidium iodide

(5 µg/ml). The cells were then analyzed with a FACSCalibur flow cytometer and

Cell Quest software (Becton Dickinson).

RT-PCR analysis of immediate-early gene expression

Total RNA (1 µg) extracted from lymph node cells (2.0 × 107) with the use of

ISOGEN (Wako) was subjected to reverse transcription (RT) with ReverTra


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Dash (Toyobo) in a final volume of 20 µl. The resulting cDNA (1 µl) was amplified

by the polymerase chain reaction (PCR) in a final volume of 50 µl of PCR

reaction mix. The respective sequences of sense and antisense primers specific

for the target genes were as follows: c-Myc, 5'-AGT GCA TTG ATC CCT CAG

TGG TCT TTC CCT A-3' and 5'-CAG CTC GTT CCT CCT CTG ACG TTC CAA

GAC GTT-3'; c-Fos, 5'-GAG CTG ACA GAT ACA CTC CAA GCG-3' and 5'-CAG

TCT GCT GCA TAG AAG GAA CCG-3'; and c-Jun, 5'-GCA TGA GGA ACC

GCA TTG CCG CCT CCA AGT-3' and 5'-CGC AAA GTC TGC CGG CCA ATA

GGC CGC T-3'. The sense and antisense primers targeted to the gene for

glyceraldehyde-3-phosphate dehydrogenase (G3PDH), used as an internal

standard, were 5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCC ACC ACC

CTG TTG CTG TA-3', respectively. PCR products were separated by

electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide

staining.

Production of recombinant proteins in bacteria

Glutathione S-transferase (GST) fusion proteins of mouse p27Kip1 and the T187A

mutant of p27Kip1 [each of which was tagged at its COOH-terminus with the

hexahistidine (His6) epitope] as well as a GST fusion protein of mouse Cks1

were expressed in Escherichia coli strain XL1-blue (Stratagene); the bacteria


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were cultured in the presence of 0.1 mM isopropyl-β-D-thiogalactopyranoside.

Bacterial cells were resuspended in phosphate-buffered saline (PBS) and lysed

by sonication, and cellular debris was removed by centrifugation for 20 min at

13,000 × g. Glutathione–Sepharose CL-4B beads (Amersham Pharmacia

Biotech) were added to the resulting supernatant, and the mixture was rotated

for 4 h at 4°C. The beads were washed with PBS, and the GST fusion proteins

attached to the beads were subjected to digestion for 4 h at 4°C with PreScission

protease (Amersham Pharmacia Biotech) in a solution containing 50 mM Tris-

HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT). Only

the processed recombinant proteins (not the GST moiety nor the GST fusion

proteins) were released from the beads, as revealed by SDS-PAGE and

Coomassie blue staining (data not shown).

Human Ubc5a tagged at its NH2- and COOH-termini with the His6 and

Flag epitopes, respectively, human Ubc3 tagged at its NH2-terminus with the His6

epitope, Saccharomyces cerevisiae Uba1 tagged at its NH2- and COOH-termini

with the Myc and His6 epitopes, respectively, and a GST fusion protein of mouse

ubiquitin were expressed in E. coli strain BL21(DE3)pLysS (Novagen); the

bacteria were cultured in the presence of 0.1 mM isopropyl-β-D-

thiogalactopyranoside. The recombinant proteins were purified with the use of

ProBond resin (Invitrogen) or glutathione–Sepharose CL-4B. After dialysis


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against a solution containing 40 mM Hepes-NaOH (pH 7.6), 60 mM NaCl, 1 mM

DTT, and 10% glycerol, the proteins were stored at –80°C.

Baculovirus expression system

Baculoviruses were generated in Sf9 cells with the use of BacPAK6 virus DNA

and the pBacPAK9-GST vector (Clontech) containing either the cDNA for a GST

fusion protein of human cyclin E tagged at its NH2-terminus with the Myc epitope

or the cDNA for human CDK2 tagged at its NH2-terminus with the hemagglutinin

epitope. The GST-cyclin E–CDK2 recombinant protein complex was purified

(according to the protocol described above for purification of GST fusion proteins

expressed in bacteria) from a lysate of Sf9 cells coinfected with the two viruses.

Only the purified recombinant proteins were detected by electrophoresis and

Coomassie blue staining.

In vitro ubiquitination assay

Primary mouse embryonic fibroblasts (MEFs) were derived from 13.5-day-

postcoitum Skp2+/+ or Skp2–/– embryos and cultured as previously described (11).

