MAP Kinase-dependent Degradation of p27Kip1 by Calpains in Choroidal Melanoma Cells

REQUIREMENT OF p27Kip1 NUCLEAR EXPORT*

Christelle Delmas, Nathalie Aragou, Sylvie PoussardDagger , Patrick CottinDagger , Jean-Marie Darbon, and Stéphane Manenti§

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 Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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-beta 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.

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 beta -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.

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 beta -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.

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 beta -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.

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 -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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.

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.


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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.

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.


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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.

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).


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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.

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.


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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).

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.


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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.

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.


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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

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).


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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-beta -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.

    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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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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