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Vol. 13, No. 9, pp. 1181-1189, May 1, 1999
1 Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York, New York 10016 USA; 2 European Institute of Oncology, 20141 Milan, Italy; 3 Technion-Israel Institute of Technology, Haifa 31096, Israel
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Abstract |
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 Top
 Abstract
 Introduction
Results
Discussion
Materials and methods
References
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The cellular abundance of the cyclin-dependent kinase (Cdk)
inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in
many types of aggressive human carcinomas. p27 can be phosphorylated on
threonine 187 by Cdks, and cyclin E/Cdk2 overexpression
can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the
ubiquitin-proteasome system or an alternative pathway is still not
known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo
and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27.
Furthermore, ubiquitination of wild-type p27, but not of p27(T187A),
can occur in G1-enriched extracts only upon addition of
cyclin E/Cdk2 or cyclin A/Cdk2. Using a
phosphothreonine 187 site-specific antibody for p27, we show that
threonine 187 phosphorylation of p27 is also cell-cycle dependent,
being present in proliferating cells but undetectable in G1
cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a
trimeric complex with the cyclin and Cdk subunits. In fact, cyclin
B/Cdk1 which can phosphorylate p27 efficiently, but
cannot form a stable complex with it, is unable to stimulate p27
ubiquitination by G1 extracts. Furthermore, another p27
mutant [p27(CK
)] that can be phosphorylated by cyclin
E/Cdk2 but cannot bind this kinase complex, is refractory
to ubiquitination. Thus throughout the cell cycle, both phosphorylation
and trimeric complex formation act as signals for the ubiquitination of
a Cdk inhibitor.
[Key Words: Ubiquitination; cell cycle; p27; Cdk; Cki; tumor suppressor]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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The eukaryotic cell cycle is regulated by the sequential activation
of cyclin-dependent kinases (Cdks) (for review, see Pines 1998
; Sheaff
and Roberts 1998). Cdk activation is regulated by phosphorylation of the catalytic subunit and by binding to activating (cyclins) and inactivating subunits (Cdk inhibitory proteins, or Ckis).
Whereas the cellular levels of Cdk subunits appear to vary little
through the cell division cycle phases, the cellular abundance of
cyclins and Cki subunits can change rapidly in response to
transcriptional or post-transcriptional events, including degradation through the ubiquitin-proteasome pathway (for review, see Pagano 1997).
The Cki p27 specifically inhibits cyclin E/Cdk2 and
cyclin A/Cdk2, two kinases necessary for DNA replication
to occur. It was shown originally that in response to mitogenic
stimuli, the levels of p27 decrease allowing Cdk2 activation and entry
into S phase. Previously, we demonstrated that the p27 half-life is longer in quiescent than in proliferating cells (Pagano et al. 1995).
Furthermore, we showed that inhibition of the proteasome in intact
cells leads to the accumulation of p27 and polyubiquitinated p27
species. Accordingly, p27 accumulates at the restrictive temperature in
the murine ts20TGR cell line that bears a mutation
in the ubiquitin-activating enzyme E1 gene. Several lines of
evidence show that degradation of p27 can be recapitulated in an in
vitro system (Pagano et al. 1995; Brandeis and Hunt 1996; Millard et
al. 1997) and that it requires both ubiquitination and the activity of
the proteasome (Pagano et al. 1995; Loda et al. 1997). First, extracts
from proliferating cells degrade p27 faster than extracts from
quiescent cells; second, incubation of p27 with proteasome-depleted
extracts results in a block of its degradation and upon readdition of
purified proteasome particles, p27 degradation is restored completely;
third, ATP depletion prevents the appearance of p27 polyubiquitinated
species and inhibits its proteolysis; fourth, addition of ATP-
-S,
an ATP analog that can be hydrolyzed by the E1 ubiquitin-activating enzyme but not by the proteasome, leads to a substantial decrease in
p27 proteolysis and to the accumulation of p27 polyubiquitinated species.
Phosphorylation plays a major role in ubiquitin-mediated proteolysis.
