From the Laboratoire de Biologie Cellulaire et Moléculaire du
Contrôle de la Prolifération, CNRS UMR 5088, IFR 109, Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse
Cedex, France and the Laboratoire Biochimie et
Technologie des Aliments USC-INRA 429, Université Bordeaux I,
Avenue des Facultés, 33405 Talence Cedex, France
Received for publication, September 17, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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We investigated the status and the regulation of
the cyclin-dependent kinases (CDK) inhibitor
p27Kip1 in a choroidal melanoma tumor-derived cell
line (OCM-1). By contrast to normal choroidal melanocytes, the
expression level of p27Kip1 was low in these cells and the
mitogen-activated protein (MAP) kinase pathway was constitutively
activated. Genetic or chemical inhibition of this pathway induced
p27Kip1 accumulation, whereas MAP kinase reactivation
triggered a down-regulation of p27Kip1 that could be
partially reversed by calpain inhibitors. In good accordance, ectopic
expression of the cellular calpain inhibitor calpastatin led to an
increase of endogenous p27Kip1 expression. In
vitro, p27Kip1 was degraded by calpains, and OCM-1
cell extracts contained a calcium-dependent
p27Kip1 degradation activity. MAP kinase inhibition
partially inhibited both calpain activity and
calcium-dependent p27Kip1 degradation by
cellular extracts. Immunofluorescence labeling and subcellular
fractionation revealed that p27Kip1 was in part localized
in the cytoplasmic compartment of OCM-1 cells but not of melanocytes,
and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase
activation triggered a cytoplasmic translocation of the protein, as
well as a change in its phosphorylation status. This
CRM-1-dependent cytoplasmic translocation was necessary for
MAP kinase- and calpain-dependent degradation. Taken
together, these data suggest that in tumor-derived cells,
p27Kip1 could be degraded by calpains through a MAP
kinase-dependent process, and that abnormal cytoplasmic
localization of the protein, probably linked to modifications of its
phosphorylation state, could be involved in this alternative mechanism
of degradation.
Progression through the cell cycle involves the sequential
activation and inhibition of cyclin-dependent kinases
(CDKs).1 CDK activities are
regulated by their association with cyclins, by
phosphorylation/dephosphorylation events, and through their direct
interaction with small inhibitory proteins called CDK inhibitors (1). Two gene families of CDK inhibitors are known: the CDK4 and CDK6
specific inhibitors of the INK4 family, and the Kip/Cip inhibitors,
which include p21Cip1 and p27Kip1.
p27Kip1 accumulates in serum-starved and density-arrested
cells, as well as in response to antiproliferative stimuli (2 ,3). Its accumulation is associated with cell cycle arrest in G1
and/or with cell entry into quiescence (4). Stimulation of quiescent cells by growth factors leads to a down-regulation of
p27Kip1 that is necessary for the G1/S
transition (5). Indeed this CDK inhibitor, first considered as a broad
specificity inhibitor, now appears as a specific inhibitor of
CDK2-cyclin E during the G1 phase. Recently, a number of
clinical studies have linked low levels and reduced half-life of the
p27Kip1 protein with a poor survival prognosis for many
tumor types (for reviews see Refs. 6 and 7). Consequently the
regulation of p27Kip1 expression has received special
attention during the last few years.
Factors modifying the stability of this protein are clearly important,
and various mechanisms of p27Kip1 degradation have been
described. The best characterized is the ubiquitin-dependent degradation by the proteasome. This
involves phosphorylation of residue Thr187 by CDK2 (8-10),
association with the F-box protein p45Skp2 (11-13),
ubiquitination (14, 15), and degradation by the proteasome in the
cytoplasmic compartment. p27Kip1 nucleocytoplasmic
translocation involves association with the protein Jab1 (16), and with
the nuclear pore-associated protein mNPAP60 (17). This pathway has been
extensively dissected and appears to be involved in a variety of
physiological processes. However, other p27Kip1 degradation
mechanisms have also been described. These include ubiquitin-independent degradation by the proteasome (18), caspase degradation in apoptotic (19, 20) or nonapoptotic conditions (21), and
degradation by calpain (22). In addition, p27Kip1 levels
can be controlled by post-transcriptional (23-25) and transcriptional (26-30) mechanisms influencing its synthesis.
The signal transduction pathways leading to p27Kip1
regulation have been partially identified. One of them is the
phosphatidylinositol 3-kinase pathway (31, 32), which acts both at the
levels of transcription (27) and of protein degradation (33). Mitogenic stimulation also activates the p42/p44 MAP kinase (indicated as MAP
kinase in the text). This pathway was also found to affect p27Kip1 regulation in several ways. In some studies, MAP
kinase activation by constitutive Raf or MEK mutants led to a
down-regulation of p27Kip1 (34-36). In contrast, similar
activation in other cases did not modify the p27Kip1 level,
but rather its association with CDKs (37-39). Finally, accumulation of
p27Kip1 following MAP kinase activation has also been
described (40 ,41). Recently, we described a MAP
kinase-dependent degradation of p27Kip1
independent of CDK2 activity in NIH3T3 cells (42).
Previous studies indicate that calpains can regulate several proteins
involved in cell cycle progression, notably p53 (44), p21 (45), cyclin
D1 (46), and p27Kip1 (22). The calpains are a family of
nonlysosomal, calcium-dependent proteases including the
ubiquitous calpains I and II, various tissue-specific isoforms, and a
28-kDa regulatory subunit (43). The activity of these proteases are
tightly regulated by a highly specific endogenous inhibitor named
calpastatin. In the present study, we demonstrate the existence of a
calpain- and MAP kinase-dependent degradation of
p27Kip1 in tumor-derived choroidal melanoma cells, and we
show that nuclear export is necessary for this process.
