Down-regulation of p27Kip1 by Two
Mechanisms, Ubiquitin-mediated Degradation and Proteolytic
Processing*
Michiko
Shirane
§,
Yumiko
Harumiya¶,
Noriko
Ishida
§,
Aizan
Hirai
**,
Chikara
Miyamoto
,
Shigetsugu
Hatakeyama
§,
Kei-ichi
Nakayama
§§§, and
Masatoshi
Kitagawa
§
From the
Department of Molecular and Cellular
Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka
812-8582, § CREST, Japan Science and Technology Corporation,
Kawaguchi 332-0012, the ¶ Department of Molecular Biotherapy
Research, Cancer Chemotherapy Center, Cancer Institute, Japanese
Foundation for Cancer Research, Tokyo 170-8455, the
Second
Department of Internal Medicine, Chiba University School of Medicine,
Chiba 260-0856, ** Chiba Prefectural Togane Hospital, Togane 283-8588, and 
Nippon Roche Research Center,
Kamakura 247-0063, Japan
 |
ABSTRACT |
The intracellular level of
p27Kip1, a cyclin-dependent kinase
(CDK) inhibitory protein, is rapidly reduced at the G1/S
transition phase when the cell cycle pause ceases. In this study, we
demonstrated that two posttranslational mechanisms were involved in
p27Kip1 breakdown: degradation via the
ubiquitin (Ub)-proteasome pathway and proteolytic processing that
rapidly eliminates the cyclin-binding domain. We confirmed that
p27Kip1 was ubiquitinated in vitro
as well as in vivo. The p27Kip1
-ubiquitination activity was higher at the G1/S boundary
than during the G0/G1 phase, and
p27Kip1 ubiquitination was reduced
significantly when the lysine residues at positions 134, 153, and 165 were replaced by arginine, suggesting that these lysine residues are
the targets for Ub conjugation. In parallel with its
Ub-dependent degradation, p27Kip1
was processed rapidly at its N terminus, reducing its molecular mass
from 27 to 22 kDa, by a ubiquitination-independent but adenosine triphosphate (ATP)-dependent mechanism with higher activity
during the S than the G0/G1 phase. This 22-kDa
intermediate had no cyclin-binding domain at its N terminus and
virtually no CDK2 kinase inhibitory activity. These results suggest
that p27Kip1 is eliminated by two independent
mechanisms, ubiquitin-mediated degradation and ubiquitin-independent
processing, during progression from the G1 to S phase.
 |
INTRODUCTION |
Cell cycle progression is controlled by a series of kinase
complexes composed of cyclins and cyclin-dependent kinases
(CDKs)1 (1). The enzymatic
activities of cyclin/CDK complexes are regulated by many mechanisms
that reflect both the diversity of the signals they integrate and the
central importance of their roles in cell cycle control. These
regulatory mechanisms include variations in cyclin levels, positive-
and negative-acting phosphorylation of the kinase subunit, and the
actions of CDK inhibitors (CKIs) (2). Of these, the CKIs appear to be
the most diverse and flexible regulators. Mammalian CKIs are classified
into two families: the Cip/Kip and Ink4 families. The former comprises
p21Cip1/Waf1, p27Kip1,
and p57Kip2, each of which has a conserved
domain, called the CDK-binding/inhibitory domain, at its N terminus.
The CKI p27Kip1 plays a pivotal role in the
control of cell proliferation (3-5). Transition from the
G1 to S phase is promoted by G1 cyclin/CDK
complexes, such as cyclin D/CDK4 and 6 and cyclin E/CDK2, and
p27Kip1 inhibits the activities of these kinases
directly by binding to them (6-9). The elimination of
p27Kip1 during the late G1 phase is
required for G1 cyclin/CDK complex activation and cell
cycle progression from the G1 to S phase in various cell
lines (10-13). Consistent with this idea is that forced expression of
p27Kip1 blocks cell cycle progression during the
G1 phase, whereas targeted p27Kip1
mRNA antisense vectors increase the fraction of cells in the S
phase. Moreover, p27Kip1 down-regulation due to
enhanced degradation in various malignant neoplasms, such as
colorectal, breast, stomach, and non-small-cell lung cancers, was
observed (14-18). Finally, we and others demonstrated that targeted
disruption of the mouse p27Kip1 gene resulted in
enhanced growth of mice, multiple organ hyperplasia, and a
predisposition to tumors (19-21). These lines of evidence support the
idea that p27Kip1 is a key molecule that
negatively regulates cell cycle progression.
A major question is: how are the intracellular levels of
p27Kip1 regulated in a precisely timed fashion?
Previous studies showed that p27Kip1 mRNA
does not fluctuate during the cell cycle, implying the existence of
posttranslational machinery that controls the
p27Kip1 expression levels (3, 22). Genetic
studies on yeast revealed that Sic1, a CKI controlling the
G1/S transition, like mammalian p27Kip1, is degraded specifically by the
ubiquitin (Ub)-proteasome system (23-25). Furthermore, the
Ub-proteasome pathway was suggested to be involved in
p27Kip1 degradation in mammals (26). The
Ub-proteasome pathway is emerging as a major and universal mechanism
that regulates selective and time-controlled elimination of short-lived
key regulatory proteins, e.g. cell cycle proteins (cyclins
(27, 28) and CKIs (29)) and transcriptional activators (I
B (30, 31),
c-Jun (32), p53 (33),
-catenin (34), and others). This pathway
requires adenosine triphosphate (ATP) and the covalent conjugation of
target proteins with multiple Ub molecules (35-37). This multistep
process involves Ub activation by a Ub-activating enzyme (E1), followed by transfer of Ub to a Ub-conjugating enzyme (E2), and the third step
is the transfer of Ub to a Ub ligase (E3), which catalyzes the
formation of isopeptide bonds between the C-terminal glycine of Ub and
the
-amino groups of lysine residues on the target proteins. During
subsequent cycles, additional Ub molecules are added to the substrate.