In Saccharomyces cerevisiae, phosphorylation of G1
regulatory proteins is necessary for their recognition by a specific
ubiquitin protein ligase and for their consequent ubiquitination and
degradation (for review, see Pagano 1997). Degradation of p27 is
stimulated by Cdk2-dependent phosphorylation (Muller et al. 1997;
Sheaff et al. 1997; Vlach et al. 1997). The vast majority, if not all, of Cdk-dependent phosphorylation of p27 is on threonine 187 (T187) (Alessandrini et al. 1997; Sheaff et al. 1997; Vlach et al. 1997). In
fact, (1) the phosphorylation site containing T187 is the only Cdk-consensus site in human and mouse p27, (2) amino acid analysis of
p27 phosphorylated in vitro by either Cdk2 or Cdk1 shows
phosphorylation exclusively on threonine, (3) p27(T187A) is no longer a
Cdk-substrate, and (4) coexpression of cyclin E/Cdk2 and
p27, stimulates T187 phosphorylation of p27. However, it is unclear
whether T187 phosphorylation acts as a signal for p27 ubiquitination or
whether it controls an alternative degradation pathway. In fact, p27
(Levkau et al. 1998) and other substrates of the ubiquitin pathway such
as p53 (Kubbutat and Vousden 1998), cyclin D1 (Choi et al. 1997), 
catenin (Brancolini et al. 1997), I
B
(Cuervo et al. 1998),
and others are also substrates of other proteolytic systems (e.g.,
caspases, calpain, lysosomal proteases, etc.), especially under
conditions of cellular stress and upon overexpression of these
substrates. In addition, for at least one protein, ornithine
decarboxylase, it has been shown that proteasome-mediated degradation
can occur in the absence of ubiquitination (for review, see Pickart
1997). Thus, understanding whether p27 phosphorylation regulates its ubiquitination is fundamental to determining which pathway regulates the degradation of p27 occurring in late G1 phase of the cell cycle. Here we present studies on p27 ubiquitination that address these
crucial points.
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Results |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Cdk-dependent phosphorylation on T187 is required for p27 ubiquitination
We have developed an in vitro p27 ubiquitination assay that requires
human cell extracts as a source of the enzymes necessary to conjugate
ubiquitin to p27 and ATP (Pagano et al. 1995). We now asked whether ATP
was necessary solely for the activation of ubiquitinating enzymes or
also for protein phosphorylation. Charging of the E1
ubiquitin-activating enzyme with ubiquitin requires the hydrolysis of
the - bond of ATP, whereas protein phosphorylation reactions
involve the transfer of the phosphate of ATP. We tested whether a
- nonhydrolyzable ATP analog (AMP-PNP), which can replace
ATP in the E1-charging reaction but not in a kinase reaction, would
allow p27 ubiquitination to occur. ATP was depleted by the addition of
hexokinase and deoxyglucose and then AMP-PNP or ATP and an ATP
regeneration system were added. In the absence of ATP, p27 was not
ubiquitinated (Fig. 1A, lanes 1,2) (Pagano et al.
1995). Readdition of ATP but not of AMP-PNP was able
to support p27 ubiquitination (lanes 3,4). To confirm that
phosphorylation is necessary for p27 ubiquitination, the reaction was
performed in the presence of staurosporine, a potent broad-specificity
kinase inhibitor, which inhibited p27 ubiquitination completely (lane
5).
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Degradation of p27 is stimulated by its phosphorylation on threonine
187 (T187), and the vast majority, if not all, of Cdk-dependent phosphorylation of p27 is on T187 (Muller et al. 1997; Sheaff et al.
1997; Vlach et al. 1997; data not shown). We therefore asked whether
Cdk activity and the integrity of T187 were also essential for p27
ubiquitination in vitro. First, we used the Cdk-specific kinase
inhibitor flavopiridol (Meijer 1995; Carlson et al. 1996) and found
that this compound inhibited p27 ubiquitination (lanes 6-8). We then
depleted Cdks from cell extracts using p13 beads (Brizuela et al.
1987). Extracts depleted with p13 beads but not with control beads were
unable to sustain p27 ubiquitination unless reconstituted with
recombinant purified cyclin E/Cdk2 complex (lanes 9-11).
Finally, we compared wild-type p27, p27(T187A), and another
proline-directed phosphorylation site p27 mutant, p27(S178A), for their
ability to be ubiquitinated in vitro. p27(T187A), but not p27(S178A),
failed to be conjugated with ubiquitin (Fig. 1B).