Cell Culture--
Primary cultures of human choroidal
melanocytes were established essentially as previously described and
their purity was assessed by immunocytochemistry using specific
markers, cytofluorometric analysis, transforming growth factor-
The melanoma cell line (spindle-shape OCM-1) was kindly provided by Dr.
S. Saornil (Valladolid Institute, Spain). These cells were cultured in
RPMI 1640 medium supplemented with 100 IU/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, and 5% fetal calf serum. The calpain inhibitors calpeptin, ALLM, PD150606, the
calpastatin peptide, and the corresponding control peptide were from
Calbiochem.
Transient Cell Transfection--
Transient transfection
experiments were performed with LipofectAMINE (Invitrogen) as
follows. Cells were plated at 106 cells per well in 6-well
culture dishes. After 24 h, the transfection was usually performed
with 1 µg of plasmid DNA mixed with 4 µl of LipofectAMINE reagent.
Cells were incubated for 3 h with this mixture in 1 ml of serum-
and antibiotic-free culture medium. At that time, 2 ml of medium
containing antibiotics and 15% serum were added, and cells were left
overnight in the presence of the transfection mixture. Cells were then
placed in the presence of fresh culture medium containing 10% serum
and then harvested after appropriate times and treatments depending on
the experiment. The dominant negative (S222A) hemagglutinin 1-tagged
MEK-1 (MEKSA) in pCDN3 was kindly provided by
Alain Eychène (Orsay, France). The
pCDNA3-calpastatin expression vector was provided by
Marc Piechaczyk (Montpellier, France).
Polyacrylamide Gel Separation and Western Blots--
For
one-dimensional electrophoresis, cells were harvested in Laemmli sample
buffer and separated on 10% SDS-PAGE gels.
For two-dimensional isoelectric focusing separations (IEF), cells were
lysed in 25 mM Tris, pH 7.4, 1% Nonidet P-40, 10 mM
Proteins from SDS-polyacrylamide gels were transferred on a
nitrocellulose membrane (20 V, 30 min). The membranes were then blocked
by 5% nonfat milk and 1% bovine serum albumin in TBS-T (TBS with
0.05% Tween 20), incubated with primary antibody for 1 h, washed
3 times for 10 min in TBS-T, incubated with the secondary horseradish
peroxidase-linked antibody for 30 min, and washed three times. All
dilutions were performed in TBS-T. The horseradish peroxidase activity
was detected by an incubation of the membrane for 1 min in ECL
(Amersham Biosciences). Quantification of Western blots was performed
by densitometric analysis with the ImageQuant software.
Immunoprecipitation--
p27Kip1 was
immunoprecipitated from OCM-1 fractions with the C-19 polyclonal
antibody (Santa Cruz). Cell fractions were prepared in low stringency
RIPA buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2,5 mM EGTA, 1 mM DTT,
0.2% Tween 20, 10% glycerol, 10 mM Nuclear Cytoplasmic Fractionation--
OCM-1 and TCM-8 cells
were harvested and resuspended in ice-cold buffer B (10 mM
Tris, pH 7.5, 10 mM NaCl, 3 mM
MgCl2, 0,1% Nonidet P-40). After 15 min incubation in ice,
cells were disrupted with several strokes of a Dounce homogeneizer.
Nuclei and unbroken cells were sedimented by centrifugation (800 × g, 4 °C) for 5 min and the supernatant was kept as the
cytoplasmic fraction. The pellets were resuspended in buffer B, placed
over a sucrose "cushion" (30% sucrose, 50 mM Tris-HCl,
pH 8,3, 5 mM MgCl2,, 0.1 mM EDTA)
and centrifuged for 10 min (800 × g, 4 °C). The
corresponding pellet containing purified nuclei was washed once with
buffer B, resuspended in buffer B, sonicated for 10 s, and heated
for 1 min at 90 °C for DNA denaturation.
Cytoplasmic and nuclear fractions were quantified by Bradford assay
(Bio-Rad). An equal amount of protein from each sample was separated by
a 12.5% SDS-PAGE and analyzed by Western blot.
Immunofluorescence Analysis--
Cells were grown on coverslips
in 4-well plates. After treatment, cells were fixed in 3%
paraformaldehyde for 30 min at room temperature, washed twice in PBS,
and permeabilized with 0.5% Triton X-100 for 5 min at room
temperature. After 3 washes in PBS, saturation was performed by
incubation for 10 min in 1% fetal calf serum in PBS at room
temperature. Immunolabeling was then performed as follows: coverslips
were incubated with primary antibody (monoclonal antibody against
p27Kip1 or polyclonal antibody against HA tag) for 1 h
at 37 °C, washed twice in 1% fetal calf serum in PBS and incubated
with fluorochrome-conjugated secondary antibody (A488 antibody against
mouse Ig or A594 antibody against rabbit Ig, respectively). Cells
nuclei were then stained with Hoechst 33342 (1 µg/ml, Sigma) and
coverslips were mounted with Mowiol. Fluorescence emission images were
obtained with a conventional Leica microscope system or with a Leica
confocal microscope system.
Antibodies--
Monoclonal anti-p27Kip1 (K25020)
from Transduction Laboratories was used for Western blotting (1:2000)
and immunofluorescence analysis (1:100). Polyclonal
anti-p27Kip1 (C-19) sc-528 was purchased from Santa Cruz
Biotechnology and used for immunoprecipitation at 5 µg/ml. The
antibody specific for Ser10-phosphorylated
p27Kip1 was from Santa Cruz Biotechnology, and the
anti-phospho-Thr187 was from Zymed Laboratories
Inc.. Active MAPK were detected by Western blotting with a
polyclonal antibody from Promega (1:5000). Anti-hemagglutinin (HA.11)
polyclonal antibody was from Babco and Alexa fluor antibodies were from
Molecular Probes. These antibodies were used for immunohistochemistry
at 1:500. Horseradish peroxidase-conjugated secondary antibodies were
from Biolabs. Antibodies against MKP-1, Raf-1, and ERK1 were from Santa
Cruz Biotechnology, and the antibody against MEK was from Biolabs. The
monoclonal antibody against calpastatin was from Biomol Research Laboratories.