Then, multi-ubiquitinated proteins are recognized by the 26 S (1500 kDa) proteasome complex and rapidly degraded into short peptides. 26 S
proteasomes are multicatalytic protease complexes containing
chymotrypsin-like, trypsin-like, and postglutamyl activities together
with ATP.
Therefore, it is important to elucidate the mechanisms responsible for
p27Kip1 breakdown, not only to improve our
understanding of cell-growth control, but also for the discovery of new
anti-cancer drugs. In this study, we found that
p27Kip1 is down-regulated at the
G1/S transition point by two pathways: Ub-mediated
degradation and a novel ubiquitination-independent processing pathway
that abrogates p27Kip1 function by eliminating
its cyclin-binding domain.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Ub and ubiquitin aldehyde (Ub-CHO) were purchased
from Sigma and Boston Biochem Inc. (Cambridge, MA), respectively. The
proteasome inhibitors lactacystin and clasto-lactacystin
-lactone were purchased from Kyowa Medics (Tokyo, Japan) and
Calbiochem, respectively, and the calpain inhibitor ALLN
(N-acetyl-Leu-Leu-norleucinal) was purchased from Roche
Molecular Biochemicals (Mannheim, Germany). The proteasome inhibitor
ZLLLal (Z-Leu-Leu-Leu-H aldehyde) and caspase inhibitors
Ac-DEVD-CHO and Ac-YVAD-CHO were purchased from Peptide Institute Inc.
(Osaka, Japan) and the protease inhibitors antipain, pepstatin,
leupeptin, E64, chymostatin, and phenylmethylsulfonyl fluoride (PMSF)
were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Cell Culture and Synchronization of Cells--
NIH3T3 cells were
cultured in Dulbecco's modified Eagle's medium containing 10% calf
serum, and FM3A cells were cultured in RPMI medium containing 10%
fetal calf serum (Life Technologies, Inc.). NIH3T3 cells were
synchronized by subjecting them to contact inhibition during culture to
confluence to arrest them at the G0/G1 phase,
then releasing them from contact inhibition, allowing them to progress
to the S phase, by replating at a density of approximately 30%. For
cell cycle analysis, bromodeoxyuridine-pulsed NIH3T3 cells were fixed
with 70% ethanol, treated with 2 N HCl containing 0.5%
Triton X-100, neutralized with borax buffer (pH 8.5), subjected to dual
color staining with an anti-bromodeoxyuridine monoclonal antibody (mAb)
conjugated with fluorescein isothiocyanate (Becton Dickinson
Immunocytometry Systems, San Jose, CA) and 5 µg/ml propidium iodide,
and then analyzed using a FACSCalibur flow cytometer and Cell Quest
software (Becton Dickinson).
Plasmids--
Mouse or human p27Kip1 was
subcloned into pGEX-6P (Amersham Pharmacia Biotech UK Ltd., Bucks,
United Kingdom) in order to produce bacterially expressed protein. A
deletion mutant of p27Kip1 was constructed by
subcloning the polymerase chain reaction fragment containing the C
terminus of the p27Kip1 sequence (38-198 amino
acids) into pGEX-6P. KR mutants were produced by replacing the lysine
residues of p27Kip1 with arginine by
site-directed mutagenesis using a QuickChangeTM
site-directed mutagenesis kit (Stratagene), according to the manufacturer's protocol. A mammalian expression vector encoding p27Kip1 was constructed by subcloning human
p27Kip1 into pcDNA3.1/Myc-His (Invitrogen,
Carlsbad, CA), and the mammalian expression vector encoding HA-tagged
ubiquitin was a gift from Dr. Dirk Bohmann (European Molecular Biology Laboratory).
Protein Expression and Purification--
Glutathione
S-transferase (GST)-tagged p27Kip1
proteins for the in vitro ubiquitination assay were
expressed in Escherichia coli XL1-blue and affinity-purified
using glutathione-Sepharose CL-4B (Amersham Pharmacia Biotech), and the
GST tag was cleaved using PreScission Protease (Amersham Pharmacia
Biotech), according to the manufacturer's instructions. Myc-tagged
p27Kip1 and HA-tagged Ubs were transiently
expressed. Each DNA was incubated with LipofectAMINE reagent (Life
Technologies, Inc.) in serum-free medium (Opti-MEM; Life Technologies,
Inc.) for 30 min at room temperature, after which the mixture was
incubated with the required cells for 16 h, followed by incubation
in complete medium for 32 h.
Preparation of Cell Extracts--
For the in vitro
ubiquitination assay, the required cells were washed with
phosphate-buffered saline), suspended in double-distilled water, and
frozen and thawed three times; the resulting lysate was subjected to
centrifugation at 100,000 × g for 4 h at 4 °C, and the supernatant (S100Pr
) was retrieved and frozen at
80 °C.
