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* INSERM, Dijon, France;
INSERM U309, La Tronche, France;
Center for Translational and Advanced Animal Research on Human Diseases, Tohoku University, Miyagi, Japan; and
Division of Cell Biology, Medical Institute of Bioregulation, Kyushu University, Japan
2Correspondence: INSERM U517, IFR100, Faculty of Medicine, 7 boulevard Jeanne d’Arc, Dijon 21000, France. E-mail: cgarrido{at}u-bourgogne.fr
ABSTRACT
Stress-inducible HSP27 protects cells from death through various mechanisms. We have recently demonstrated that HSP27 can also enhance the degradation of some proteins through the proteasomal pathway. Here, we show that one of these proteins is the cyclin-dependent kinase (Cdk) inhibitor p27Kip1. The ubiquitination and degradation of this protein that favors progression through the cell cycle was previously shown to involve either a Skp2-dependent mechanism, i.e., at the S-/G2-transition, or a KPC (Kip1 ubiquitination-promoting complex)-dependent mechanism, i.e., at the G0/G1 transition. In this work, we demonstrate that, in response to serum depletion, p27Kip1 cellular content first increases then progressively decreases as cells begin to die. In this stressful condition, HSP27 favors p27Kip1 ubiquitination and degradation by the proteasome. A similar observation was made in response to stress induced by the NO donor glyceryl trinitrate (GTN). HSP27-mediated ubiquitination of p27Kip1 does not require its phosphorylation on Thr187 or Ser-10, nor does it depend on the SCFSkp2 ubiquitin ligase E3 complex. It facilitates the G1/S transition, which suggests that, in stressful conditions, HSP27 might render quiescent cells competent to re-enter the cell cycle.—Parcellier, A., Brunet, M., Schmitt, E., Col, E., Didelot, C., Hammann, A., Nakayama, K., Nakayama, K., Khochbin, S., Solary, E., Garrido, C. HSP27 favors ubiquitination and proteasomal degradation of p27Kip1 and helps S-phase re-entry in stressed cells.
Key Words: stress proteins • proteasome • ubiquitin • cell death • cell proliferation
EXPOSURE OF CELLS to stressful stimuli enhances the expression of several heat shock (or stress) proteins, which permits the cell to survive otherwise lethal conditions. One of the most strongly induced stress proteins is HSP27, a small heat shock protein family member. HSP27 monomers form oligomers of various sizes, ranging from 100 to 1000 kDa. At least in vitro, this highly dynamic process is modulated by phosphorylation of serine residues, i.e., the phosphorylated protein forms small oligomers only, whereas the dephosphorylated HSP27 also forms large complexes. This oligomerization process modulates the protein functions: small oligomers interact with actin cytoskeleton and may be responsible for the thermoresistance of the cells (1)
, whereas large oligomers decrease the cellular content in reactive oxygen species and prevent caspase-dependent apoptotic cell death (2
, 3)
. Several mechanisms account for HSP27-mediated prevention of cell death, including interaction with cytochrome c when released from the mitochondria to the cytosol to prevent the formation of the apoptosome and caspase activation (4)
. When expressed at very high levels, HSP27 also inhibits premitochondrial steps in apoptotic death pathways (5)
.
In vitro, large oligomers of HSP27 behave as powerful, ATP-independent chaperones that inhibit protein aggregation (2)
. In vivo, the protein is found in aggresomes and inclusion bodies that characterize neurodegenerative diseases, in combination with the 26S proteasome (6)
. This multi-subunit complex, which consists of a 20S proteolytic core and two 19S (also called PA700) regulatory complexes, is involved in ubiquitin-dependent degradation of cellular proteins. Schematically, a target protein is conjugated with a multimer of ubiquitin residues, then transferred to the 26S proteasome to be degraded. Colocalization of HSP27 and the 26S proteasome suggested a role for HSP27 in protein degradation. Accordingly, we have recently demonstrated that, in stressful conditions, HSP27 overexpression could enhance the degradation of ubiquitinated I-
B
by the 26S proteasome (7)
.