Calpain Purification--
Calpains I and II were purified from 2 kg of fresh rabbit muscle using phenyl-Sepharose and anionic exchange
chromatographic steps according to Garret et al. (48), and
stored in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 1 mM NaN3 at 2 °C.
Zymograms--
For zymographic detection of calpains, cells were
harvested in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1%
Triton X-100, 1 mM DTT, 1 µM pepstatin, 1 µM aprotinin, 100 µM phenylmethylsulfonyl
fluoride). Separation of proteins was performed in nondenaturing
conditions, in 10% polyacrylamide gels containing 0.2% casein as
described (49). After the migration, gels were incubated for 20 h
in 25 mM MOPS, pH 7.5, 5 mM CaCl2, 5 mM In Vitro Degradation Assays--
Radiolabeled
35S-p27Kip1 proteins used for in
vitro assays was produced by in vitro
transcription-translation (TNT T7 Quick System, Promega).
To obtain cell lysates, cell pellets were resuspended in ice-cold
hypotonic buffer (1:1) and incubated for 15 min in ice. After Dounce
homogenization, lysates were centrifuged at 100,000 × g for 30 min at 4 °C. The supernatants were quantified for protein concentration (Bradford assay, Bio-Rad)) and stored at
MAP Kinase Activity and p27Kip1 Status Are Altered in
OCM-1 Cells--
We wished to evaluate the effect of mitogenic stimuli
(growth factors and cell adhesion) on MAP kinase-mediated signaling and
on the cell cycle regulatory role of p27Kip1 in OCM-1
melanoma cells. To this end, the effects of serum starvation (24 h) or
suspension (24 h) on these cells were examined. Western blot analysis
of the corresponding fractions revealed that p27Kip1 levels
were not significantly modified by these treatments (Fig. 1A, upper panel).
Similarly, the activity of the p42/44 MAP kinase, detected after
Western blot with a phospho-specific antibody, was not reduced by serum
starvation nor by the absence of adhesion (Fig. 1A,
lower panel). Neither serum starvation nor cell suspension modified the MAP kinase protein levels (not shown). By contrast, serum
starvation of primary culture melanocytes led to a MAP kinase inhibition as measured by the active, phosphorylated form, as well as
significantly increased p27Kip1 levels (Fig.
1B). This up-regulation was further increased after 48 h of serum starvation (not shown). In asynchronous cultures, p27Kip1 expression was much higher in melanocytes than in
melanoma cells (Fig. 1C). Interestingly, the electrophoretic
pattern of the protein was different in the two cell lines.
p27Kip1 appeared as a doublet in melanocytes whereas a
single band was detected in OCM-1 cells in these electrophoretic
conditions. The doublet observed in melanocytes does not correspond to
the difference of migration observed between various phosphorylated
populations (see Fig. 4, A-C) and will be
discussed later (Fig. 4D). In asynchronous cultures, the MAP
kinase activity and protein levels were similar in both cell types, as
was also the case for the expression levels of MEK and Raf-1 (not
shown). By contrast, the expression of the MAP kinase phosphatase 1 (MKP-1) was reduced in OCM-1 cells (Fig. 1C), suggesting a
possible explanation to MAP kinase deregulation. To further compare the
status of p27Kip1 in transformed and normal cells, we
examined protein accumulation and localization in asynchronous
conditions by indirect immunofluorescence (Fig. 1D). Higher
levels of p27Kip1 expression were again detected in
melanocytes, confirming the Western blot analysis. Furthermore,
subcellular localization of p27Kip1 was apparently
different, with diffuse cytoplasmic and nuclear localization in
transformed OCM-1 cells, versus major nuclear labeling in
50% of primary melanocytes. Together, these data suggest that MAP
kinase is constitutively activated in OCM-1 cells, and that
p27Kip1 regulation by mitogenic signals is different in
normal melanocytes and in transformed melanoma cells.
The MAP Kinase Pathway Regulates p27Kip1
Expression--
We then investigated whether the low levels of
p27Kip1 expression in OCM-1 melanoma cells might reflect a
regulatory effect of the constitutive MAP kinase activity. To modulate
the level of activated MAP kinase, OCM-1 cells were treated with the
MEK inhibitor U0126. The effect of this treatment on
p27Kip1 expression was followed by Western blot (Fig.
2A). MAP kinase inhibition was
accompanied with p27Kip1 accumulation that was maximal
after 16 h. On removing the MEK inhibitor from the culture medium,
a progressive down-regulation of p27Kip1 was observed
beginning after 8 h (Fig. 2A). By contrast, in
melanocytes the effect of U0126 on the p27Kip1 level was
modest as shown by Western blot (Fig. 2B) and
immunofluorescence (Fig. 2C). These data suggest that MAP
kinase activity can influence p27Kip1 levels to a higher
extent in OCM-1 cells than in TCM-8 normal melanocytes. To confirm this
result, OCM-1 cells were transfected with the dominant negative form of
MEK (MEKSA). The effect on endogenous p27Kip1
was investigated by double immunofluorescence labeling. We observed five times more cells with a strong nuclear labeling of
p27Kip1 (55%) in cells expressing MEKSA than
in MEKSA-negative cells (10%), which show weaker and more
diffuse labeling both in the cytoplasm and nucleus (Fig. 2,
D and E). When transfection was performed with
empty pCDNA3 vector, the localization status of p27Kip1 was not modified by comparison with nontransfected
cells (not shown). Taken together, these data demonstrate that MAP
kinase activity regulates p27Kip1 levels and possibly
affect its subcellular localization in OCM-1 cells.
p27Kip1 Localization Is Dependent on MAP Kinase
Activity--
In the immunofluorescence experiments shown in Figs.