For the in vivo ubiquitination assay and analysis of
endogenous p27Kip1 levels, the required cells
were incubated in lysis buffer containing 0.1% Nonidet P-40 on ice for
15 min, cleared by centrifugation at 15,000 rpm for 15 min at 4 °C,
and the protein concentration of the supernatant was determined by the
Bradford method (Protein Assay; Bio-Rad). For the in vitro
degradation assay, NIH3T3 cells were homogenized in phosphate-buffered
saline followed by centrifugation at 100,000 × g for
1 h at 4 °C and the resulting supernatant (S100) was retrieved
and frozen at
80 °C.
Immunoblotting Analysis--
Each reaction mixture or cell
lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred onto Immobilon-P membranes
(Millipore, Bedford, MA), which were probed with the required
anti-p27Kip1 (N-20, C-19; Santa Cruz
Biotechnology Inc., Santa Cruz, CA, or clone 57, hereafter designated
TDL; Transduction Laboratories, Lexington, KY), anti-c-Myc (9E10; Santa
Cruz) or anti-
-tubulin (TU-01; Funakoshi, Tokyo, Japan) antibody.
The Renaissance blotting system (NEN Life Science Products) was used to
visualize the proteins.
In Vitro Ubiquitination Assay--
Mouse recombinant
p27Kip1 was incubated with the FM3A or NIH3T3
cell extract in the presence of an ATP-regenerating system (50 mM Tris (pH 8.3), 5 mM MgCl2, 5 mM ATP, 10 mM creatine phosphate, 0.2 unit/ml
creatine kinase) together with 1 mg/ml Ub, 100 µg/ml Ub-CHO, 2 mM dithiothreitol, a protease inhibitor mixture (10 µg/ml
each of antipain, pepstatin, chymostatin, leupeptin, and PMSF) and a
proteasome inhibitor mixture (250 µM ALLN, 250 µM ZLLLal, and 25 µM
clasto-lactacystin
-lactone). The reactions were carried
out at 37 °C for 1 h and terminated by adding SDS sample
buffer, and each reaction mixture was subjected to SDS-PAGE on a 10%
gel, followed by immunoblotting analysis with the anti-p27 mAb TDL. In
order to confirm that p27Kip1 had been
ubiquitinated, identical reaction mixtures to those described above
were subjected to in vitro ubiquitination in the presence of
biotinylated Ub (produced using EZ-Link Sulfo-NHS-LC-Biotin (Pierce),
according to the manufacturer's protocol) instead of Ub. Each reaction
mixture was incubated with the TDL mAb, and the resulting
immunoprecipitate was analyzed by immunoblotting with Neutravidin-HRP
conjugate (Pierce) as the probe.
In Vivo Ubiquitination Assay--
NIH3T3 cells were incubated
with or without 100 µM ALLN for 12 h in a
CO2 incubator, lysed, and then subjected to Western blotting with the TDL mAb. COS7 cells were transiently transfected with
myc-tagged p27 and HA-tagged Ub and 36 h later, 50 µM lactacystin was added to the culture, which was
incubated for another 12 h in a CO2 incubator. Then,
the cells were lysed, as described above, and subjected to
immunoblotting with the anti-c-myc antibody 9E10.
In Vitro Degradation Assay--
Mouse recombinant
p27Kip1 was incubated with the NIH3T3 cell
extract (S100) at 37 °C for 30 or 60 min and subjected to
immunoblotting with the required anti-p27Kip1
antibody (N-20, C-19, or TDL).
In Vivo Degradation Assay--
NIH3T3 cells were metabolically
labeled with 100 µCi/ml Tran35S-label (ICN
Pharmaceuticals Inc., Costa Mesa, CA) in methionine-free medium for
3 h, and then chased in complete medium for 0 or 3 h in a
CO2 incubator Cell lysates were immunoprecipitated with the
TDL mAb, followed by Protein G-Sepharose (Amersham Pharmacia Biotech)
affinity purification and SDS-PAGE, as described above, autoradiography, and quantification using a BAS-2000 imaging analyzer (Fuji Film, Kanagawa, Japan).
CDK2 Kinase Inhibition Assay--
Purified baculovirus-expressed
cyclin E/CDK2 was incubated with 100 µg/ml histone H1, 100 µM cold ATP, and 70 µCi/ml [
-32P]ATP
(Amersham Pharmacia Biotech, 3000 Ci/mmol) with recombinant intact
p27Kip1 or its deletion mutant p27
22k) for 30 min at 30 °C (38, 39). Phosphorylated histone H1 was separated by
SDS-PAGE and detected using a BAS2000 image analyzer.
 |
RESULTS |
Ubiquitination of p27Kip1 in Vivo and in
Vitro--
The results of the previous study suggested that
p27Kip1 is down-regulated by the Ub-proteasome
pathway (26). Originally, we developed an assay system for estimating
p27Kip1 ubiquitination activity in
vitro. Careful manipulation was required to detect the unstable
intermediates; the key factors for detection of ubiquitinated
p27Kip1 were inhibition of isopeptidase
activity, a supply of excess Ub, and the exclusion of proteasomes.