In summary, our results show that in vitro ubiquitination of p27 requires its Cdk-dependent phosphorylation on T187.
p27 ubiquitinating activity is cell-cycle regulated
Compared to proliferating or S-phase cells, G0 and
G1 cells contain much lower levels of p27 proteolytic
activity when assayed either in vitro (Pagano et al. 1995; Brandeis and
Hunt 1996; Millard et al. 1997) or in vivo (Pagano et al. 1995; Hengst
and Reed 1996). We asked whether this difference in degradation ability
reflects a difference in ubiquitination-specific degradation activity. We incubated p27 with extracts made from either proliferating HeLa
cells or from HeLa cells enriched in the G1 population with lovastatin, or alternatively with extracts made from either
G1 or quiescent (G0) human diploid fibroblasts.
Extracts from proliferating cells were able to sustain p27
ubiquitination, whereas the G0 or G1 extracts were
not active in p27 ubiquitin conjugation (Fig. 2, lanes
1,2,10,11). In contrast, both the proliferating cell extract and the G1 extract were active in a cyclin B in vitro ubiquitination assay (Fig. 2, lanes 8,9), in agreement with fact that
the cyclin B-specific ligase is active in G1 (Amon et al. 1994). The addition of recombinant purified cyclin E/Cdk2
to G1 extracts enabled ubiquitination of wild-type p27 to an
extent similar to that observed in proliferating extracts (Fig. 2,
lanes 3,12). Similarly, cyclin E/Cdk2 stimulated the in
vitro degradation of p27 by G1 extracts (data not shown). In
contrast, p27(T187A) mutant was not ubiquitinated (Fig. 2, lanes 6,7)
or degraded (data not shown) even in the presence of recombinant
purified cyclin E/Cdk2. Staurosporine inhibited the
stimulation of ubiquitination by cyclin E/Cdk2 (Fig. 2,
lane 4). This effect was likely caused by the inhibition of cyclin
E/Cdk2 kinase activity, because when cyclin
E/Cdk2 was first incubated with p27 and then added to the reaction in the presence of staurosporine, p27 ubiquitination in a
G1 extract still occurred efficiently (Fig. 2, lane 5).
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These results show that the enzymatic activity missing from G1 extracts can be provided by cyclin E/Cdk2. In contrast, quiescent-cell extracts were rescued very poorly by the addition of cyclin E/Cdk2 (Fig. 2, lane 13) indicating that yet another activity, perhaps that of a ubiquitin ligase, is missing from those extracts.
p27 phosphorylation on T187 and p27 ubiquitination are cell-cycle regulated
As the half life of p27 is longer in G1 cells than in
proliferating ones (Pagano et al. 1995; Hengst and Reed 1996), and its ubiquitination requires its phosphorylation on T187, we asked whether
p27 phosphorylation on T187 and p27 ubiquitination were cell-cycle
regulated. We generated a phospho-T187 site-specific p27 antibody using
a phosphopeptide that spans the phosphorylated T187 residue of p27.
After affinity chromatography using both phospho- and nonphosphopeptide
columns, the purified antibody recognized recombinant p27 only when
phosphorylated by cyclin E/Cdk2 (Fig. 3A, lanes
1,2). To determine whether p27 is phosphorylated on
T187 in a cell-cycle-regulated manner, we immunoprecipitated total p27
from G1-enriched, proliferating, and G1-depleted
HeLa cells (prepared as described in Materials and Methods), and then we immunoblotted with the phospho-T187 site-specific p27 antibody. The
phospho-T187-specific p27 antibody detected phosphorylated p27 only in
the immunoprecipitates from proliferating and G1-depleted cells (Fig. 3A, top panel, lanes 3-5), whereas an anti-p27 antibody detected p27 in all three samples, with the highest levels found in the
G1-enriched population (Fig. 3A, bottom panel, lanes 3-5). In a parallel experiment, the G1-enriched, proliferating, and G1-depleted HeLa cell extracts were denatured to dissociate
p27 from any interacting proteins, and immunoprecipitated with a
mixture of the phospho-T187-specific p27 antibody and an anti-p27
antibody. Immunoblotting of the immunoprecipitates with an antibody to
ubiquitin detected ubiquitinated p27 in G1-depleted but not
in G1-enriched cells (Fig. 3B).