The ubiquitin/proteasome pathway regulates the cellular content in many cellular proteins, including regulatory proteins that control cell-cycle progression such as the Cdk inhibitor p27Kip1. In normal cells, the protein accumulates in G0-phase of the cell cycle and its degradation by the proteasome is required for G1/S transition and subsequent progression through the cell cycle. It was initially shown that, in response to mitogenic stimuli, p27Kip1 was phosphorylated on Thr187 by the cyclin E-Cdk2 complex (8
, 9)
and consequently recognized by Skp2 (S-phase kinase interacting protein), also called Fbl1 (10
11
12)
. A factor known as Cks1 is required for contact between Skp2 and p27Kip1 (13
, 14)
. Other subunits (Skp1, Cul1, and Roc1) of the so-called SCF (Skp1/Cul1/F-box) ubiquitin–protein isopeptidase ligase complex are then recruited through the F-box of Skp2, and p27Kip1 is ubiquitinated to be targeted for proteasomal destruction. Experiments in Skp2–/– mice suggested that the same ubiquitin ligase complex could degrade cyclin E (15
, 16)
.
Several observations have indicated that p27Kip1 could be degraded also by an Skp2-independent system. First, Skp2 is expressed at low concentration in G0 and early to mid-G1 phases while it accumulates during S- and G2-phases (17)
, indicating that Skp2 protein concentration does not correlate with p27Kip1 degradation at G0/G1 transition. Secondly, p27Kip1 is exported from the nucleus to the cytoplasm at G0/G1 transition, whereas Skp2 is restricted to the nucleus (18
19
20)
. Third, in the absence of Skp2, down-regulation of p27Kip1 occurs normally at the G0/G1 transition, while it is impaired in S- and G2-phases. Thus, p27Kip1 degradation is promoted by the SCFSkp2 complex during progression from G2- to M-phase but is independent of this complex at the G1/S transition. It was recently demonstrated that the cytoplasmic ubiquitin ligase KPC (Kip1 ubiquitination-promoting complex) controlled the degradation of p27Kip1 after export from the nucleus in G1-phase (21)
.
In the present study, we demonstrate that, in some stressful conditions, HSP27 favors p27Kip1 degradation by the proteosomal machinery, an effect that does not depend on p27Kip1 phosphorylation at Thr187 or Ser-10, nor does it involve the SCFSkp2 ubiquitin ligase complex. This HSP27-mediated pathway of p27Kip1 degradation may render quiescent cells competent to re-enter the cell cycle.
MATERIALS AND METHODS
Culture and reagents
The U937 human leukemic cell line was grown in RPMI 1640 medium (BioWhittaker, Fontenay-sous-bois, France) supplemented with 10% (v/v) FBS and 2 mM L-glutamine. Hela and COS cell lines were grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Saint Quentin Fallavier, France) supplemented with 10% (v/v) FBS. Rat colon carcinoma REG cells were grown in Ham’s F-10 medium supplemented with 10% (v/v) fetal calf serum. We have already described stable transformants of human U937 cells carrying the gene encoding human HSP27 (22)
. One of the characterized clones (clone 4) was used in this work. Another clone carrying the hygromycin resistance gene and the empty vector was used as control. We have also described the mutations performed in HSP27 as well as REG cells transfected with these mutants (4)
. Transformants carrying the plasmid pCI-neohsp27 containing the full-length HSP27 cDNA cloned in an antisense orientation (5)
, as well as transient transfections using pcDNA3-HSP27-hemagglutinin, pcDNA3-p27kip1-hemagglutinin or pcDNA3-Skp2 plasmids were performed with Superfect transfection reagent (Qiagen, Courtaboeuf, France), following the manufacturer instructions. Cotransfection of a ß-galactosidase expressing vector demonstrated that neither HSP27- nor p27-expressing vectors affected the expression of cotransfected constructs. Glyceryl trinitrate (GTN) was purchased from Merck (Merck, Germany), Z-leu-leu-leu-H-(aldehyde) (MG132) from Peptide Institute (Osaka, Japan) and lactacystin from Calbiochem (San Diego, CA). N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN), 3,4 dichloroisocoumarin (DCIC) and leptomycin B were purchased from Sigma-Aldrich.
siRNA preparation and transfection
Sense and antisense HSP27 (sense strand DNA sequence 5'-AAAATCCGATGAGACTGCCGC-3') oligonucleotides were used to inhibit hsp27 gene expression by RNA interference. A siRNA for the luciferase gene was used as control (sense strand DNA sequence 5'-AACTTACGCTGAGTACTTCGATT -3'). Oligonucleotides were purchased from Operon (Cologne, Germany). Sense and antisense oligonucleotides were annealed to generate the double-stranded siRNAs at the final concentration of 100 µM. HeLa cells were plated in serum-containing medium without antibiotics the day before transfection and then were transfected by adding 5 µl of oligofectamine (Invitrogen, Cergy Pontoise, France) to 1 µl of 20 µM siRNAs (final concentration 20 nM). Cells were rinsed with medium 24 h after transfection and then maintained in culture for one day before analysis.