Isopeptidase, which associates with 26 S proteasomes and hydrolyzes
multi-Ub chains, made it difficult to detect ubiquitinated
p27Kip1. In this study, the addition of Ub-CHO,
an isopeptidase inhibitor, to the reaction mixture dramatically
improved the amount of ubiquitinated p27Kip1
detected in vitro (compare lanes 4 and
8 in Fig. 1A). The
addition of exogenous Ub to the reaction mixture also increased the
formation of Ub-p27Kip1 conjugates (compare
lanes 6 and 8 in Fig. 1A),
suggesting that Ub was a limiting factor in this reaction. When GST-Ub
(34 kDa) was added to the reaction mixture instead of Ub (8 kDa), a
species with a higher molecular mass appeared (compare lanes
8 and 10 in Fig. 1A). Thus, this
result confirms that the bands with lower electrophoretic mobilities
were ubiquitinated p27Kip1, not aggregates of
p27Kip1 or nonspecific products resulting from
cross-reaction with the mAb. Proteasomes were removed from the cell
cytoplasmic extract by differential centrifugation at 100,000 × g for 4 h, and the proteasome activity of the resulting
proteasome-depleted supernatant (S100Pr
) was very low. The exclusion
of proteasomes also dramatically increased
Ub-p27Kip1 conjugate formation (data not
shown).

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Fig. 1.
Identification of ubiquitinated
p27Kip1 in vitro and in
vivo. A, exogenous
p27Kip1 protein (exo-p27) was incubated with or
without the FM3A cell lysate from which the proteasomes had been
removed by ultracentrifugation (S100Pr ), as described under " Experimental procedures," with an ATP-regenerating system. Ub,
GST-Ub, and Ub-CHO were added as indicated. The reaction mixtures were
analyzed by immunoblotting with the anti-p27Kip1
mAb TDL. B, exo-p27 was incubated (lanes
1 and 3) or not incubated (lane
2) in ubiquitination mixture with biotinylated Ub instead of
Ub. The reaction mixtures were immunoprecipitated with the TDL mAb
(lanes 1 and 2), or the mock mAb
(anti-mouse IgG; lane 3) and then immunoblotted
and probed with avidin-HRP. The bands marked n.s. represent
nonspecific bands, which were also observed in the control preparations
(not immunoprecipitated or immunoprecipitated with anti-mouse IgG).
C, NIH3T3 cells synchronized at the G1/S phase
were cultured with (lane 2) or without
(lane 1) 100 µM ALLN and the cell
lysates were analyzed by immunoblotting with the TDL mAb. The levels of
ubiquitinated p27Kip1 and a truncated product
(p27 22k) were augmented by ALLN treatment. D, COS7 cells
were transiently transfected with an expression vector encoding
myc-tagged p27Kip1 with or without an expression
vector encoding Ub, treated with or without 50 µM
lactacystin (as indicated by plus and minus signs
above the lanes) and analyzed by immunoblotting with the
anti-myc mAb 9E10. Lactacystin treatment increased the amount of
ubiquitinated p27Kip1.
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|
In order to confirm that the protein with the higher molecular mass we
detected was ubiquitinated p27Kip1, identical
reaction mixtures containing biotinylated-Ub instead of Ub were
subjected to immunoprecipitation-immunoblotting analysis. The resulting
products were immunoprecipitated with the TDL mAb, blotted, and probed
with an avidin-HRP conjugate. Then mono-ubiquitinated p27Kip1 and smeared bands of multi-ubiquitinated
p27Kip1 were detected (Fig. 1B,
lane 1).
Ubiquitinated p27Kip1 was also detected in
vivo. ALLN, which inhibits proteasome and calpain activities,
induced the accumulation of Ub-p27Kip1
conjugates in NIH3T3 cells (Fig. 1C). Ubiquitinated
p27Kip1 was also observed in the transient
p27Kip1 cDNA transfection assay using COS7
cells (Fig. 1D), in which co-expression of Ub and
p27Kip1 led to moderately enhanced
ubiquitination of p27Kip1 (Fig. 1D,
lane 2) and the selective proteasome inhibitor
lactacystin further enhanced the ubiquitinated
p27Kip1 level (Fig. 1D,
lane 3). These data indicate that
p27Kip1 is ubiquitinated in vivo as
well as in vitro. Of note, mono- and di-ubiquitinated
p27Kip1 was also accumulated in the ALLN- or
lactacystin-treated cells (Fig. 1, C and D,
respectively), suggesting that the degradation of the
mono-ubiquitinated p27Kip1 might also be
dependent on the proteasome. We also observed accumulation of a
p27Kip1 fragment with a lower molecular mass
(approximately 22 kDa, referred to hereafter as p27
22k; Fig. 1,
C and D). The proteasome and calpain inhibitor
ALLN enhanced the accumulation of both p27Kip1
and p27
22k (Fig. 1C), whereas the specific proteasome
inhibitor lactacystin resulted in the decrease in p27
22k (Fig.
1D), suggesting that this smaller p27 fragment was produced
by proteasomes and subsequently degraded by a calpain-like protease.