1 and 2, we detected an atypical cytoplasmic localization of
p27Kip1 and an effect of the dominant negative MEK mutant
on this localization. To further define this relationship, we performed
the following immunofluorescence experiments. Cells were first treated
with the MEK inhibitor for 24 h, then cultured in the absence of
the inhibitor for increasing times. The cellular localization of
p27Kip1 was then examined by indirect immunofluorescence.
As shown in Fig. 3A, in
untreated cells, diffuse p27Kip1 staining was observed in
both cytoplasmic and nuclear compartments. In U0126-treated cells, this
staining became mostly nuclear, although reduced but still
evident cytoplasmic p27Kip1 remained in many cells.
The intensity of the whole immunolabeling was increased in
U0126-treated cells, in good correlation with the corresponding Western
blot analysis (see Fig. 2A). When U0126 was removed, a clear
decrease of the nuclear signal was observed after 6 h, concomitant
with a discrete increase of the cytoplasmic fluorescence. Because the
p27Kip1 down-regulation observed by Western blot was modest
at that time (see Fig. 2A), this likely reflected a
nucleocytoplasmic translocation of the protein. These data thus suggest
that MAP kinase activity induces p27Kip1 cytoplasmic
relocalization in OCM-1 cells. To further test this hypothesis,
subcellular fractionation was performed on cells treated by the MEK
inhibitor. As can be seen in Fig. 3B, about 80% of cellular
p27Kip1 was cytoplasmic in asynchronous cells, whereas only
50% of the protein was cytoplasmic in cells treated with U0126 for
24 h. As a control, we employed cyclin H, described as a nuclear
protein. Indeed, cyclin H was mostly detected in the nuclear fractions. When U0126 was removed from the cells to allow MAP kinase reactivation, a clear translocation of the protein from the nuclear to the
cytoplasmic compartment was observed (Fig. 3C). Taken
together, these data show that p27Kip1 is largely
cytoplasmic in OCM-1 cells, and demonstrate that this atypical
localization is at least partially because of constitutive MAP kinase
activity.
Phosphorylation States of p27Kip1 Vary According to MAP
Kinase Activity--
Recent works have focused on the importance of
p27Kip1 phosphorylation for its localization and its
degradation. For this reason, we employed bidimensional electrophoresis
to investigate the phosphorylation state of the protein in our
experimental conditions. In asynchronous OCM-1 cells, the
two-dimensional pattern revealed five major spots (numbered 1 to 5)
ranging from pH 6.8 to 5.8, as seen in Fig. 4A (upper panel).
Spots 1-3 showed higher mobility in the second dimension than spots 4 and 5. The corresponding doublet was not observed in the other figures
(see for instance Fig. 1A) because smaller polyacrylamide
gels with lower resolution were used for the monodimensional
electrophoresis. The higher mobility species do not represent
degradation products of p27Kip1, because both N-terminal
and C-terminal directed antibodies recognized all isoforms by Western
blot (not shown). Following phosphatase treatment (Fig. 4A,
lower panel), only spots 1 and 2 were conserved, demonstrating that the others were because of different phosphorylated populations of the protein. It is not yet clear whether spot 2 represents a phosphatase-resistant phosphorylated form or another type
of covalent modification.
Because we observed that p27Kip1 from TCM-8 melanocytes had
a different electrophoretic pattern (see Fig. 1C), we
performed a two-dimensional analysis of this fraction (Fig.
4D). The two-dimensional pattern of p27Kip1 from
TCM-8 cells was similar to the one observed in OCM-1, except that each
spot was duplicated, giving rise to a lower mobility form with
identical isoelectric points. These data suggest that a covalent
modification of p27Kip1 occurs in melanocytes, which
increases the apparent molecular weight of the protein, and is absent
in melanoma cells.
In OCM-1 cells where MAP kinase activity was inhibited by U0126, the
pattern of p27Kip1 was significantly modified, with the
intensity of spot 5 clearly increased (Fig. 4B) and the
appearance of an additional phosphoisoform (spot 6). When the MEK
inhibitor was removed for 4 h, spot 5 was significantly decreased
while spot 6 disappeared. These data demonstrate that MAP kinase
activity modulates the phosphorylation status of p27Kip1.
These results thus suggest that reduced MAP kinase activity relieves
some inhibition and permits the initiation of a phosphorylation process
of p27Kip1.
We then compared the two-dimensional electrophoretic pattern of the
nuclear and cytoplasmic fractions of p27Kip1 from cells
treated with U0126. As observed in Fig. 4C, the
phosphorylation pattern was significantly different in the two
compartments, with spots 1 and 4 being the principal forms in the
nucleus. These data thus suggest that phosphorylation events are
involved in regulating the cellular localization of
p27Kip1, and that spot 4 corresponds to the main
phosphorylated population of p27Kip1 in this compartment.
Because phosphorylation of Ser10 and Thr187 has
been implicated in p27Kip1 localization and/or degradation,
we investigated the phosphorylation of these residues in the
cytoplasmic and nuclear compartments of OCM-1 cells. To discriminate
the low and high mobility forms of the protein, we used more resolutive
gels (13 cm) for this experiment. As seen in Fig. 4E, P-S10
and P-T187 antibodies only recognized the lower mobility forms of
p27Kip1, suggesting that spots 2 and 3 were because of
other phosphorylation events. Comparison of Fig. 4, C and
E, suggests that the upper band observed in the nuclear
fraction in Fig. 4E corresponds essentially to spot 4 in
Fig. 4C. Importantly, p27Kip1 phosphorylated on
Ser10 and/or on Thr187 was present in both
cellular compartments, with the Thr187 population slightly
more abundant in the nuclear fraction. Altogether these data suggest
that phosphorylations on these residues do not trigger, by themselves,
the nucleocytoplasmic translocation of p27Kip1.