Immunoblot analysis
Whole cell lysates were prepared by lysing the cells in radio-immuno-precipitation assay buffer [50 mM Tris-HCl, pH 8; 150 mM NaCl; 0.5% sodium deoxycholate; 0.1% SDS; 1% Nonidet P-40; protease inhibitor cocktail (Roche Diagnostic, Basel, Switzerland)]. Cells were kept on ice for 30 min and were vortexed 3 times. After centrifugation (14000 g, 15 min, 4°C), supernatant was used to measure protein concentration by means of the micro-bicinchoninic acid protein assay (Bio-Rad, Ivry sur Seine, France). Proteins were separated in SDS-polyacrylamide gel and electroblotted onto PVDF membranes (Bio-Rad). Immunoblot analysis was performed using specific antibodies and enhanced chemoluminescence-based detection (Santa Cruz, Tebu, Le Perray-en-Yvelines, France). The antibodies used were the rabbit polyclonal anti HSP27; HSP70 and HSP90 from StressGen (Victoria, Canada); Thr187-phospho-p27, Ser-10-phospho-p27, Cul1, and Rbx1 from Zymed Laboratories (San Francisco, CA); p21 from Santa Cruz; the mouse monoclonal antip27, cyclin A, D1, E, and ß-catenin from Pharmingen (San Diego, CA); Skp2 from Zymed; ubiquitin and HSC70 from Santa Cruz. All monoclonal antibodies used were of the IgG1 isotype.
Immunoprecipitation
Cells (107) were lysed in immunoprecipitation buffer (50 mM HEPES, pH 7.6; 150 mM NaCl; 5 mM EDTA; 0.1% Nonidet P-40). After centrifugation during 10 min at 15,000 g, the supernatant was incubated with the indicated antibody (Ab) (1:100) with constant agitation at 4°C. Then, the immunocomplexes were precipitated with protein A-Sepharose (Amersham, Les Ullis, France). The pellet was used for immunoblotting after five successive washings, and the supernatant was used for immunodepletion.
Determination of proteasome activity
Proteasome activity was determined as described previously (23)
. Briefly, cells (4x106 in 200 µl PBS, pH 7.4) were incubated for 30 min at 37°C with 100 µM of the cell-permeant fluorogenic substrate N-succinyl-L-leucyl-L-leucyl-L-tyrosine-7-amido-4-methyl coumarin (Bachem, Basel, Switzerland). Fluorescence generated by its cleavage was quantified by using a Kontron SFM 25 spectrofluorometer (Kontron AG., Zurich, Switzerland).
Quantitative polymerase chain reaction (PCR) analysis
Of the total RNA, 500 ng was reverse-transcribed using MMLV-RT (Promega, Charbonnières, France) in presence of oligodT, according to the manufacturer’s instructions. Each sample was tested in duplicate and standardized to the ß2-microglobulin mRNA. Briefly, 0.5 µl cDNA (5 ng) of p27kip1 and ß2-microglobulin were used as template in 1x buffer (QuantitectSYBR Green PCR master mix, Qiagen) and 0.3 µM of each primer was used in a total reaction volume of 25 µl. Forwards primers p27kip1: 5'-CCA TTT gAT Cgg AgA computed tomography (CT)-3' and ß2 microglobulin: 5'-CTC ACG TCA TCC automatic gain control AGA GA-3'. Reverse primers p27kip1: 5'- CAC TCg CAC gTT TgA chloroamphenicol acetyltransferase CT-3'; ß2 microglobulin: 5'-TCT TTT TCA GTG GGG GTG AA-3'. Analyses were performed in an Abprism 7700 Detector system (Applied Biosystems, Les Ulis, France) with sequence detector system software.
Pulse-chase
Control and HSP27-U937 cells were methionine-starved for 15 min then pulsed for 30 min with [35S] methionine (1 mCi/mL; Amersham-Pharmacia Biotech). Cells were then washed and chased with excess of unlabeled methionine. At the indicated chase time points, cells were collected at –80°C prior to lysis and immunoprecipitation. Gels were dried and exposed to Kodak Biomax MR Film.