Cell Cycle-dependent Ubiquitination of
p27Kip1--
During the G0/G1
phase, p27Kip1 accumulates and then its level
decreases as cells progress toward the S phase (10). We examined whether the p27Kip1 ubiquitination activity is
regulated in a cell cycle-dependent manner. First, the
correlation between the cell cycle stages and expression levels of
p27Kip1 in NIH3T3 cells was examined. The cell
cycle was synchronized at the G0/G1 phase by
contact inhibition, the cells were released and allowed to progress to
the S phase by replating, and the subsequent progress of the cell cycle
was monitored by flow cytometry (Fig. 2A). Almost all the cells were
arrested at the G0/G1 phase by contact
inhibition, and, about 12 h after release, the cells progressed from the G1 to S phase almost synchronously. The number of
cells in the S phase increased 12 h after release and decreased
after 18 h, and the number in the M phase was maximal after about
21 h. The p27Kip1 expression level was high
in the G0/G1 phase (from 0 to 9 h), declined rapidly at the G1/S transition point (between 9 and 12 h), and was minimal after about 18 h (Fig.
2B).

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Fig. 2.
Cell cycle-dependent expression
and ubiquitination of p27Kip1. A,
NIH3T3 cells were synchronized at the G0/G1
phase, released so they progressed to the S phase, as described under
"Experimental Procedures," and the cell cycle was analyzed by flow
cytometry at the indicated times after release. The proportions of
cells in the G0/G1 (squares), S
(solid circles), and G2/M
(triangles) phases (as percentages of the total number of
cells) are shown. B, the p27Kip1
expression level at each time point was analyzed by immunoblotting with
the TDL mAb. The -tubulin expression level was also shown as an
internal control. C, in vitro ubiquitination
assay of p27Kip1 in lysates prepared from NIH3T3
cells cultured for 0, 10, and 20 h after release.
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Cell lysates were prepared from cells harvested 0, 10, and 20 h
after release, roughly corresponding to the
G0/G1, G1/S, and G2/M
phases, respectively. The p27Kip1 ubiquitination
activities of these lysates were determined by the in vitro
ubiquitination assay, as described above under "Experimental Procedures" and in the legend to Fig. 1. The ubiquitination activity was higher in cells at the G1/S boundary (10 h) than during
any other phase (Fig. 2C, lane 2).
Therefore, we concluded that p27Kip1
ubiquitination activity is regulated in a cell
cycle-dependent manner, increases at or near the
G1/S boundary stage, and declines thereafter.
Determination of Ubiquitination Site(s) on
p27Kip1--
In an attempt to determine which lysine
residue(s) in p27Kip1 is/are the target(s) for
Ub conjugation, the 13 lysine residues in human
p27Kip1 were substituted by arginine in
clusters, as shown in Fig. 3A: KR1 (K25R, K47R, K59R), KR2 (K68R, K73R, K81R), KR3 (K96R, K100R), KR5
(K134R, K153R, K165R), and KR6 (K189R, K190R). These mutants were
subjected to the in vitro ubiquitination reaction followed by immunoblotting analysis and probing with the TDL mAb, which recognizes the amino acid (aa) stretch around position 60. The ubiquitination level of KR5 was significantly lower than those of
wild-type (wt) p27Kip1 and KR2, -3, and -6 (Fig.
3B, lanes 1-12). As KR1, which
contained mutations at position 59, did not react with this mAb (Fig.
3B, lanes 3 and 4), another
anti-p27Kip1 mAb, C-19, was used to probe this
mutant (Fig. 3B, lanes 13-16). The
ubiquitination level of KR1 was comparable to that of wt
p27Kip1 (Fig. 3B, lanes
14 and 16). A time-course experiment confirmed that the ubiquitination of KR5 was impaired markedly in comparison with
that of wt p27Kip1 (Fig. 3C). As
cleavage of the GST tag was incomplete as a result of the purification
of recombinant p27Kip1,
GST-p27Kip1 remained. The possibility that Ub
was conjugated to the GST portion of the
GST-p27Kip1 fusion protein was excluded by the
reduction in ubiquitination of KR5 mutant. These results indicate that
Ub conjugation of p27Kip1 targets some of or all
the lysine residues at positions 134, 153, and 165.

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Fig. 3.
In vitro ubiquitination
of p27Kip1 KR mutants. A,
lysine residues in p27Kip1 were substituted by
arginine in clusters, as indicated. B, in order to locate
the ubiquitination site(s) in p27Kip1,
recombinant KR mutants (KR1, -2, -3, -5, and -6) and wt
p27Kip1 proteins were subjected to the in
vitro ubiquitination assay, as described in the legend to Fig.
1A. The TDL mAb was used for analysis of KR2, -3, -5, and
-6, the epitopes of which are located near aa 60; and the
anti-p27Kip1 antibody C-19, which recognizes the
C terminus of p27Kip1, was used to analyze KR1.
C, time-course analysis of ubiquitination with wt
p27Kip1 and the KR5 mutant in vitro
.
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Proteolytic Processing of p27Kip1--
We also
observed that bands with higher electrophoretic mobilities (p27
22k
in Fig. 1, C and D) appeared in parallel with p27Kip1 ubiquitination in vivo and
in vitro, suggesting the activity responsible for processing
p27Kip1 in the cell lysates that contributes to
the down-regulation of p27Kip1 in parallel with
that responsible for Ub-dependent degradation. Therefore,
we investigated this processing activity. The small fragment shown in
Fig. 1D seemed to be a truncated product lacking the
N-terminal portion, because the myc epitope attached to the C terminus
of p27Kip1 was retained. We investigated the
processing mechanism by incubating recombinant
p27Kip1 protein with the NIH3T3 cell extract and
analyzing the reaction product (p27
22k in Fig.