Calpains Are Involved in MAP Kinase-dependent
p27Kip1 Regulation--
Because MAP kinase activation can
down-modulate p27Kip1 expression, we investigated the
degradation mechanisms involved. The atypical cytoplasmic localization
of p27Kip1 in OCM-1 cells led us to test whether calpains,
a family of cytoplasmic calcium-dependent proteases, could
be involved in p27Kip1 down-regulation. For this, MAP
kinase activity was modulated as in Fig. 1, except that various calpain
inhibitors were added to the medium during the relevant period after
removal of the MEK inhibitor. Indeed, the down-regulation process was
partially or totally reversed by addition of the chemical calpain
inhibitors ALLM, calpeptin, and PD150606, as seen in Fig.
5A. As a control, ERK2
expression was not affected by these compounds (lower
panel). To further confirm these data, we also used a peptide (CS
peptide) corresponding to the inhibitory domain of calpastatin, the
ubiquitous cellular inhibitor of calpains. As shown in Fig.
5B (upper panel), this peptide also impaired the
MAP kinase-dependent response, again indicating a role for
calpains in the regulated degradation of p27Kip1. A control
peptide composed of scrambled residues from the CS peptide, did not
modify the down-regulation process (lower panel).
To reinforce our interpretation, we transfected OCM-1 cells with
calpastatin, the physiological inhibitor of calpains. As can be seen in
Fig. 5C, expression of this protein induced an increase of
endogenous p27Kip1, thus confirming our initial conclusion
that calpain activity regulates p27Kip1 levels in these
cells. As observed for the chemical inhibitors (Fig. 5A),
this overexpression did not affect the ERK2 levels. Taken together,
these data indicate that calpains are involved in the MAP
kinase-dependent down-regulation of p27Kip1 in
transformed melanoma cells.
p27Kip1 Is a Substrate for Calpain in Vitro--
If
p27Kip1 is indeed directly regulated by calpains, then it
should be a substrate for specific cleavage by calpains in
vitro. In a first test of this possibility, in vitro
translated p27Kip1 was incubated with purified m- and
µ-calpains (calpains I and II). As can be seen in Fig.
6A, this led to
calcium-dependent degradation of p27Kip1, and
to generation of a cleavage product of 25 kDa. A similar experiment was
then performed replacing purified calpains with OCM-1 cell
extracts. A calcium-dependent proteolytic activity was
detected in cytoplasmic extracts from asynchronous cells (Fig. 6B). To test whether this degradation was MAP
kinase-dependent, we followed the degradation of endogenous
p27Kip1 from control or U0126-treated cell extracts. As can
be seen in Fig. 6C, p27Kip1 was not degraded in
the fraction from cells treated with U0126, suggesting that MAP kinase
activity somehow regulates this degradation. Together these experiments
demonstrate that p27Kip1 is an in vitro
substrate for calpains, and that a calcium- and MAP
kinase-dependent proteolytic activity is able to degrade
the protein in OCM-1 cell extracts.
We then investigated whether p27Kip1 from U0126-treated
cells was still a substrate for calpains. To this end,
p27Kip1 from control cells or from cells treated for
24 h with U0126 was immunoprecipitated, then subjected to
degradation by purified calpain. Indeed, p27Kip1 from
U0126-treated and control cells were degraded to a similar extent by
purified calpain (Fig. 6D). Thus the inhibition of
calpain-dependent p27Kip1 degradation observed
in U0126-treated cells does not appear to reflect the modification
state of p27Kip1 protein in these cells.
The MAP Kinase Pathway Regulates Calpain Activity--
We then
investigated whether MAP kinase signaling could be involved in calpain
activity regulation in OCM-1 cells. For this, we performed zymographic
detection of calpain proteolytic activities from cell extracts of
U0126-treated cells. As can be seen in Fig. 7A, the calpain activity
detected in asynchronous cells was partially reduced in U0126-treated
cells. Upon U0126 removal, this activity increased progressively over a
7-h period. This activity probably corresponds to calpain II, as judged
by identical migration with this purified isoform (see Fig.
7B). When the gel was incubated in the presence of EGTA
instead of calcium, the proteolytic activities were not detected
(lower panel), confirming that calpains are involved. These
data demonstrate that the MAP kinase pathway may regulate the calpain
activity in OCM-1 cells. Surprisingly, when we performed zymographic
detection of calpains in TCM-8 cells, we could not observe any
proteolytic activity in these conditions (Fig. 7B). This
suggests a lower activity of calpain II in these cells, but does not
exclude the existence of other isoforms not detected by this
method.
Nuclear Export Is Necessary for p27Kip1 Degradation by
Calpains--
Because MAP kinase activation induced both
calpain-dependent p27Kip1 degradation and its
nucleocytoplasmic translocation, we wished to know whether degradation
was functionally linked to nucleocytoplasmic transport. To answer this
question, we sought to sequester p27Kip1 in the nucleus,
using leptomycin B, an inhibitor of CRM-1-dependent nuclear
export, described as an inhibitor of p27Kip1 nuclear export
(16). In the presence of this inhibitor, the MAP kinase- and
calpain-dependent down-regulation of p27Kip1
was inhibited (Fig. 8A).
Furthermore, the nuclear localization of p27Kip1 observed
in U0126-treated cells was maintained during MAP kinase reactivation in
the presence of leptomycin B (Fig. 8B). A similar effect was
observed when leptomycin B was added 4 h after U0126 removal,
confirming that p27Kip1 translocation occurs after that
time. These data demonstrate that MAP kinase-dependent
nuclear export of p27Kip1 is CRM-1 dependent, and that this
export is necessary for the degradation by calpains occurring after MAP
kinase reactivation.