In vitro transcription/translation
Synthesis of Skp2 and HSP27 proteins was performed using the TNT Coupled Reticulocyte Lysate Systems from Promega. Briefly, pcDNA3-HSP27 or pcDNA3-Skp2 plasmids (1 µg) was added to a mix reaction containing TNT rabbit reticulocyte lysate, TNT reaction buffer, TNT RNA polymerase, amino acid mixture minus leucine (1 mM), amino acid mixture minus methionine (1 mM), and RNasin (40 µg/µl). The reaction is incubated during 90 min at 30°C. Then, the lysates were frozen at –80°C.
Ubiquitination reaction
Of the protein extracts 25 µg was resuspended in a buffer containing 50 mM Tris/HCl, pH 7.5; 5 mM MgCl2; 0.2 mM DTT; 4 mM ATP; and 7 µg/µl ubiquitin in a final vol of 50 µl. The reaction was incubated 30 min at room temperature and stopped by the addition of loading buffer. To perform ubiquitination reaction on the reticulocyte lysate, we added 25 µg of protein extracts to 20 µl of reticulocyte lysate in the ubiquitination reaction buffer.
Nickel pull-down
COS cells were plated in 10 cm dishes and transfected (Ca-phosphate method) with 4 µg of pSGHis-ubiquitin, 5 µg hemagglutinin (HA)-p27, and/or 4 µg of Flag-HSP27. In cells analyzed in the absence of MG132 treatment, 4 µg of Flag-HSP27 and 8 µg of HA-p27 were cotransfected to compensate for HSP27-stimulated p27 down-regulation. Cells were lysed 48 h after transfection in 1 ml of buffer A (6 M guanidium-HCl; 100 mM Na2HPO4/NaH2PO4, pH 8.0; 10 mM imidazole). Lysates were sonicated (100 J) to reduce the viscosity. After centrifugation, extracts were incubated with 50 µl of Nickel-NTA-Agarose beads (Qiagen, Courtaboeuf, France) for 3 h at room temperature. Beads were then washed twice in buffer A; twice in buffer A diluted five times in 50 mM Tris, pH 6.8, 20 mM imidazole; and twice in 50 mM Tris, pH 6.8, 20 mM imidazole. Beads were then eluted with 30 µl of 2x Laemmli sample buffer supplemented with 20 mM imidazole, and supernatants were subjected to SDS-PAGE and Western blotting.
Gel-filtration analyses
Cells, 5 x 108, were lysed in 300 µl buffer (25 mM HEPES, pH 7.5; DTT 0.2 mM; CHAPS 1%) by vortex for 1 min in ice. After centrifugation for 15 min at 14000 g, 1 mg of the supernatant was fractionated at room temperature by fast protein liquid chromatography using a Superose-6 column (Pharmacia, Uppsala, Sweden) at a flow rate of 0.5 ml min–1. Fractions of 1 ml were collected on ice.
Immunofluorescence staining
Cells were fixed with PBS-paraformaldehyde 4% during 15 min and permeabilized by incubation with PBS-Triton 0.1% for 3 min. After washing with PBS, samples were saturated with PBS-BSA 3% during 30 min before incubation overnight at 4°C with the antibodies anti-HSP27 (StressGen) or anti-P27 (BD Pharmingen). After 4 washes with PBS-BSA 1%, appropriate secondary antibodies coupled with fluorochromes (Alexa 486 and 568 nM; Molecular Probe, Leiden, Netherlands) were added during 1 h at room temperature in the dark. The analysis was performed with a microscope Axiovert 200M (Zeiss, Germany).
Cell cycle analysis
Cells were washed in ice-cold PBS 1x, resuspended in 500 µl of ice-cold PBS 1x, and diluted by dropwise addition of 1.5 ml of 100% ethanol. After fixation in ethanol, samples were stored at 4°C for at least 1 h. Ethanol-fixed cells was resuspended in PBS 1x/dye: PBS 1x with 100 µg/ml ribonuclease A (Sigma-Aldrich) and 10 µg/ml propidium iodide (Sigma-Aldrich). Cells were incubated for 1 h at 37°C. Propidium iodide-stained nuclei were then analyzed using a Becton Dickinson LSR II cytometer. The percentage of cells in each phase of cell cycle was determined using Modfit software (Verity Software House, Topshame, ME).