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Our results demonstrate that endogenous cellular p27 is phosphorylated on T187 and that this phosphorylation, along with p27 ubiquitination, is cell-cycle regulated, being absent in G1 and present in proliferating cells.
p27 ubiquitination requires its stable binding to a cyclin/Cdk complex
The experiments described above show that G1-enriched
extracts are unable to sustain p27 ubiquitination unless cyclin
E/Cdk2 is added to the reaction. Surprisingly, cyclin E
in a complex with an inactive catalytic mutant of Cdk2
[Cdk2m (Desai et al. 1992)] was able to stimulate p27
ubiquitination although to a lesser extent than wild-type cyclin
E/Cdk2 (Fig. 4A, lanes 2,3). This
stimulation was not caused by the presence of cyclin E in the cyclin
E/Cdk2m complex, as cyclin E alone did not
stimulate conjugation of ubiquitin to p27 (Fig. 4A, lane 5). We
speculated that the stimulation of p27 ubiquitination by cyclin
E/Cdk2 is caused by both p27 phosphorylation and the
formation of a p27/cyclin/Cdk complex,
which allows its recognition by the ubiquitin ligase. This is the case
for other proteins that require phosphorylation and assembly with other subunits for ubiquitination [e.g., IB (Yaron et al. 1997)]. If this hypothesis is correct, the stimulation of p27 ubiquitination by
cyclin E/Cdk2m could be caused by complex
formation and phosphorylation by low levels of the cyclin
E/Cdk2 present in G1 extracts.
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All p27 ubiquitin conjugation reactions were performed in the presence
of okadaic acid, a potent phosphatase inhibitor able to stabilize p27
phosphorylated forms (data not shown). Removal of okadaic acid from the
reaction mix completely blocked the stimulation of p27 ubiquitination
by cyclin E/Cdk2m (Fig. 4A, lane 4). We then
performed a p27 phosphorylation reaction with cyclin
E/Cdk2m followed by a ubiquitination reaction
using G1 cell extracts that had been preincubated with
flavopiridol. Under these conditions cyclin
E/Cdk2m was unable to induce p27 ubiquitination
even in the presence of okadaic acid (Fig. 4A, lanes 6-8), indicating
that cyclin E/Cdk2m stimulation of p27
ubiquitination requires a kinase present in the cellular extract. Cdk2
activity in these G1-enriched extracts is present in
sufficient amounts to phosphorylate some p27 in the presence of okadaic
acid (data not shown). As 0.3 µM flavopiridol shows high
specificity towards Cdk2 and Cdk4 (Meijer 1995), it is very likely that
the kinase present in the G1 extract necessary to stimulate
p27 ubiquitination in the presence of cyclin
E/Cdk2m is a G1 Cdk, and is most
likely Cdk2 as Cdk4 is inactive in HeLa cell extracts (Tam et al.
1994).
To demonstrate that p27 ubiquitination requires both Cdk-dependent
phosphorylation as well as its binding to the kinase complex, we used
cyclin E/Cdk2, cyclin B/Cdk1, and cyclin
A/Cdk2 complexes which, although having different
affinities for p27 (Polyak et al. 1994; Toyoshima and Hunter 1994),
were all able to phosphorylate wild-type p27 on T187 (as detected with
a phospho-T187 site-specific p27 antibody) but not p27(T187A) (data not
shown). We tested cyclin E/Cdk2, cyclin
E/Cdk2m, cyclin B/Cdk1, and
cyclin A/Cdk2 for their ability (1) to phosphorylate p27,
(2) to bind p27, and (3) to induce p27 ubiquitination. All three of
these kinases were able to phosphorylate p27 (Fig. 4B, second panel)
but cyclin B/Cdk1 was unable to stably interact with p27
(Fig. 4B, third panel). These results are in agreement with published
reports showing that the affinity of p27 for cyclin B/Cdk1 is lower than that for cyclin E/Cdk2
and cyclin A/Cdk2 (Polyak et al. 1994; Toyoshima and
Hunter 1994). In contrast to cyclin A/Cdk2 and cyclin E/Cdk2, cyclin
B/Cdk1 did not bind or stimulate p27 ubiquitination at
kinase concentrations which allowed efficient p27 phosphorylation (Fig.