In all experiments of this study, we used nonsenescent MEFs (no more than

passage 2). In some experiments, the cell cycle of MEFs was synchronized

either at G0-G1 by serum deprivation for 96 h in medium supplemented with 0.1%


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fetal bovine serum, or at S-G2 by arrest at the G1-S boundary induced by

incubation with aphidicolin (1 µg/ml) for 14 h followed by culture for 3 h in

aphidicolin-free medium. The cells (1.0 × 108) were washed with PBS and then

suspended in 500 µl of buffer A [40 mM Hepes-NaOH (pH 7.6), 1 mM DTT, and

protease inhibitor mixture (antipain, pepstatin, chymostatin, leupeptin, and

phenylmethylsulfonyl fluoride each at 10 µg/ml)] containing 60 mM NaCl. For

preparation of nuclear and cytoplasmic extracts, the cell suspension was frozen

and thawed, and then subjected to centrifugation at 2000 x g for 5 min at 4°C.

The resulting nuclear pellet was resuspended in 300 µl of ice-cold buffer

A containing 300 mM NaCl, rotated for 30 min at 4°C, and centrifuged at 100,000

x g for 4 h at 4°C. The new supernatant was dialyzed against buffer A containing

60 mM NaCl, centrifuged for 10 min at 20,000 x g to remove debris, and then

mixed with 800 µl of DE52 resin (Whatman) that had been equilibrated with

buffer A containing 60 mM NaCl. After incubation for 30 min at room temperature,

the slurry was transferred to a 2-cm-diameter column and washed with buffer A.

Proteins were eluted from the column in a stepwise manner with buffer A

containing 150 and 300 mM KCl, and 160-µl fractions were collected.

The cytoplasmic supernatant resulting from the centrifugation of the MEF

lysate at 2000 × g was subjected to further centrifugation at 100,000 × g for 4 h

at 4°C. The new supernatant was mixed with 800 µl of DE52 that had been


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equilibrated with buffer A containing 60 mM NaCl, and then subjected to column

chromatography as described above.

For preparation of whole-cell extracts, MEFs were suspended in buffer A

containing 300 mM KCl, frozen and thawed, rotated for 30 min at 4°C, and then

centrifuged at 100,000 × g for 4 h at 4°C. The resulting supernatant was dialyzed

against buffer A containing 60 mM NaCl, centrifuged for 10 min at 20,000 x g to

remove debris, and then mixed with 800 µl of DE52 that had been equilibrated

with buffer A containing 60 mM NaCl. Column chromatography was then

performed as described above.

DE52 column fractions (3 µl) were mixed with 50 ng of Uba1, 100 ng of

Ubc5a or Ubc3, 3 µg of GST-ubiquitin, and 50 ng of wild-type p27Kip1 or the

T187A mutant in a 10-µl reaction mixture containing 40 mM Hepes-NaOH (pH

7.6), 60 mM NaCl, 2 mM DTT, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, and

1.5 mM ATP. In some experiments, 100 ng of GST-cyclin E–CDK2 complex or

100 ng of Cks1 were added to the reaction mixture. After incubation for 30 min at

26°C, the reaction mixtures were subjected to SDS-PAGE and immunoblot

analysis with a monoclonal antibody to p27Kip1.


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RESULTS

Normal degradation of p27Kip1 at the G0-G1 transition in Skp2–/– cells

Analysis of Skp2–/– mice revealed that phenotypic characteristics such as cellular

accumulation of p27Kip1 were less evident in lymphocytes than in liver, kidney,

and lung (31) (data not shown). With the use of immunoblot analysis, we

examined the mitogen-induced down-regulation of p27Kip1 in mature lymphocytes,

almost all of which reside in the G0 phase of the cell cycle in the absence of

stimulation (6). Mitogenic stimulation with the combination of PDBu and

ionomycin resulted in the almost complete disappearance of p27Kip1 from wild-

type lymphocytes within 12 h (Fig. 1A). Unexpectedly, Skp2–/– lymphocytes

exhibited similar kinetics of p27Kip1 down-regulation, suggesting that the

decrease in the abundance of p27Kip1 in the early phase (up to 12 h) of mitogenic

stimulation occurs independently of Skp2-mediated proteolysis. However, the

abundance of p27Kip1 increased again to substantial levels by 36 to 48 h in the

Skp2–/– cells, whereas the amount of this protein remained low in wild-type

lymphocytes (Fig. 1B).