Detection of apoptosis
Cells were seeded at 1.5 x 106 cells/ml in 75 cm2 plastic tissue culture flasks. At different times, cells were incubated in 5 mM EDTA for 5 min and washed with PBS. Cells (105) were incubated with 100 µl of 1x binding buffer (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2), 5 µl annexin V-FITC (Pharmingen, Becton Dickinson), and 1 µl propidium iodide (50 µg/ml). After 15 min at 4°C in the dark, cells were resuspended in 400 µl of binding buffer and analyzed by flow cytometry (FACScan, Becton Dickinson) using Cell Quest software (Becton Dickinson).
RESULTS
HSP27 overexpression enhances p27Kip1 proteasomal degradation
We have previously reported that HSP27 overexpression enhanced the proteasome activity in different cell lines exposed to heat or anticancer drugs (7)
. Here, we expanded this observation by showing that, in the U937 human leukemic cell line exposed to other stressful stimuli, including treatment with the NO donor GTN (500 µM) for 24 h and serum depletion for 48 h, HSP27 overexpression enhanced the ability of cell lysates to cleave the substrate Suc-LLVY-AMC that measures a chemotrypsin-like activity (Fig. 1
B). This HSP27-mediated effect was abolished in the presence of the proteasome inhibitors MG132 and lactacystin (Fig. 1B, C
), while it was not affected by the cathepsin inhibitor DCIC or the calpain I inhibitor ALLN (Fig. 1C
). These observations suggested that the measured chemotrypsin-like activity was mainly due to the proteasome. Down-regulation of HSP27 by expression of an antisense construct in control cells or by transient expression of a siRNA in HSP27 overexpressing cells (Fig. 1A
) prevented cells from enhancing their Suc-LLVY-AMC cleavage activity in response to GTN or serum depletion (Fig. 1B
). Similar effects of HSP27 were observed by using Z-LLE-MCA as a substrate to measure a trypsin-like activity, another catalytic activity of the proteasome (not shown).
|
The main identified function of the proteasome 26S is the degradation of ubiquitinated proteins. Therefore, we studied whether HSP27 expression levels could affect the cellular content of ubiquitinated proteins. Multi-ubiquitinated proteins accumulated in the cells exposed to the proteasome inhibitors lactacystin (Fig. 1D
) or MG132 (not shown). HSP27 protein expression concentration did not significantly affect the concentration of ubiquitinated proteins in nontreated cells (Fig. 1D
). In contrast, HSP27 overexpression decreased the content of ubiquitinated proteins in GTN-treated or serum-deprived cells. This decrease in ubiquitinated proteins was prevented by antisense-mediated decrease in HSP27 cellular content by a proteasome inhibitor like lactacystin, but it was not affected by a cathepsin inhibitor (Fig. 1D
).
Then, we analyzed whether, in these stressful conditions, HSP27 overexpression could facilitate the degradation of well-characterized targets of the proteasome, including ß catenin, cyclin A, cyclin D1, cyclin E, and p27Kip1. In response to GTN exposure, the Cdk inhibitor p27Kip1 was the only one among the five studied proteins whose proteasomal degradation was affected by HSP27 overexpression (Fig. 1E
). The decrease in the content of p27kip1 observed in HSP27 overexpressing cells exposed to stress was abolished by antisense-mediated HSP27 depletion (Fig. 1F
).
Quantitative PCR analyses indicated that p27kip1 gene transcription was not significantly different in control- and HSP27-transfected cells after GTN treatment (Fig. 1G
). A pulse-chase experiment showed that HSP27 overexpression did not significantly affect p27kip1 protein synthesis while it reduced its half-life under stress conditions such as GTN treatment (Fig. 1H
). These results indicate that HSP27 provokes a decrease in the content of p27kip1 by affecting mainly the protein half-life and not its synthesis or the gene transcription.
A kinetic study of p27Kip1 expression in serum-deprived U937 cells indicated that, in accordance with previous reports (24)
, the amount of p27Kip1 increased within 6 h, as cells accumulated in the G0-/G1-phase of the cell cycle, peaked at 12–24 h and subsequently decreased at 48 h as cells began to die (Fig. 2
A). HSP27 overexpression enhanced this serum starvation-induced degradation of p27Kip1 without modifying the initial accumulation of the protein (Fig. 2A
). Similarly, in Hela cells, HSP27 overexpression enhanced the degradation of p27Kip1 after serum deprivation (Fig. 2B
) or exposure to GTN (Fig. 2C
). In all these situations, the proteasome inhibitor MG132 prevented this enhanced degradation of p27Kip1 (Fig. 2B, C
), suggesting again that HSP27 overexpression favored the proteasomal degradation of p27Kip1 in response to stress.