We have previously characterized major cell cycle deregulations in
choroidal melanoma cell lines by comparison with their nontransformed
counterparts, choroidal melanocytes. Modifications of cyclin expression
were identified in these cells, but also an underexpression of
p27Kip1 (47). Here we have shown that the MAP kinase
activity is constitutive in these tumor-derived cells, independent of
growth factors and cell adhesion. Constitutive activity of the MAP
kinase pathway exists in many other cancer cells, and may reflect
activating mutations in one or more of the many components from the
receptor to the MAP kinase itself. Recent work demonstrates that the
isoform B-Raf is mutated in 66% of melanoma tumors (50), with
subsequent constitutive MAP kinase activity. In OCM-1 cells, we
observed a down-regulation of MKP-1, the phosphatase responsible for
MAP kinase dephosphorylation and hence inhibition. Although reduced MKP-1 may contribute to constitutive MAP kinase activity, as observed in p59Fyn1-dependent transformation of murine
melanocytes (51), the molecular event(s) leading to constitutive
activation in OCM-1 cells remains to be identified.
In this work, we demonstrate that chemical (U0126) and genetic
(MEKSA) MAP kinase inhibition influences the cellular level and/or localization of p27Kip1. The potential existence of
a link between the MAP kinase pathway and p27Kip1
regulation has been a controversial subject during the last few years
(34-36, 38-41). Recently, we described a MAP
kinase-dependent and CDK2-independent regulation of
p27Kip1 degradation in NIH 3T3 cells (42), in good
accordance with results demonstrating that p27Kip1
degradation during G0 exit and G1 was
CDK2-independent (52). A much more pronounced effect of U0126 on
p27Kip1 was observed in transformed OCM-1 cells compared
with primary cultures of melanocytes. This may reflect the constitutive
activity of this pathway in OCM-1 cells relative to mitogenic signals. This may also indicate that the MAP kinase-dependent
degradation pathway is more active in transformed cells, or that
additional MAP kinase proteolytic pathways exist in these cells.
In good correlation with this hypothesis, we identified a
calpain-dependent degradation of p27Kip1 in
OCM-1 cells, and we established that this process was dependent on MAP
kinase activity. This is not the first report of p27Kip1
degradation by calpains, because Patel and Lane (22) described a
similar process during mitotic clonal expansion of preadipocytes. However, this is the first evidence that this mode of degradation is
utilized in a tumor-derived cell line. This suggests a possible link
between this activity and the transformation process that takes place
in melanoma cells. In this respect, the possible implication of calpain
activities in the transformation process induced by v-Src was
previously reported (53), and calpain degradation of
p21Cip1, another member of the p27Kip1 family
of CDK inhibitor, was recently described in response to human
cytomegalovirus infection (45). Further analysis will be required to
confirm a possible implication of calpains in the transformation of
melanoma cells.
Our data suggest that MAP kinase activity regulates
calpain-dependent p27Kip1 turnover in OCM-1
cells. This might reflect either MAP kinase-dependent enhancement of calpain activity toward specific targets including p27Kip1 in these cells or conversely, MAP
kinase-dependent modification(s) of p27Kip1
that make it sensitive to this proteolytic pathway. We were able to
establish that MAP kinase inhibition indeed led to partially decreased
calpain activity, in good accordance with previous reports that
described a MAP kinase-dependent regulation of calpain
(54). Furthermore, MAP kinase activity also regulates
p27Kip1 localization and phosphorylation status in OCM-1
cells, two parameters known to be involved into p27Kip1
degradation. Based on these data, we propose that MAP kinase likely
regulates both calpain activity and p27Kip1 modification(s)
and that p27Kip1 accumulation occurring upon MAP kinase
inhibition is mediated by these two complementary effects (Fig.
9).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
responsiveness, and cell proliferation behavior (47). Cell culture was
performed in Hams' F-12 medium supplemented with 100 IU/ml penicillin,
100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, 0.1 mM isobutylmethylxanthine, 10 ng/ml cholera toxin, 5 ng/ml
fibroblast growth factor-2, and 10% fetal calf serum. Cells were
replated at 1:3 dilution when they reached confluence.
-glycerophosphate, 1 mM NaF, 0.1 mM Na orthovanadate in the presence of protease inhibitors
(Complete tablet, Amersham). One volume of these fractions was mixed
with 4 volumes of IEF sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 24 mM spermine) and 2% ampholine buffer was added just before
the run. These fractions were loaded on immobilized linear pH (pH 3 to
10) 13-cm strips, and focused for 50,000 V/h, using the IPGphor
apparatus from Amershan Biosciences. The IEF strips were either
kept at
80 °C before use, or equilibrated in 50 mM
Tris, pH 6.8, 6 M urea, 30% glycerol, 10 mM
DTT for 30 min before loading for SDS-polyacrylamide gel electrophoresis. For phosphatase treatment, immunoprecipitated p27Kip1 (see below) was incubated with 30 units of calf
intestinal phosphatase (Biolabs) in 20 µl of reaction buffer (50 mM Tris, pH 8.0, 10% glycerol) for 4 h at
37 °C.
-glycerophosphate,
1 mM NaF, 0.1 mM Na orthovanadate). One ml of
these fractions were incubated for 3 h with 5 µg of the
polyclonal antibody. After adding 40 µl of protein A-Sepharose, the
fractions were incubated overnight at 4 °C. Sepharose beads were
then washed three times with 1 ml of RIPA buffer, twice with 1 ml of
PBS, and then processed for Western blot or calpain degradation assays.
mercaptoethanol, after two initial washes of 30 min in this same buffer. Gels were then stained with Coomassie Blue, and destained to reveal the zymographic bands corresponding to calpains. As a control, a second gel was incubated in the same buffer
with 5 mM EGTA instead of calcium.
80 °C. In vitro assays were performed in a 30-µl
final volume of PBS containing in vitro translated
p27Kip1 and 100 µg of cell lysate. Reactions were
achieved at 30 °C, stopped by the addition of SDS sample buffer,
then analyzed by SDS-PAGE and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Low p27Kip1 level and
constitutive MAP kinase activity in OCM-1 melanoma cells.