4), which was readily detected,
suggesting this processing reaction was probably rapid and further
degradation of the p27
22k fragment was rate-limiting. The molecular
masses of the product of exogenous p27Kip1 and
the derivative of endogenous p27Kip1 in NIH3T3
cells were both approximately 22 kDa, indicating that the reaction
in vitro faithfully reproduced
p27Kip1 processing in vivo (Fig.
4A, lanes 1 and 3). The
amount of intact p27Kip1 decreased as the amount
of the processed product increased. The sum of
p27Kip1 expression level was also reduced,
indicating that the further degradation process from the 22-kDa
intermediate occurred. This processing reaction was
ATP-dependent, because the addition of an ATP-regenerating
system to the reaction mixture promoted it, resulting in a reduction in
the amount of wt p27Kip1 and an increase in
p27
22k protein production (Fig. 4B, lane 3). In contrast, ATP-
S, which suppresses ATP
regeneration, inhibited the processing activity, as the amount of wt
p27Kip1 protein did not decreased relative to
the control level (Fig. 4B, lanes 2 and 4). Furthermore, the proteasome-specific inhibitor clasto-lactacystin
-lactone significantly suppressed the
breakdown of p27Kip1 (Fig. 4B,
lane 5), leading to accumulation of the 27-kDa
protein, whereas the proteasome inhibitor ZLLLal, which also inhibits
calpain, resulted in the accumulation of both 27- and 22-kDa proteins
(Fig. 4B, lane 6). The effects of
clasto-lactacystin
-lactone and ZLLLal in
vitro corresponded with the effects of lactacystin and ALLN in vivo, respectively (Fig. 1, D and
C). These results suggest that
p27Kip1 is processed to the p27
22k fragment
by 26 S proteasomes and then degraded to smaller peptides by a
calpain-like protease.

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Fig. 4.
Characterization of the proteolytic
processing of p27Kip1. A,
exogenous p27Kip1 was subjected to the in
vitro degradation assay in the presence of the NIH3T3 cell lysate
(14 µg, lane 3) and electrophoresed together
with the lysate of asynchronous NIH3T3 cells (90 µg, lane
1), followed by immunoblotting with the TDL mAb. Exogenous
p27Kip1 before the degradation reaction is shown
in lane 2. The reaction time is indicated.
B, in vitro degradation assay of
p27Kip1 performed in the presence of an
ATP-regenerating system (ATP, phosphocreatine, and creatine
phosphokinase), ATP S, clasto-lactacystin -lactone, or
ZLLLal (lanes 2-6, respectively). The sample
before the degradation reaction is shown in lane
1. C, in vitro degradation assay of
p27Kip1 in the presence of dimethyl sulfoxide
(DMSO; vehicle control), 250 µg/ml antipain, pepstatin,
leupeptin, and E64 (lanes 3-6, respectively),
10, 50, or 250 µg/ml chymostatin (lanes 7-9)
or PMSF (lanes 10-12). The sample before the
degradation reaction is shown in lane 1.
D, in vitro degradation assay of
p27Kip1 in the presence of dimethyl sulfoxide
(vehicle control), 250 µM YVAD, or 250 µM
DEVD (lanes 2-4, respectively). The sample
before the degradation reaction is shown in lane
1.
|
|
The effects of some other protease inhibitors on the breakdown of
p27Kip1 were tested (Fig. 4C). At
high concentrations (250 µg/ml), antipain, pepstatin, leupeptin, and
E64 had no significant effects on degradation or processing (Fig.
4C, lanes 3-6, respectively), whereas
chymostatin (50 µg/ml) and PMSF (250 µg/ml) inhibited processing
reactions (Fig. 4C, lanes 7-9 and
10-12, respectively) leading to accumulation of 27-kDa
protein. Chymostatin is a specific inhibitor of chymotrypsin-type serine proteases, whereas PMSF inhibits several serine proteases, such
as chymotrypsin, trypsin, and thrombin. These results suggest that
p27Kip1 was processed by chymotrypsin-like
protease activity within the proteasomes (37).
We also tested caspase inhibitors, because
p27Kip1 and p21Cip1 have
been reported to be cleaved by caspase-3 (40) and
p21Cip1 was found to be cleaved by caspase-1
(41) during the progression of apoptosis. However, neither the
caspase-1 inhibitor YVAD nor the caspase-3 inhibitor DEVD affected the
proteolytic processing of p27Kip1 (Fig.
4D).
Collectively, our results suggest that p27Kip1
was degraded by at least two pathways: Ub-proteasome-mediated
degradation and proteolytic processing, by which
p27Kip1 was first processed by proteasomes in an
ATP-dependent manner to produce an approximately 22-kDa
fragment (p27
22k) and then degraded to small peptides by a
calpain-type protease. These results indicate the existence of a novel
proteolytic processing pathway, in addition to the Ub-proteasome
pathway, that regulates intracellular p27Kip1
expression levels.
We carried out further experiments to determine whether the rapid
processing of p27Kip1 to p27
22k affects to
the function of p27Kip1. First, in an attempt to
locate the processing site, three antibodies that recognize different
positions of p27Kip1 were used. The epitope
recognized by the anti-p27Kip1 mAb TDL may be
located near the aa at position 60, as discussed above, and the
anti-p27Kip1 Abs N-20 and C-19 were raised
against the N (aa 2-21) and C (aa 181-198) termini (Fig.