|
The ability of HSP27 to enhance p27Kip1 degradation was further confirmed in COS cells transiently transfected with contructs encoding HSP27 and p27Kip1 (Fig. 2D
). Altogether, these results indicated that HSP27 overexpression could enhance the proteasomal degradation of p27Kip1 in stressful conditions.
p27Kip1 phosphorylation on Thr187 or Ser-10 is not required for interaction with HSP27
Coimmunoprecipitation experiments, performed with an equal amount of Ab and non-saturating conditions, in control and HSP27-transfected U937 cells demonstrated that HSP27 could associate with p27Kip1 while other HSPs, including HSP70 (Fig. 3
A) and HSP90 (not shown), did not. The HSP27 interaction with p27Kip1 was increased by HSP27 overexpression. Exposure of U937 cells overexpressing HSP27 to 500 µM GTN for 24 h (Fig. 3A
) or serum starvation for 48 h (not shown) enhanced the HSP27/p27Kip1 interaction (Fig. 3A
). Since phosphorylation on Thr187 has been shown to be a signal for p27Kip1 recognition by Skp2 protein, we analyzed whether HSP27 interaction with p27Kip1 required its phosphorylation on Thr187. Interestingly, HSP27 did not interact with Thr187-phosphorylated p27Kip1 (Fig. 3A
). Degradation of p27Kip1 by the recently described KPC ubiquitin ligase complex requires its phosphorylation on Ser-10 (21)
. Again, HSP27 did not interact with Ser-10 phosphorylated p27Kip1 (Fig. 3A
). In accordance with these observations, in response to GTN, HSP27 overexpression stimulated the degradation of nonphosphorylated p27Kip1 without affecting the degradation of Ser-10 or Thr187 phosphorylated forms (Fig. 3B
).
|
Serum depletion for 48 h and exposure to GTN for 24 h were observed to modify the subcellular location of p27Kip1 in U937-HSP27 cells (Fig. 3C
). In accordance with previous observations (25)
, HSP27 was located mainly in the cytosol. p27Kip1 was identified mainly in the nuclear compartment of nontreated cells and accumulated in the cytosol in response to GTN exposure or serum deprivation. Thus, the degradation of p27Kip1 observed in these cells under stressful conditions may occur in the cytosol.
HSP27 induced p27Kip1 degradation does not depend on the SCFSkp2 complex
The observation that HSP27 stimulates the degradation of a nonphosphorylated p27Kip1 suggested its involvement in a Skp2- and KPC-independent pathway of p27Kip1degradation. To check for the lack of involvement of Skp2 in the process, we first compared the effect of HSP27 and Skp2 on cellular protein ubiquitination. We performed in vitro ubiquitination experiments by adding ubiquitin and ATP to cellular extracts. Extracts from cells overexpressing HSP27 increased cellular protein ubiquitination compared with extracts from control cells. Conversely, antisense-mediated HSP27 depletion or immunodepletion of HSP27 from cell extracts decreased cellular protein ubiquitination (Fig. 4
A). An in vitro ubiquitination experiment was also performed by adding recombinant HSP27 or recombinant Skp2 or both to control cell extracts (Fig. 4B
). Recombinant HSP27 increased protein ubiquitination in a manner similar to that obtained by addition of Skp2 protein (Fig. 4B
). Surprisingly enough, simultaneous addition of HSP27 and Skp2 had no effect on cellular protein ubiquitination in this assay, suggesting an antagonistic effect (Fig. 4B
). These observations were confirmed by transient transfection of U937 cells with either HSP27 or Skp2 or both. Whereas HSP27 and Skp2 increased p27Kip1 degradation when overexpressed alone; their simultaneous overexpression did not (Fig. 4C
).
|
HSP27 interacts with Skp2 without recruiting the ubiquitin ligase complex
Coimmunoprecipitation experiments identified an interaction of HSP27 with Skp2 (Fig. 5
A). Two other HSPs, namely HSP70 and HSP90, did not interact with Skp2 (Fig. 5B
). Immunoprecipitation with an anti-HSP27 Ab failed to detect any other protein of the SCFSkp2 ubiquitin ligase complex, including Cul1 and Rbx1, whereas these proteins could interact with Skp2 in both control and HSP27-overexpressing cells (Fig. 5A
). These results suggested that HSP27 could interact with Skp2 when this latter protein was not part of the ubiquitin ligase complex.