A, OCM-1 choroidal melanoma cells were serum starved for
24 h ( Ser) or placed in suspension for the same time
(Susp) and further analyzed by Western blot for
p27Kip1 expression (p27) and MAP kinase activity (P-MAPK).
B, TCM-8 melanocytes were serum-starved for 24 h and
analyzed for the expression of p27Kip1 and the MAP kinase
activity. C, asynchronous TCM-8 melanocytes and OCM-1
melanoma cells were directly compared by Western blot for MAP kinase
activity, and for p27Kip, ERK2, and MAP kinase phosphatase
(MKP-1) levels. D, p27Kip1 expression and
localization was compared by indirect immunofluorescence in
asynchronous TCM-8 and OCM-1 cells. Nuclei were stained with Hoechst
33342 reagent.
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Fig. 2.
MAP kinase pathway regulates
p27Kip1 expression. A, OCM-1 cells were treated
with 20 µM MEK inhibitor U0126 (U0) (Ctr,
untreated cells) for 24 h, further maintained for different times
in the absence of the inhibitor ( U0) and analyzed for
p27Kip1 expression by Western blot. The MAP kinase activity
was followed in parallel (P-MAPK). B and
C, the effect of the MEK inhibitor U0126 on
p27Kip1 expression and localization in TCM-8 melanocytes
was investigated by Western blot (B) and immunofluorescence
(C). D, OCM-1 melanoma cells were transiently
transfected with a dominant negative mutant of MEK (MEKSA).
24 h after transfection, cells were processed for
double-immunodetection of MEKSA with an anti-hemagglutinin
antibody (red) and endogenous p27Kip1
(green). Arrows indicate cells expressing
MEKSA with modified expression of p27Kip1.
E, quantification of p27Kip1 nuclear
localization in MEKSA positive (MEKSA)
versus MEKSA negative cells (Ctr).
,
p27Kip1 labeling excluded from the nucleus. +, diffuse
labeling in both compartments. ++, cytoplasmic labeling weak or absent,
with strong labeling of the nucleus.
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Fig. 3.
MAP kinase activation mediates
p27Kip1 nuclear export. A, after treating OCM-1
cells with U0126 for 24 h, the drug was removed for different
times (4, 6, 8, 10, or 16 h), then p27Kip1
localization was observed by immunofluorescence. B, OCM-1
cells were treated with U0126 for 24 h (U0), and
nuclear (N) and cytoplasmic (Cy) fractions were
prepared as described under "Materials and Methods." An equal ratio
of total cytoplasmic and nuclear fractions was applied to the gel, to
directly compare the ratio of p27Kip1 present in each
fraction. As a control for nuclear fractions, we used cyclin H
(CycH) expression. C, cytoplasmic and nuclear
fractions were prepared from cells treated with U0126 for 24 h
(U0) or from cells released for 7 h from a 24-h U0126
block ( U0). In that case, equal amounts of proteins were
applied for each fraction.
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Fig. 4.
p27Kip1 phosphorylation states.
A, p27Kip1 was immunoprecipitated from
asynchronous OCM-1 cells, and further treated (+PPase) or
not ( PPase) with calf intestinal phosphatase.
Two-dimensional electrophoresis separation was then performed as
described under "Materials and Methods," and the
p27Kip1 profile was analyzed by Western blot with a
monoclonal antibody from Transduction Laboratories. Major spots were
numbered from 1 to 5. B, OCM-1 cells were left asynchronous
(Ctr), treated for 24 h with U0126 (U0), or
released from U0 block for 4 h (
U0 4h) and the
two-dimensional profile of p27Kip1 from the corresponding
fractions was analyzed. C, OCM-1 cells were treated with
U0126 and the two-dimensional electrophoretic patterns of
p27Kip1 from nuclear (N) and cytoplasmic
(Cy) compartments were compared. D, Western blot
analysis of p27Kip1 from TCM-8 melanocytes following
two-dimensional separation. Duplicated spots were numbered 1' to 5'.
E, expression of p27Kip1 and its phosphorylated
forms on serine 10 (P-S10) and threonine 187 (P-T187) were compared in the cytoplasmic (Cy)
and nuclear (N) fractions of asynchronous OCM-1 cells.
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Fig. 5.
Calpain inhibitors impair the MAP
kinase-dependent p27Kip1 down-regulation.
A, OCM-1 cells were treated with U0126 for 24 h
(U0), and released from this inhibition for 12 h
( U0) in the presence or absence of three different calpain
inhibitors: 50 µM calpeptin (Calp.), 10 µM PD 150606 (PD), or 50 µM ALLM
(ALLM). The effect of these treatments on
p27Kip1 expression was examined by Western blot. The
expression of the MAP kinase ERK2 was followed as a control
(lower panel). B, same experiment as in
A, with a synthetic peptide (10 µM)
corresponding to an inhibitory domain of the physiological calpain
inhibitor calpastatin (CSp., upper panel) or with
a control peptide (Cp., lower panel).
C, OCM-1 cells were transfected with empty
pCDNA3 (Ctr) or
pCDNA3/calpastatin (Calpast.).
p27Kip1 and calpastatin expressions were then analyzed by
Western blot. As in A, ERK2 expression was used as a
control.
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Fig. 6.
p27Kip1 is an in vitro
substrate for calpain. A, in vitro
translated 35S-p27Kip1 was incubated for 30 or
60 min with purified calpain I or II, in the presence or absence of 5 mM EGTA. p27Kip1 variations were analyzed by
autoradiography. The arrow indicates a cleavage product of
p27Kip1. B, in vitro translated
p27Kip1 was incubated for 30 min with an OCM-1 cell
cytoplasmic extract in the presence or absence of calcium (± EGTA). p27Kip1 behavior was followed by
autoradiography. C, cell extracts were prepared from
asynchronous (Ctr) or from U0126-treated (U0)
OCM-1 cells, and incubated for 60 min at 30 °C. The degradation of
endogenous p27Kip1 was followed by Western blot.