4B, bottom panel, lanes 1,3). Similarly, cyclin
E/Cdk2m which bound, but did not phosphorylate
p27, was unable to stimulate p27 ubiquitination efficiently (Fig. 4B,
bottom panel, lane 5) unless incubated in the presence of either cyclin
E/Cdk2 or cyclin B/Cdk1 (data not shown).
To study further the possibility that a stable interaction of cyclin
E/Cdk2 with p27 was required for ubiquitination, we used a p27 mutant that cannot stably interact with both cyclins and Cdks
[p27(CK)], but is still a substrate of cyclin
E/Cdk2 (Vlach et al. 1997). We confirmed that p27(CK)
could be phosphorylated by cyclin E/Cdk2 but did not bind
stably to this complex (Fig. 4C, top and middle panels). Importantly, p27(CK) was refractory to ubiquitin conjugation (Fig. 4C, bottom panel).
In conclusion, using three different approaches we demonstrate that efficient p27 ubiquitination requires phosphorylation on T187 and its stable interaction with a cyclin/Cdk complex.
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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In agreement with the notion that Cdk2 needs to be active for DNA
replication to occur (Pagano et al. 1992, 1993; van den Heuvel and
Harlow 1993; Ohtsubo et al. 1995) the levels of the Cdk inhibitor p27
are high in G1 and decrease as cells approach S phase.
Transcription of p27 occurs at a similar rate throughout the different
phases of the cell-cycle and, although there is translational control
of p27 levels (Hengst and Reed 1996), this does not appear to be the
main mechanism for the reduction in p27 abundance observed at the
G1/S transition (Millard et al. 1997). Rather
we have shown that the half-life of p27 is cell-cycle regulated, being
much longer in quiescent fibroblasts than in proliferating cells
(Pagano et al. 1995). Likewise, the half-life of p27 in
contact-inhibited and lovastatin-blocked cells is longer than in
asynchronous and thymidine-blocked cells, respectively (Hengst and Reed
1996). In addition, we have demonstrated that p27 is ubiquitinated both
in vivo and in vitro (Pagano et al. 1995).
p27 is phosphorylated on threonine 187 by cyclin E/Cdk2
and overexpression of this complex in mammalian cells induces
degradation of transfected p27 (Muller et al. 1997; Sheaff et al. 1997;
Vlach et al. 1997). However, it had not been clear whether T187
phosphorylation targets p27 for ubiquitin-mediated degradation or for
other degradation pathways. In fact, it has been shown that, in
addition to cell-cycle arrest, p27 overexpression induces apoptosis
(Katayose et al. 1997) and that during apoptosis p27 is cleaved by
caspases (Levkau et al. 1998). We now have demonstrated that
Cdk-mediated phosphorylation of p27 on threonine 187 is indeed required
for p27 ubiquitination and consequent degradation. Accordingly, it has
been shown recently that the cell-free degradation of p27 is dependent
on Cdk2 activity (Nguyen et al. 1999). Furthermore, we have established
that ubiquitination of p27 bound to a cyclin/Cdk complex
is more efficient than the ubiquitination of uncomplexed p27.
Importantly, the phosphorylation on threonine 187 and the
ubiquitination of cellular p27, as well as p27 ubiquitinating activity
(as assayed in vitro) were found to be regulated collectively during
the cell cycle, being present in proliferating cells but not in
G0- and G1- arrested cells.
Increased turnover of p27 starts in late G1 and continues
until mitosis. It is conceivable, therefore, that different Cdks are
responsible for p27 phosphorylation on T187 at different points of the
cell cycle. Cdk2 and Cdk1 are the candidate kinases for p27
phosphorylation/binding and subsequent ubiquitination.
Cdk3 is also a good candidate as its function is requested for the G1/S transition and has several properties
similar to those of Cdk2 and Cdk1 (van den Heuvel and Harlow 1993). In
contrast, Cdk4 does not seem to play a role in inducing p27 proteolysis
since co-expression of cyclin D1 and Cdk4 does not result in p27
elimination (Sheaff et al. 1997); however, the cyclin
D1/Cdk4 complex does regulate p27 activity by
sequestration (Blain et al. 1997; Cheng et al. 1998). In agreement with
these data, we found that the cyclin D3/Cdk4 complex does
not stimulate p27 ubiquitination (Montagnoli and Pagano, unpubl.). The
published p27/cyclin A/Cdk2 X-ray
crystallography data show that p27 inserts itself within the Cdk
catalytic cleft, likely preventing ATP transfer (Russo et al. 1996).