The abundance of cyclin D, cyclin E, and Skp2 was also examined in

wild-type lymphocytes in the early (Fig. 1C) and late (Fig. 1D) phases of

mitogenic stimulation. Consistent with previous results (32), cyclin D1 was not


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detected in the lymphocytes (data not shown). Cyclin D2 was detected as early

as 3 h after stimulation, whereas the amount of cyclin E (which is expressed in

late G1 phase and degraded in S phase) increased 18 to 24 h after stimulation.

The expression of Skp2 was first evident 18 to 24 h after stimulation and was

maximal at 30 to 36 h.

The cell cycle profile did not differ substantially between Skp2+/+ and

Skp2–/– cells for up to 48 h after stimulation (Fig. 1E). In both instances, cells

began to enter S phase 18 to 24 h after stimulation and to enter G2 phase at 36 h.

Given that neither Skp2 nor cyclin E was expressed in the early phase (G0-G1) of

p27Kip1 degradation, and that the down-regulation of p27Kip1 occurred in Skp2–/–

cells, this process appears to require neither Skp2 nor cyclin E–mediated

phosphorylation of p27Kip1. In contrast, Skp2 is indispensable for the late phase

(S-G2) of p27Kip1 degradation.

Prevention of p27Kip1 degradation at G0-G1 by a proteasome inhibitor

We next examined whether the early phase of p27Kip1 degradation induced by

mitogenic stimulation is dependent on the ubiquitin-proteasome pathway. We

incubated wild-type lymphocytes in the presence of lactacystin, a specific

inhibitor of the proteasome, during mitogenic stimulation. The down-regulation of

p27Kip1 was markedly inhibited by lactacystin (Fig. 2A). Short-term inhibition of


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the proteasome by incubation of cells with the quick-acting compound MG132

for the last 2 h before cell harvest yielded similar results. MG132 thus markedly

inhibited the further degradation of p27Kip1 after its addition to the culture (Fig.

2B). To exclude the possibility that proteasome inhibition affects transduction of

the mitogenic signal, we also examined the expression of the immediate-early

genes c-Myc, c-Jun, and c-Fos by RT-PCR analysis (Fig. 3). The levels and

kinetics of expression of these genes were not substantially affected by

treatment of the wild-type cells with lactacystin, indicating that mitogenic

signaling was intact in lymphocytes exposed to this agent. These data suggest

that the early phase of degradation of p27Kip1 at the G0-G1 transition is mediated

by the ubiquitin-proteasome pathway.

Impairment of the second cell cycle in Skp2–/– cells

We previously demonstrated that the growth of Skp2–/– lymphocytes was slightly

reduced 4 days and substantially reduced 6 days after mitogenic stimulation

compared with the growth of wild-type lymphocytes (31). We thus examined the

cell cycle progression of Skp2–/– lymphocytes in greater detail (Fig. 4). Although

the cell cycle profile of Skp2–/– lymphocytes was similar to that of wild-type cells

for up to 2 days after stimulation, the proportion of cells in S phase after 3 days

was markedly lower for Skp2–/– lymphocytes (15%) than for the wild-type cells


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(40%). These results suggest that, although the G1-S transition of the first cell

cycle occurs normally in Skp2–/– cells (as a result of the degradation of p27Kip1

during the G0-G1 transition), the subsequent accumulation of p27Kip1 during S-G2

phases of the first cycle (caused by the absence of Skp2) results in G1 arrest

during the second cell cycle. The putative machinery responsible for p27Kip1

degradation at the G0-G1 transition of the first cell cycle thus does not appear to

function at the G1-S transition of subsequent cycles (see Fig. 8).

Skp2-dependent and -independent polyubiquitination of p27Kip1

We fractionated total extracts of Skp2+/+ and Skp2–/– MEFs by DE52 ion-

exchange column chromatography with stepwise elution with KCl. We then

examined the in vitro ubiquitination of p27Kip1 in the presence of the ubiquitin-

activating enzyme (E1) Uba1, the ubiquitin-conjugating enzyme (E2) Ubc5a, a

GST fusion protein of ubiquitin, and each column fraction (Fig. 5A). The Skp2+/+

cell extract yielded a pronounced peak of polyubiquitination activity

encompassing fractions 6 and 7, whereas the corresponding activity peak

derived from Skp2–/– cells spanned fractions 6 to 9. To determine whether

ubiquitination of p27Kip1 was dependent on phosphorylation of Thr187 at the

COOH-terminus of the protein, we subjected a p27Kip1 mutant in which Thr187 was

replaced by Ala (T187A) to the in vitro ubiquitination assay (Fig. 5B). A large


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peak of polyubiquitination activity was detected mostly in fractions 5 to 8 and a

smaller peak of activity was apparent in fractions 14 and 15 derived from both

Skp2+/+ and Skp2–/– cells. These data suggest that a mediator of p27Kip1

polyubiquitination is present in the Skp2–/– cells, and that the polyubiquitination

activity conferred by this factor is independent of phosphorylation of p27Kip1 on

Thr187.