D, p27Kip1 was immunoprecipitated from control
(Ctr) and U0126-treated OCM-1 cells (U0) with a
polyclonal antibody against the C-terminal domain (Santa Cruz).
Immunoprecipitated p27Kip1 was then subjected to
degradation by purified calpain as in A, and detected by
Western blot with a monoclonal antibody (Transduction
Laboratories).
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Fig. 7.
The MAP kinase pathway modulates calpain
activity. A, OCM-1 cells were treated with U0126 for 24 h (U0) or not (Ctr), and further maintained in
the absence of the inhibitor for 3 h ( U0 3h) or
7 h (
U0 7h). Calpain activity was assessed by
zymographic analysis in the presence of calcium (upper
panel) or with 5 mM EGTA (bottom panel).
After Coomassie Blue staining and destaining, bands corresponding to
calcium-dependent proteolytic activity were visualized (and
indicated by an arrow). B, calpain activity of
purified calpain II, OCM-1, or TCM-8 extracts were analyzed by
zymographic detection.
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Fig. 8.
p27Kip1 cytoplasmic translocation
is necessary for MAP kinase-dependent calpain
degradation. OCM-1 cells were treated with U0126 for 24 h.
The inhibitor was then removed for 12 h in the presence or absence
of 2 ng/ml leptomycin B (LMB), an inhibitor of
CRM-1-dependent nuclear export. Leptomycin B was added
immediately after removal of U0126 (12h) or 4 h later
(8h). Cells were analyzed for p27Kip1 expression
by Western blot (A) or immunofluorescence analysis
(B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
A model of regulation of MAP
kinase-dependent p27Kip1 degradation by
calpain. Active MAP kinase induces both an increase of calpain
activity and a nucleocytoplasmic translocation of p27Kip1.
Both events participate to the increased accessibility of
p27Kip1 to calpains and its subsequent degradation.
One of the striking features of p27Kip1 status in OCM-1
cells is its cytoplasmic localization and the effect of MAP kinase on it. p27Kip1 nucleocytoplasmic translocation was recently
reported during exit of fibroblasts from quiescence in response to
growth factors (55). p27Kip1 cytoplasmic localization was
also reported by others in transformed cell lines (56), and in
choroidal tumors (57). More recently, this was also observed in
transforming growth factor--resistant cells (58), and in response to
Akt phosphorylation (59). Although this was essentially discussed in
terms of altered function of the protein, this may also have an
influence on its regulation. In particular, the compartment in which
proteasome-mediated p27Kip1 degradation occurs has been
controversial during the last few years (55, 60). This state of affairs
likely reflects a multiplicity of regulation mechanisms depending on
the cell type, the cell cycle phase, and the extracellular signal. We
provide evidence that in OCM-1 cells, the abnormal and MAP
kinase-dependent localization of p27Kip1 in the
cytoplasm is probably one of the reasons for degradation by calpains.
Indeed, the degradation process triggered by MAP kinase activation was
inhibited by the CRM-1-dependent export inhibitor
leptomycin B, demonstrating that nuclear export is necessary for
calpain degradation. This suggests, but does not definitively demonstrate that this proteolytic process occurs in the cytoplasmic compartment. Calpain activity was mainly described in the cytoplasmic and plasma membrane compartments, although some nuclear activities have
also been reported (61). In OCM-1 cells, and possibly in other
transformed cell types, the cytoplasmic localization of p27Kip1 may facilitate its interaction with calpains and
subsequent degradation by this pathway. Further experiments will be
needed to identify unambiguously the cellular compartment where this
degradation occurs.
We also observed a difference of phosphorylation status of the nuclear and cytoplasmic populations. Phosphorylation of p27Kip1 on Thr187 (10), Ser10 (62), and more recently Thr198 (59) was found to regulate its localization and degradation by the proteasome. In our cell system, Ser10 and Thr187 phosphorylations do not appear as key events regulating p27Kip1 localization, because Thr187- and Ser10-phosphorylated p27Kip1 was present in both compartments. In consequence, the precise function and space-time regulation of these phosphorylations remain to be clarified. Although MAP kinase is unlikely to be a direct kinase for p27Kip1, our data suggest that its activation participates in the phosphorylation/dephosphorylation events involved into the localization and the degradation of this protein.
These data are summarized in our model depicted in Fig. 9. MAP kinase
activation can induce a nucleocytoplasmic translocation of
p27Kip1, according to a still to be defined molecular
mechanism. This p27Kip1 localization allows its degradation
by calpains, which are themselves sensitive to MAP kinase activity. In
OCM-1 melanoma cells, and possibly in other transformed cell types, the
constitutive activation of the MAP kinase pathway could amplify this
mechanism and lead to low levels of p27Kip1 expression that
may help account for the transformed phenotype of the cells. Further
experiments will be needed to identify the molecular links between MAP
kinase activity and p27Kip1 degradation, and to test this
model in other transformed cell types.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to David Cribbs for helpful reading of the manuscript. We thank Alain Eychène (Orsay, France) and Marc Piechaczyk (Montpellier, France) for providing expression vectors. We also thank Karine Pelpel for helpful suggestions concerning two-dimensional electrophoresis analysis.
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FOOTNOTES |
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* This work was supported by the Centre National de la Recherche Scientifique and the Université Paul Sabatier.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: Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, CNRS UMR 5088, Université Paul Sabatier, Batiment 4R3B1, 118 Route de Narbonne, 31062 Toulouse Cedex, France. Tel.: 33-561-55-69-19; Fax: 33-561-55-81-09; E-mail: manenti@cict.fr.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M209523200
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; MAP kinase, mitogen-activated protein kinase; TBS, Tris-buffered saline; DTT, dithiothreitol; ALLM, N-acetyl-Leu-Leu-Met-CHO; MKP-1, mitogen-activated protein kinase phosphatase; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase.
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