This would exclude therefore that p27 can be phosphorylated in an
intracomplex reaction. Our results that p27 bound to an inactive cyclin
E/Cdk2m complex can be phosphorylated by an
unbound cyclin B/Cdk1 complex (Montagnoli and Pagano,
unpubl.), rather demonstrate that an intercomplex phosphorylation of
p27 is possible.
p27 inhibits different Cdks with different affinities (Polyak et al.
1994; Toyoshima and Hunter 1994; Blain et al. 1997). We have shown here
that the abilities of Cdk2 and Cdk1 to phosphorylate p27 on T187 and to
induce p27 ubiquitination do not correlate. Therefore, although
phosphorylation is necessary for p27 ubiquitination, it is not
sufficient. In fact, cyclin B/Cdk1 in amounts sufficient to phosphorylate but not bind efficiently p27, does not stimulate p27
ubiquitination. Similarly, p27(CK), a mutant unable to bind cyclin
E/Cdk2 complex is still phosphorylated by this kinase but
cannot be ubiquitinated. Finally, a cyclin E/Cdk2m complex that binds p27 but cannot
phosphorylate it, stimulates p27 ubiquitination by relying on a Cdk
(Fig. 4A, lanes 6-8, flavopiridol-sensitive) present in the
G1 extract. It is conceivable that p27 phosphorylation by and
binding to a cyclin/Cdk complex expose a domain in p27 that is recognized by a specific ubiquitin ligase, or, alternatively, the cyclin/Cdk complex binds and targets this ligase to p27.
The inability of cyclin E/Cdk2 to stimulate p27
ubiquitination in extracts from quiescent (G0) cells suggests
that another factor, perhaps a cell-cycle-regulated ubiquitin ligase
necessary for p27 ubiquitination, is limiting. In yeast,
phosphorylation of G1 regulators (e.g., Ckis, cyclins) allows
their recognition by ubiquitin ligases called SCFs because they are
formed by three subunits: S-phase kinase-associated protein 1 (Skp1), CulA, and one of many F-box
proteins (for review, see Pagano 1997). Whereas CulA interacts directly
with a ubiquitin-conjugating enzyme, the F-box protein subunit recruits
specific phosphorylated substrates. We and others have shown that, as
in yeast, the human homolog Skp1/Cul1 complex forms a
scaffold for multiple F-box proteins, including Skp2 (for review, see
Patton et al. 1998), -transducin repeat-containing protein Trcp,
also called Slimb or Fbp1 (Latres et al. 1999; Winston et al. 1999),
and other novel F-box proteins (C. Cenciarelli, D.S. Chiaur, S. Murthy,
D. Guardavaccaro, M. Loda, G. Inghirani, W. Parks, M. Vidal, D. Demetrick, and M. Pagano, in prep.). These different SCF complexes
potentially target different substrates for ubiquitin-mediated
degradation. We are currently assessing whether a specific F-box
protein targets p27 for ubiquitination.
The p27/cyclin/Cdk complex is very stable
and even when phosphorylated on T187, p27 does not appear to dissociate
from the cyclin/Cdk complex. We have reported here that
Cdk-dependent phosphorylation of p27 at T187 together with the
formation of a stable p27/cyclin/Cdk trimeric complex are both required for ubiquitination to occur. Thus,
it is possible that p27, in response to specific mitogenic stimuli, is
removed physically by its ubiquitination and/or
degradation from the complex which it inhibits. p27 could be regulated
similarly to IB, whose ubiquitination requires both its
phosphorylation and its stable binding to NF-B (Yaron et al.
1997).
In summary, the timely regulation of p27 cellular abundance and activity derives from a combination of several factors, which include the ability of certain cyclin/Cdk complexes to interact stably with p27, the cellular abundance and specific activity of Cdk complexes which phosphorylate p27, as well as the availability of yet-to-be-identified p27-specific ubiquitin ligase.