We previously showed that Skp2 is predominantly localized to the

nucleus (33,34). We thus also subjected nuclear extracts prepared from Skp2+/+

and Skp2–/– MEFs to ion-exchange chromatography. Skp2 was detected mostly

in fractions 5 and 6 of the column eluate (Fig. 6A). Fraction 5 of the nuclear

extracts of Skp2+/+ and Skp2–/– cells was then included in the in vitro

ubiquitination assay with wild-type p27Kip1, E1, the E2s Ubc3 or Ubc5a, and

GST-ubiquitin (Fig. 6B). With the fraction from Skp2+/+ cells, polyubiquitination of

p27Kip1 was evident in the additional presence of cyclin E and CDK2 and was

further enhanced by Cks1. In contrast, polyubiquitinated species of p27Kip1 were

not detected with fraction 5 from Skp2–/– cells.

To confirm that the polyubiquitination of p27Kip1 mediated by Skp2 is

dependent on phosphorylation of p27Kip1 on Thr187, we subjected the T187A

mutant of p27Kip1 to the in vitro ubiquitination reaction in the presence of fraction

5 of the nuclear extracts of Skp2+/+ and Skp2–/– cells (Fig. 6C). In contrast to


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wild-type p27Kip1 (which underwent polyubiquitination in the presence of fraction

5 from wild-type cells), theT187A mutant did not undergo polyubiquitination in

the presence of fraction 5 from either Skp2+/+ or Skp2–/– cells. These data

suggest that polyubiquitination activity in the nucleus requires Skp2, and that

phosphorylation of p27Kip1 on Thr187 by the cyclin E–CDK2 complex is necessary

for Skp2-mediated polyubiquitination.

Given that p27Kip1 is translocated from the nucleus to the cytoplasm by

Jab1-mediated nuclear export (35), we next examined whether molecular

machinery for polyubiquitination of p27Kip1 is present in the cytoplasm.

Cytoplasmic extracts were prepared from Skp2+/+ and Skp2–/– cells and were

fractionated by ion-exchange chromatography. The resulting fractions 5 were

then included in the in vitro ubiquitination assay with wild-type p27Kip1 or the

T187A mutant as the substrate. Similar extents of polyubiquitination were

apparent with the fractions derived from Skp2+/+ and Skp2–/– cells as well as with

wild-type p27Kip1 and the T187A mutant (Fig. 6D). Thus, this cytoplasmic

polyubiquitination activity is independent both of Skp2 and of phosphorylation of

p27Kip1 on Thr187. The putative ubiquitin ligase responsible for this Skp2-

independent polyubiquitination appears to be located predominantly in the

cytoplasm, given that the corresponding fraction derived from nuclear extracts

did not contain such an activity. Together, our results suggest the existence of


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two pathways of p27Kip1 ubiquitination: a Skp2- and Thr187

phosphorylation–dependent pathway in the nucleus, and a Skp2- and Thr187

phosphorylation–independent pathway in the cytoplasm.

Finally, we examined the cell cycle dependence of the polyubiquitination

activities in the nucleus and cytoplasm. Nuclear and cytoplasmic extracts were

prepared from Skp2+/+ and Skp2–/– MEFs synchronized at G0 phase (Fig. 7A) or

at S-G2 phases (Fig. 7B), and were subjected to the in vitro ubiquitination assay.

The polyubiquitination activity in the nuclear extract of Skp2+/+ cells was greater

at S-G2 than at G0. In contrast, the activity in the cytoplasmic extracts of both

Skp2+/+ and Skp2–/– cells at G0 was similar to that at S-G2. Thus, the Skp2- and

Thr187 phosphorylation–dependent pathway of p27Kip1 polyubiquitination in the

nucleus is active when cells enter successive rounds of the cell cycle. In contrast,

the cytoplasmic polyubiquitination pathway is active at both G0 and S-G2 phases.

Given that signal-dependent nuclear export of p27Kip1 occurs at the G0-G1

transition,2 exported p27Kip1 may be polyubiquitinated by the constantly active

ubiquitination machinery in the cytoplasm.