5B) of
p27Kip1. The p27
22k fragment was detected by
C-19 and TDL, but not by N-20 (Fig. 5A), which suggests that
the processing site was located on the N-terminal side some distance
from position 60. In view of its deduced molecular mass, the processing
site seemed to lie near positions 35-40. Therefore, this processing
reaction eliminates the cyclin-binding domain.

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|
Fig. 5.
Location of the processing site and
functional analysis of p27Kip1.
A, the in vitro degradation assay was performed
for 0 (lanes 1, 4, and 7),
30 (lanes 2, 5, and 8), and
60 (lanes 3, 6, and 9) min
and the degradation products were subjected to immunoblotting with the
anti-p27Kip1 mAbs N-20 (lanes
1-3), TDL (lanes 4-6), and C-19
(lanes 7-9). B, diagram of the
deduced cleavage sites in p27Kip1 and the
processed product p27 22k (22 kDa). The numerals represent
the aa positions, and the portions recognized by N-20, TDL, and C-19
are denoted by bold lines. C, in
vitro phosphorylation of histone H1 by cyclin E/CDK2 with
[ -32P]ATP was performed without (lane
1) or with 0.1, 1, 10, 100, or 1000 nM p27 22k
(lanes 2-6), or wt
p27Kip1 (lanes 7-11); the
reaction mixtures were separated by SDS-PAGE; and the products were
detected and quantified using a BAS2000 imaging analyzer.
|
|
In order to examine the function of the processed
p27Kip1, the CDK2-inhibitory activities of
recombinant p27Kip1 and N terminus-deletion
mutant p27Kip1 (p27
22k, Fig. 5B) were tested.
Intact p27Kip1 inhibited the catalytic activity
of CDK2 in a concentration-dependent manner (Fig. 5C,
lanes 7-11), whereas the inhibitory activity of
p27
22k was approximately 100 times lower than that of wt
p27Kip1 (Fig. 5C, lanes
2-6). Thus, the conversion of native
p27Kip1 to p27
22k by proteolytic processing
may interfere with the CDK inhibitory activity of
p27Kip1 .
Cell Cycle-dependent, Ubiquitination-independent
Processing of p27Kip1--
We also examined whether the
activity that degraded endogenous p27Kip1 in
NIH3T3 cells in vivo was cell cycle-dependent.
As expected, the p27Kip1-degradative activity
was higher in the S than the G0/G1 phase (Fig.
6, A and B). In the
S phase, the amount of p27
22k produced increased markedly in
parallel with the amount of p27Kip1 degraded.
This result suggests the processing activity probably contributes to
the down-regulation of p27Kip1 as the cell cycle
progresses from the G1 to the S phase. In vitro experiments suggested that the degradative processing was executed by
26 S proteasomes, as this rapid reaction was ATP-dependent and inhibited by proteasome inhibitors.

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|
Fig. 6.
Cell cycle-dependent processing
of endogenous p27Kip1 in vivo.
A, NIH3T3 cells were synchronized at the
G0/G1 or S phase then metabolically labeled
with Tran35S-label and chased, as described under
"Experimental Procedures." B, the levels of
p27Kip1 (solid bar) and
p27 22k (shaded bar), as percentages of the
intact p27Kip1 level before degradation (0 h),
in each phase are shown. C, NIH3T3 cells were transiently
transfected with myc-tagged wt p27Kip1 and the
KR5 mutant in parallel with mock transfection, metabolically labeled
with Tran35S-label, separated by SDS-PAGE, and analyzed
using a BAS2000 imaging analyzer. The molecular masses of myc-p27 and
myc-p27 22k were 28 and 23 kDa, respectively.
|
|
In order to determine whether this degradative processing reaction was
mediated through ubiquitination, wt p27 and its
ubiquitination-deficient mutant KR5 were transiently expressed in
NIH3T3 cells, resulting in comparable levels of p27
22k in both
groups, suggesting that the reaction that produced p27
22k was
ubiquitination-independent. Collectively, our data suggest the
existence of a novel mechanism that negatively regulates intracellular
p27Kip1 levels, i.e. a Ub-independent
proteolytic processing pathway, in addition to the Ub-mediated
degradation pathway.
 |
DISCUSSION |
The p27Kip1 expression level alters
dramatically during the cell cycle, particularly from the
G1 to the S phase, suggesting that rapid elimination of
p27Kip1 is probably a prerequisite for the
activation of cyclin/CDK kinase complexes and cell cycle progression.