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Materials and methods |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Protein extraction for in vitro ubiquitination assay
Approximately 4 ml of HeLa S3 cell pellets were suspended in 6 ml
of ice-cold buffer consisting of 20 mM Tris-HCl (pH 7.2), 2 mM DTT, 0.25 mM EDTA, 10 µg/ml
leupeptin, and 10 µg/ml pepstatin. The suspension was
transferred to a cell nitrogen-disruption bomb (Parr, Moline, IL) that
had been rinsed thoroughly and chilled on ice before use. The bomb
chamber was connected to a nitrogen tank and the pressure was brought
slowly to 1000 psi. The chamber was left on ice under the same pressure
for 30 min, and the pressure was released slowly. The material was
transferred to an Eppendorf tube and centrifuged in a microcentrifuge
at 10,000g for 10 min. The supernatant (S-10) was divided into
smaller samples and frozen at 
80°C. This method of extract
preparation based on the use of a cell nitrogen-disruption bomb extract
preserves the activity of in vitro ubiquitinate p27 better than the
method described previously (Pagano et al. 1995) (data not shown).
In vitro ubiquitination assay
In vitro-translated 35S-labeled p27 (1 µl) was
incubated at 30°C for different times (0-75 min) in 10 µl of
ubiquitination mix containing 40 mM Tris at pH 7.6, 5 mM MgCl2, 1 mM DTT, 10% glycerol, 1 µM ubiquitin aldehyde, 1 mg/ml methyl
ubiquitin, 10 mM creatine phosphate, 0.1 µg/ml creatine kinase, 0.5 mM ATP, 1 µM okadaic acid, and 20 µg of HeLa cell extract. In
the indicated samples, purified active His-cyclin E/Cdk2
was added. In the experiments in which flavopiridol effects were
tested, ATP was used at the concentration of 5 µM.
Reactions were stopped with Laemmli sample buffer containing
-mercaptoethanol and their products were run on protein gels under
denaturing conditions. Polyubiquitinated p27 forms were identified by
autoradiography. Ubiquitin aldehyde was added to the ubiquitination
reaction to inhibit the isopeptidases that would remove the chains of
ubiquitin from p27. Methyl ubiquitin was added because it competes with
the ubiquitin present in the cellular extracts and terminates p27
ubiquitin chains that can be appreciated as discrete bands instead of a
high molecular smear. These shorter polyubiquitin chains have lower
affinity for the proteasome and therefore are more stable. In the
absence of methyl ubiquitin, p27 degradation activity, instead of p27
ubiquitination activity, could be measured (data not shown). In
addition to the fact that p27 mutant lacking all 13 lysines (a gift of
B. Amati, Swiss Institute for Experimental Cancer Research,
Switzerland) was not ubiquitinated, a further formal demonstration that
p27 high-mobility species are ubiquitinated was obtained using an immunoprecipitation with an antibody to p27 followed by a subsequent immunoprecipitation with an anti-ubiquitin antibody (data not shown).
Reagents and antibodies
Ubiquitin aldehyde (Hershko and Rose 1987), methyl-ubiquitin
(Hershko and Heller 1985) and p13 beads (Brizuela et al. 1987) were
prepared as described. AMP-PNP, staurosporine, hexokinase, and
deoxyglucose were from Sigma; lovastatine form Merck; flavopiridol was
from Hoechst Marion Roussel. The preparation, purification, and
characterization of a mouse monoclonal antibody (mAb) to human p27
(clone 4FIIDII) and a polyclonal against human p27 were performed in
collaboration with Zymed. In the indicated case, a goat anti-p27 from
Santa Cruz was used. Ubi-1-1510 mAb to ubiquitin was from Zymed and Rat
anti-HA antibody was from Boehringer Mannheim. The phospho-site
p27-specific antibody was generated in collaboration with Zymed by
injecting rabbits with the phospho-peptide NAGSVEQT*PKKPGLRRRQT, corresponding to the carboxyl terminus of the human p27 with a phosphothreonine at position 187 (T*). The antibody was then purified from serum with two rounds of affinity chromatography using both phospho- and nonphosphopeptide chromatography.
p27 mutants
All p27 constructs were derived from the human cDNA
sequence. Point mutations described in the text were generated by
oligonucleotide-directed mutagenesis using the polymerase chain
reaction of the QuikChange site-directed mutagenesis kit (Stratagene).
All mutants were sequenced in their entirety. Mouse p27 wild type and
mouse p27(CK) mutant (R30A, L32A, F62A, F64A) (Vlach et al. 1997)
used in Figure 3C were obtained from B. Amati.