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DISCUSSION

The amount of p27Kip1 is relatively high in quiescent (G0) cells, decreases on

entry of cells into the cell cycle, and is controlled predominantly by the rate of

p27Kip1 degradation (16,17). This CKI undergoes ubiquitination and is degraded

in a proteasome-dependent manner (5). Phosphorylation of p27Kip1 on Thr187 is

mediated by the cyclin E–CDK2 complex and has been shown to be required for

ubiquitination of p27Kip1 (23-25). Previous studies indicate that Skp2 specifically

interacts with the extreme COOH-terminus of p27Kip1 only when Thr187 is

phosphorylated by cyclin E–CDK2 (26-28). This association of Skp2 with p27Kip1

results in recruitment of the latter to the SCF core complex, thereby promoting its

ubiquitination and degradation. This ubiquitination process was recently shown

to be markedly enhanced by Cks1 associated with Skp2 (29,30).

In parallel with Skp2-mediated ubiquitination, p27Kip1 is exported from the

nucleus and degraded. This export step appears to be dependent on Jab1, a

component of the 450-kDa COP9 (signalosome) complex (35). Our unpublished

data indicate, however, that the Jab1-mediated export of p27Kip1 from the nucleus

at the G0-G1 transition occurs in a Skp2-independent manner.2 Furthermore,

given that Skp2 is localized predominantly to the nucleus, the ubiquitination of

p27Kip1 at this stage of the cell cycle is likely mediated by a ubiquitin ligase (other


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Hara et al. 21

than SCFSkp2) that is located in the cytoplasm. We therefore propose that p27Kip1

is degraded by at least two distinct mechanisms, one of which is dependent on

Skp2 and one of which is not (Fig. 8). The Skp2-independent mechanism

operates in the cytoplasm and degrades p27Kip1 at the G0-G1 transition. Jab1-

mediated nuclear export of p27Kip1 may transfer this protein to the degradation

machinery in the cytoplasm. The Skp2-dependent mechanism, mediated by the

SCFSkp2 ubiquitin ligase, serves to degrade p27Kip1 that is phosphorylated on

Thr187 in the nucleus during S-G2 phases in successive rounds of the cell cycle.

In addition to Thr187, phosphorylation of other residues of p27Kip1 may be

important in the control of its stability. We recently showed that phosphorylation

of Ser10 accounted for ~70% of the total phosphorylation of p27Kip1, and that the

extent of phosphorylation at this site was ~75-fold greater than that at Thr187 (36).

The extent of Ser10 phosphorylation is markedly increased in cells in G0 phase of

the cell cycle compared with that apparent in cells in S or M phase. The stability

of p27Kip1 phosphorylated on Ser10 is also substantially greater than that of the

unphosphorylated form of the protein. These observations suggest that Ser10 is

the major site of phosphorylation of p27Kip1, and that phosphorylation at this site

(like that at Thr187) contributes to the regulation of p27Kip1 stability (36). The

increased stability of the Ser10-phosphorylated form of p27Kip1 suggests that

dephosphorylation of this residue might play an important role in progression of


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Hara et al. 22

the cell cycle from G0 to G1. It is possible that phosphorylation of Ser10 may be a

determinant of the nuclear export of p27Kip1 or of the ubiquitination of this protein

in the cytoplasm.

The most obvious cellular phenotype of Skp2–/– mice is the presence of

markedly enlarged, polyploid nuclei and multiple centrosomes (31). However,

there is substantial tissue variability in the penetrance of this phenotype. It is

most prominent in liver, kidney, lung, and testis, whereas hematopoietic cells and

neurons appear normal. Although the reason for such tissue differences has

been unclear, they might result from variability in dependence on the two distinct

mechanisms of p27Kip1 degradation. Purification of the activity responsible for the

ubiquitination of p27Kip1 in the cytoplasm and molecular identification of the

corresponding putative ubiquitin ligase should provide insight into these issues.

Mutations in the p27Kip1 gene appear to be rare in human cancers.

However, a reduced abundance of p27Kip1 in a subset of colon and breast

cancers correlates well with poor prognosis (37-40). Furthermore, the loss of

p27Kip1 alleles in mice increases the sensitivity of these animals to cancer-

inducing agents (14). Identification of components of the protein degradation

machinery that determine the turnover rate of p27Kip1 may thus provide insight

into the altered expression of this protein in tumor cells as well as into whether

such altered expression is a cause or a consequence of cell transformation.


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Hara et al. 23

Indeed, Skp2 is overexpressed in many human cancer cell lines (41), suggesting

that p27Kip1 degradation mediated by Skp2 may be related to carcinogenesis. It is

thus possible that the putative ubiquitin ligase responsible for the Skp2-

independent pathway of p27Kip1 degradation is also deregulated in cancer cells.