The mechanism responsible for the regulation of
p27Kip1 expression remains elusive. By analogy
with CKIs in Saccharomyces cerevisiae (Sic1) and
Schizosaccharomyces pombe (Rum1), protein degradation via
the Ub-proteasome pathway has been speculated to be critical for
controlling the expression levels of mammalian CKIs (23-25). Pagano
et al. showed that p27Kip1 was
ubiquitinated and that its ubiquitination was augmented by a
Ub-conjugating enzyme (E2), Ubc3, the human homologue of Cdc34 in
S. cerevisiae (26). Thus, the regulatory system that
controls CKI expression seems to be evolutionally well conserved. We
demonstrated ubiquitination of p27Kip1 using the
system we originally designed to assay ubiquitination in
vitro. Inhibition of isopeptidase activity (by Ub-CHO), an excess
supply of Ub (exogenous Ub or GST-Ub), and elimination of proteasomes
(S100Pr
) were critical for the detection of unstable ubiquitinated-p27Kip1. After these experimental
modifications, multimeric Ub chains conjugated with
p27Kip1 were visualized clearly and
immunoprecipitation/immunoblotting analysis of the reaction mixtures
verified that the high molecular weight forms were ubiquitinated
p27Kip1. We used this assay system to locate the
ubiquitination sites and demonstrated that the ubiquitination activity
was controlled in a cell cycle-dependent manner. In many
biological systems, phosphorylation is a signal for ubiquitination. The
phosphorylation of Sic1 CKI in S. cerevisiae is necessary
for binding to Cdc4, an F-box protein that recruits the substrate
(Sic1) to the Skp1/Cdc53/Cdc34 complex, leading to subsequent
ubiquitination. Similarly, phosphorylation of Rum1 CKI in S. pombe was shown to be essential for CKI binding to
Pop1+, the homologue of Cdc4 in S. cerevisiae
(25). Furthermore, p27Kip1 was reported to be
degraded in a phosphorylation-dependent manner (42, 43). Taken
together with our findings, these lines of evidence led us to
hypothesize that phosphorylation precedes
p27Kip1 ubiquitination, which is mediated by an
Skp1/Cul1/F-box protein complex. This hypothesis remains to be tested.
In this study, we demonstrated the presence of an alternative
p27Kip1 degradation pathway operating at the
G1/S transition point of the cell cycle. The Ub-independent
p27Kip1 processing activity produced the 22-kDa
fragment of C-terminal p27Kip1 rapidly, and was
ATP-dependent and sensitive to proteasome-specific and
chymotrypsin-specific inhibitors, suggesting that 26 S proteasomes conduct this processing reaction, because most of the chymotrypsin-like activity present in the cytosol fractions appears to be attributable to
proteasomes (37). This is highly similar to the case of cyclin B1 (49 kDa) that is processed at the N terminus, producing a 42-kDa truncated
form, by 26 S proteasome in an ATP-dependent and
Ub-independent manner (44). Similarly, the rate-limiting enzyme
ornithine decarboxylase is down-regulated by 26 S proteasomes in a
Ub-independent manner (45), whereas the transcription factor NF-
B
underwent Ub-dependent proteolytic processing by 26 S
proteasomes during cellular maturation (46). The C terminus of the p105
precursor of NF-
B is destroyed by ATP-dependent
processing, leaving its N-terminal p50 fragment active. We located the
site of p27Kip1 processing using three mAbs, the
recognition sites of which were known, at approximately aa positions
35-40. This processing reaction eliminated almost the entire
cyclin-binding domain from p27Kip1 and reduced
the cyclin/CDK-inhibitory activity of p27Kip1.
Furthermore, the processing activity increased during the S phase. Uren
et al. (47) reported that the intracellular levels of a
variant of p27Kip1, a C-terminal 22-kDa form,
increased in parallel with the multiplication of the DNA content.
Therefore, it is highly likely that rapid processing of the intact
p27Kip1 molecule to an inert 22-kDa fragment
promotes the progression of the cell cycle from the G1 to
the S phase. It remains to be tested whether this processing allows
p27Kip1 to be ubiquitinated for further
destruction by Ub-dependent activity of the 26 S proteasome.
The transcriptional and translational efficiencies of
p27Kip1 expression, as well as
post-translational proteolysis, may regulate the intracellular
expression levels of p27Kip1. In cells near the
G1/S border stage, the Ub-proteasome and Ub-independent processing pathways may cooperatively promote
p27Kip1 degradation and reduce its level below
the threshold necessary to restrain the cell cycle from progressing
from the G1 to S phase. Alternatively, these proteolytic
mechanisms may play individual roles in specific pathways. As several
studies have indicated that deregulation of
p27Kip1 expression in a variety of malignant
neoplasms is due to disturbed proteolytic activity, it is important to
elucidate the proteolytic pathways that control
p27Kip1 expression. Better understanding of such
pathways may lead to the discovery of anti-cancer drugs with novel
modes of action.
 |
ACKNOWLEDGEMENTS |
We thank Dr. D. Bohmann for the plasmid used
in this study, Dr. T. Tanaka for fruitful discussion, N. Nishimura and
other laboratory members for technical assistance, and M. Kimura and A. Takimoto for secretarial assistance.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the Ministry
of Education, Science, Sports and Culture of Japan (to M. K.) and
by grants from Toray Science Foundation, Sagawa Cancer Research Foundation, and Inamori Foundation (to K. N.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§§
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. Tel.:
81-92-642-6815; Fax: 81-92-642-6819; E-mail:
nakayak1{at}bioreg.kyushu-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
CDK, cyclin-dependent kinase;
CKI, CDK inhibitor;
Ub, ubiquitin;
GST, glutathione S-transferase;
Ub-CHO, ubiquitin aldehyde;
HRP, horseradish peroxidase;
mAb, monoclonal antibody;
wt, wild-type;
aa, amino acid(s);
PMSF, phenylmethylsulfonyl fluoride;
PAGE, polyacrylamide gel electrophoresis;
ATP
S, adenosine
5'-O-(thiotriphosphate);
ZLLLal, Z-Leu-Leu-Leu-H
aldehyde;
ALLN, N-acetyl-Leu-Leu-norleucinal;
E1, ubiquitin-activating enzyme;
E2, ubiquitin carrier protein;
E3, ubiquitin-protein isopeptide ligase.
 |
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