Recombinant proteins
Baculoviruses expressing human His-tagged cyclin A, His-tagged
cyclin B, His-tagged cyclin E, HA-tagged Cdk2, HA-tagged Cdk1, HA-tagged catalytic inactive Cdk2 (K33T, K34S) (Cdk2m) were
supplied by D. Morgan (Desai et al. 1992). Recombinant viruses were
used to infect 5B cells as described (Hannon 1995) and assayed for
expression of their encoded protein by immunoblotting. Cyclin-Cdk
complexes and uncomplexed cyclin subunits were purified by
nickel-agarose chromatography (Invitrogen) according to the manufacturer's instructions.
Cell synchronization and extract preparation
HeLa S3 cells and human lung fibroblasts, IMR-90, were obtained
from the American Type Culture Collection. G1-enriched HeLa cells were obtained with a 24-hr lovastatin treatment (O'Connor and
Jackman 1995), and G1-depleted cells with an 11-hr treatment with nocodazole (40 ng/ml). Synchronization was monitored
by flow cytometry (percent of cells in G1, S and
G2/M is reported in the Fig. 3 legend). IMR-90
were synchronized in G0 by serum starvation for 48 hr, and in
G1 by serum starvation followed by serum readdition for 8-10
hr. Cell-cycle analysis by flow cytometry showed that after serum
starvation, ~90% of the cells had a 2N DNA content. Conditions for
protein extraction have been described previously (Pagano et al. 1993).
Immunoprecipitation and immunoblotting
Cell extracts were prepared with a 0.1% Triton X-100 lysis buffer
as described (Pagano et al. 1995). Proteins were first denatured with
1% SDS and then diluted with 1% Triton X-100 lysis buffer prior to
addition of the antibody. Protein extract (3 mg) was immunoprecipitated
as described (Pagano et al. 1995) with either 15 µg/ml affinity purified (AP) anti-p27 monoclonal
antibody or a mixture of AP anti-p27 antibody (5 µg/ml) and AP phosphosite p27-specific antibody (15 µg/ml). Conditions for immunoblotting have been
described previously (Pagano et al. 1995).
Kinase assay
In vitro-translated p27 (1 µl) immunoprecipitated with an
anti-p27 antibody or 1 µg of recombinant purified p27 was incubated for 1 hr at 30°C in the presence of 50 mM Tris-Cl at pH
7.5, 10 mM MgCl2, 1 mM DTT, 30 µM ATP, 5 µCi [-32P]ATP, and
50-200 ng of recombinant purified cyclin B/Cdk1 or cyclin E/Cdk2 complex.
| |
Acknowledgments |
|---|
We thank B. Amati, D. Bohmann, J. Bolen, M. Garabedian, and D. Morgan for reagents, J. Dominguez and C. Mercurio for their contribution to this work; and J. Bloom and L. Yamasaki for reading the manuscript critically. M.P. is grateful to L. Yamasaki and T.M. Thor for their continuous support. A.M. is supported in part by an Italian Foundation for Cancer Research (FIRC) fellowship; A.C.C. by a supplement for under-represented minorities to the National Institutes of Health (NIH) RO1 grant no. GM57587; G.F.D. by grants from the Italian Association for Cancer Research (AIRC)/FIRC, from the Italian National Research Council (Progetto Strategico `Ciclo Cellulare e Apoptosi' e Progetto Finalizzato `Biotecnologie'); A.H. by a HFSPO grant (RG0229/98-M); M.P. by a Human Frontiers Science Program Organization (HFSPO) grant (RG0229/98-M) and by NIH RO1 grants CA76584 and GM57587.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked `advertisement' in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received January 25, 1999; revised version accepted March 10, 1999.
4 Corresponding author.
E-MAIL paganm02{at}med.nyu.edu; FAX (212) 263 8211.
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References |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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-Trcp associates with the Cul1/Skp1 complex and regulates the stability of -catenin.
Oncogene
18:
849-854[CrossRef][Medline].
-TRCP ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IB and -catenin and stimulates IB ubiquitination in vitro.
Genes & Dev.
13:
270-283B cellular function via specific targeting of the IB-ubiquitin ligase.
EMBO J.
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6486-6494[CrossRef][Medline].
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the IgG heavy chains (Ig).