During revision of this manuscript, Malek et al. (42) demonstrated the

existence of two pathways of p27Kip1 degradation, one dependent on Thr187

phosphorylation and one not, with the use of a mouse "knock-in" model.

Although our present results are mostly consistent with those of Malek et al.,

these latter researchers suggested that the degradation of p27Kip1 at the G0-G1

transition is dependent on Skp2. This discrepancy might be attributable to a

difference in the number of passages of the MEFs studied. We have thus

observed that the Skp2-independent ubiquitination activity apparent at the G0-G1

transition is prominent in lymphocytes and in MEFs subjected to a small number

of passages, whereas senescent MEFs are prone to loss of this activity with

successive passages.2


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Hara et al. 24

ACKNOWLEDGMENTS

We thank M. Matsumoto and N. Ishida for helpful discussion; R. Yasukochi, K.

Shimoharada, S. Matsushita, N. Nishimura, and other laboratory members for

technical assistance; and M. Kimura for help in preparing the manuscript. This

work was supported in part by a grant from the Ministry of Education, Science,

Sports, and Culture of Japan, by Nissan Science Foundation, and by a research

grant from the Human Frontier Science Program.


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Hara et al. 25

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FOOTNOTES

1Abbreviations: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; PDBu,

phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; GSK-3β,

glycogen synthase kinase–3β; BrdU, bromodeoxyuridine; FITC, fluorescein

isothiocyanate; RT-PCR, reverse transcription–polymerase chain reaction;

G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-

transferase; PBS, phosphate-buffered saline; DTT, dithiothreitol; MEF, mouse

embryonic fibroblast.

2Ishida, N., and Nakayama, K. I., in preparation.


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Hara et al. 31

FIGURE LEGENDS

Fig. 1. Skp2-independent degradation of p27Kip1. (A and B) Lymphocytes derived

from Skp2+/+ and Skp2–/– mice were stimulated with the combination of 10 nM

PDBu and 300 nM ionomycin for 0 to 12 h (A) or 0 to 48 h (B). The intracellular

abundance of p27Kip1 was examined by immunoblot analysis of cell extracts with

a monoclonal antibody to p27Kip1. The same blots were also probed with

antibodies to GSK-3β as a control. (C and D) Skp2+/+ lymphocytes were

stimulated with 10 nM PDBu and 300 nM ionomycin for 0 to 12 h (C) or 0 to 48 h

(D), after which the abundance of p27Kip1, cyclins D2 and E, Skp2, and GSK-3β

(control) was examined by immunoblot analysis. (E) Skp2+/+ and Skp2–/–

lymphocytes were stimulated with PDBu and ionomycin for the indicated times,

labeled with 10 µM BrdU for 30 min before harvesting, stained with antibodies to

BrdU and propidium iodide, and analyzed by flow cytometry. The percentages of

cells in G0-G1, S, and G2-M phases of the cell cycle are shown.

Fig. 2. Effects of proteasome inhibitors on degradation of p27Kip1 at Go-G1 in

wild-type lymphocytes. (A) Cells were stimulated with 10 nM PDBu and 300 nM

ionomycin in the absence (–) or presence (+) of 10 µM lactacystin for the

indicated times, after which cell extracts were subjected to immunoblot analysis


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Hara et al. 32

with antibodies to p27Kip1 or to GSK-3β (control). (B) Cells stimulated with 10 nM

PDBu and 300 nM ionomycin were treated with 10 µM MG132 (lower panels) or

vehicle (upper panels) for the last 2 h of the indicated times, after which cell

extracts were subjected to immunoblot analysis as in (A).

Fig. 3. Effect of lactacystin on the expression of immediate-early genes induced

by mitogenic stimulation in wild-type lymphocytes. Cells were stimulated with 10

nM PDBu and 300 nM ionomycin in the absence or presence of 10 µM

lactacystin for the indicated times, after which total RNA was isolated and

subjected to RT-PCR analysis with primers specific for c-Myc, c-Fos, or c-Jun as

well as with G3PDH-specific primers as a control. The PCR products were

separated by agarose gel electrophoresis and stained with ethidium bromide.

Fig. 4. Extended cell cycle profiles of lymphocytes derived from Skp2+/+ and

Skp2–/– mice. Cells stimulated with PDBu and ionomycin for the indicated times

were labeled with BrdU for 30 min before harvesting, stained with antibodies to

BrdU (and FITC-conjugated secondary antibodies) and propidium iodide, and

analyzed by flow cytometry. The percentages of cells in G0-G1, S, and G2-M

phase are shown below each panel.


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Hara et al. 33

Fig. 5. In vitro ubiquitination of p27Kip1 in the presence of fractions derived from

total extracts of Skp2+/+ and Skp2–/– MEFs. Extracts were fractionated by

stepwise elution with KCl from a DE52 column, and portions of the resulting

fractions were assayed for the ability to mediate the ubiquitination of either wild-

type p27Kip1 (A) or the T187 mutant of this protein (B) in the presence of Uba1,

Ubc5a, and GST-ubiquitin. Reaction products were detected by immunoblot

analysis with antibodies to p27Kip1. The positions of the unmodified,

monoubiquitinated [(GST-Ub)1], and polyubiquitinated [(GST-Ub)n] p27 proteins

are indicated.

Fig. 6. Skp2-dependent and -independent ubiquitination of p27Kip1. (A) Nuclear

extracts prepared from Skp2+/+ and Skp2–/– MEFs were fractionated by stepwise

elution with KCl from a DE52 column. Portions of the resulting fractions were

subjected to immunoblot analysis with antibodies to Skp2. (B) Portions of

fraction 5 from the DE52 column eluate of the nuclear extracts were assayed for

their ability to mediate p27Kip1 ubiquitination in the absence or presence of cyclin

E–CDK2 or of Cks1. Reaction mixtures also included Uba1, Ubc3 (left panel) or

Ubc5a (right panel), and GST-ubiquitin. Reaction products were detected by

immunoblot analysis with antibodies to p27Kip1. (C) In vitro ubiquitination assays

were performed with wild-type p27Kip1 or the T187A mutant in the absence or


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Hara et al. 34

presence of DE52 column fraction 5 from nuclear extract of Skp2+/+ or Skp2–/–

MEFs, Cks1, cyclin E–CDK2, and the combination (Reaction) of Uba1, Ubc5a,

and GST-ubiquitin, as indicated. (D) Cytoplasmic extracts from Skp2+/+ and

Skp2–/– MEFs were fractionated by stepwise elution with KCl from a DE52

column, and portions of the resulting fractions 5 were subjected to the in vitro

ubiquitination assay as in (C) in the absence of Cks1 and cyclin E–CDK2.

Fig. 7. Constant activity of the Skp2-independent p27Kip1 polyubiquitination

pathway in the cytoplasm. Nuclear and cytoplasmic extracts were prepared from

Skp2+/+ and Skp2–/– MEFs synchronized either at G0 phase (A) or at S-G2 phases

(B). The extracts were fractionated by stepwise elution with KCl from a DE52

column, and the resulting fractions were subjected to in vitro ubiquitination

assays with wild-type p27Kip1 or the T187A mutant in the presence of Cks1, cyclin

E–CDK2, and the combination (Reaction) of Uba1, Ubc5a, and GST-ubiquitin,

as indicated. Reaction products were detected by immunoblot analysis with

antibodies to p27Kip1.

Fig. 8. Model for cell cycle regulation through p27Kip1 degradation by two distinct

mechanisms. The degradation of p27Kip1 in the nucleus during successive

phases (G1-S-G2-M) of the cell cycle appears to be regulated by the SCFSkp2


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Hara et al. 35

ubiquitin ligase, which targets p27Kip1 phosphorylated on Thr187 (T187-P). In

contrast, p27Kip1 appears to be ubiquitinated in the cytoplasm at the G0-G1

transition by an as-yet-unidentified ubiquitin ligase that functions independently

both of Skp2 and of phosphorylation of p27Kip1 on Thr187.


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B Time (h)

0 12 18 24 36 48

p27

Time (h)

0 3 6 9 12

A

30

Time (h)

0 12 18 24 36 48

Time (h)

0 3 6 9 12 30

GSK-3βGSK-3β

p27

C0 3 6 9 12

Time (h) Time (h)

Cyclin E

Cyclin D2

Skp2

p27

GSK-3β

D

0 12 18 24 30 4836

Cyclin E

Cyclin D2

Skp2

p27

GSK-3β

E Skp2+/+

Skp2-/-

Time (h)

G2-MS

1009080706050403020100

1009080706050403020100

0 12 24 36 48 0 12 24 36 48

G0-G1

Skp2+/+

Skp2+/+

Skp2-/-

Skp2+/+

Skp2-/-

Skp2-/-

Skp2+/+

Skp2-/-

Per

cen

tag

e o

f ce

lls in

eac

h p

has

e

Hara et al., Figure 1


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0 3 6 9 12

Time (h)

Lactacystin

(+)p27

( )

Time (h)

0 3 6 9 12Lactacystin

GSK-3β( )

(+)


0 3 5 7 9

Time (h)

p27

MG132 (+)

0 3 5 7 9

Time (h)

GSK-3β

MG132 (+)

A

B


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c-Fos

c-Jun

c-Myc

G3PDH

0 5 15 30 60 120 180 0 5 15 30 60 120 180

(+) Lactacystin( ) Lactacystin

Time (min):



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Skp2+/+

Skp2-/-

Time of stimulation (days)

Brd

U in

corp

ora

tio

n (

FIT

C)

DNA Content (Propidium Iodide)

0 1 2 3

0.1% 15.2%41.4%11.7%98.8%

1.1%

87.6%

0.4%

50.8%

6.8%

78.8%

4.9%

0.2%G0/G1

10.4% 45.4% 40.5%SG2/M

G0/G1S

G2/M

G0/G1S

G2/M

G0/G1S

G2/M

G0/G1S

G2/M

G0/G1S

G2/M

G0/G1S

G2/M

G0/G1S

G2/M

99.3%

0.5%

88.5%

0.8%

47.6%

6.1%

55.8%

3.4%



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p27-(GST-Ub)1

p27-(GST-Ub)1

p27-(GST-Ub)n

p27-(GST-Ub)n

p27 (T187A)

p27 (T187A)

p27-(GST-Ub)1

p27-(GST-Ub)1

p27-(GST-Ub)n

p27-(GST-Ub)n

p27

p27

B

Skp2+/+

Skp2-/-

Skp2+/+

Skp2-/-

0.15 M0.3 M

KCl

A

Fraction:

0.15 M

0.3 M

KCl

Fraction:4 5 6 7 8 9 10 11121314151617

43 5 6 7 8 9 1011121314151617



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4 5 6 7 8 9 10 11 12 13kDa62kDa51

kDa38

kDa62kDa51

kDa38

0.15 M0.3 M

Skp2

Skp2

A

B

Fraction:

KCl

--

--

Cks1:Cyclin E-CDK2: Cyclin E-CDK2:+

- ++

-- +

- ++

- + + + + + +Cks1: -

--- +

- ++

-- +

- ++

- + + + + + +

Ubc3 Ubc5a

p27 Fr:p27

Fraction 5: Fraction 5:

Skp2+/+

Skp2-/-

Skp2 +/+ Skp2

-/- Skp2 +/+ Skp2

-/-

p27-(GST-Ub)np27-(GST-Ub)n

Hara et al., Figure 6A, B


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C

Reaction: - --

Cks1:

Cyclin E-CDK2:

+ +

+

+++-

- +- ++

+ ++

++

- -- + +

+

+++-

- +- ++

+ ++

++Fraction 5:

p27

p27-(GST-Ub)1

Wild-type p27 Mutant p27 (T187A)

Mutant p27 (T187A)

Skp2 +/+Skp2 -/-Skp2 +/+ Skp2 -/-

Fraction 5:

Reaction: - -- + + - ---

+ ++ + + +- + + + +

Wild-type p27

Skp2 +/+Skp2 -/-Skp2 +/+ Skp2 -/-

p27-(GST-Ub)1

p27-(GST-Ub)n

p27-(GST-Ub)n

p27

D

Hara et al., Figure 6C, D


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Mutant p27 (T187A)Wild-type p27

Skp2 +/+Skp2 -/-Skp2 +/+ Skp2 -/-

Reaction: - -+ + - -+ +


Skp2 +/+Skp2 -/-Skp2 +/+ Skp2 -/-

- -+ + - -+ +


Skp2 +/+Skp2 -/-Skp2 +/+ Skp2 -/-

Reaction: - -+ + - -+ +


Skp2 +/+Skp2 -/-Skp2 +/+ Skp2 -/-

- -+ + - -+ +

Nucleus Cytoplasm

Nucleus Cytoplasm

p27-(GST-Ub)1

p27-(GST-Ub)n

p27

p27-(GST-Ub)1

p27-(GST-Ub)n

p27

A

B



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G1

S

G2

M G0

p27 P

p27

Skp2-independentT187-P-independentCytoplasm

Skp2-dependentT187-P-dependentNucleus



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Hatakeyama and Kei-Ichi NakayamaTaichi Hara, Takumi Kamura, Keiko Nakayama, Kiyotaka Oshikawa, Shigetsugu

ubiquitination pathwayDegradation of p27Kip1 at the G0-G1 transition mediated by a Skp2-independent

published online October 26, 2001J. Biol. Chem.

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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M107274200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/10/26/jbc.M107274200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2001/10/26/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/10/26/jbc.M107274200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

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