|
HSP27 favors p27Kip1 ubiquitination
We next determined whether p27Kip1 ubiquitination was affected by HSP27 by using ubiquitin nickel beads. We observed that, in the presence of MG132 to inhibit ubiquitinated p27Kip1 degradation, HSP27 overexpression in Cos cells was associated with accumulation of mono-ubiquitinated p27Kip1 (Fig. 6
A), the only form of ubiquitinated p27Kip1 detected in our experimental conditions. In the absence of MG132, this mono-ubiquitinated p27Kip1 was rapidly degraded (Fig. 6B
).
|
HSP27 induces the degradation of p27Kip1 in the form of small oligomers
Gel filtration experiments have demonstrated that HSP27 could form oligomers of various sizes, ranging from dimers to 
1000 kDa large oligomers (2
, 26)
. This was confirmed here by showing that, in nonstressed normal cells, HSP27 could be recovered in the form of both small and large oligomers (Fig. 7
A). As described previously (2
, 3)
, HSP27 mutant Ala-HSP27, in which the three phosphorylatable serines have been replaced by alanine residues, forms oligomers of small and large size, as observed with the wild-type (WT) protein (Fig. 7A
). Whereas, HSP27 mutant Asp-HSP27, in which the three serine residues have been replaced by aspartates to mimic phosphorylation (27)
, forms only small oligomers (Fig. 7A
) (3)
. We stably expressed these two mutated forms of HSP27 and the WT protein in rat colon carcinoma REG cells that do not express detectable levels of endogenous HSP27 (28)
(Fig. 7A
) and exposed the cells to serum deprivation for 48 h or 500 µM GTN for 24 h. Expression of Asp-HSP27 mutant increased more efficiently Suc-LLVY-AMC cleavage activation induced by serum depletion (Fig. 7B
) or GTN (not shown) than expression of the mutant Ala-HSP27 or the WT HSP27. More specifically, the Asp-HSP27 mutant induced a stronger degradation of p27Kip1 than the Ala-HSP27 mutant or the WT HSP27 (Fig. 7C
). These data suggested that HSP27 activated the proteasome and favored Skp2-independent p27Kip1 degradation in the form of small oligomers.
|
Since a mutated HSP27 that forms exclusively large oligomers has never been identified, we performed gel-filtration analyses in an attempt to confirm our previous assumption. Cell extracts from HSP27-transfected cells, either untreated or serum depleted in the presence of MG132, were fractionated through a Superose 6 column (Fig. 8
A). We detected HSP27 oligomers in various eluted cellular fractions, which ranged from 100 to almost 1000 kDa. Serum depletion for 48 h induced a shift of HSP27 toward small oligomers, as described previously with other stimuli (29)
. p27Kip1 was found in a unique fraction, namely fraction 15, in which HSP27 is in the form of small oligomers, and was never identified in cellular fractions that contain HSP27 large oligomers. Coimmunoprecipitation analyses performed in fraction 15 identified an interaction between HSP27 and a nonphosphorylated form of p27Kip1 (Fig. 8B
), whereas phosphorylated p27Kip1 was not identified in the complex (not shown). Together with the results shown on Fig. 3B
, these observations suggest that small oligomers that accumulate in response to stress in HSP27-overexpressing cells are the form of HSP27 that preferentially interacts with p27Kip1. These observations enforced our conclusions, which indicate that small HSP27 oligomers were most probably the form of HSP27 involved in p27Kip1 ubiquitination and proteasomal degradation.
|
HSP27 affects the G0-/G1-phase of the cell cycle
p27Kip1 has been involved in maintenance of the G0-/G1-phase of the cell cycle, whereas its down-regulation is required to progress from G1 to S-phase, then from S-phase to G2. This observation led us to test whether and how the HSP27-mediated p27Kip1 degradation was involved in cell-cycle progression. Overexpression of HSP27 in U937 human leukemic cells and expression of this stress protein in REG colon carcinoma cells did not affect their progression in the cell cycle when grown in complete medium (Figures 9
A,C). After 48 h of serum starvation, we observed that control cells remained in the G0-/G1-phase of the cell cycle whereas HSP27-overexpressing cells progressed into S-phase (Fig. 9B,D
). The fraction of cells into S-phase was higher in those expressing the Asp-HSP27 mutant that forms only small oligomers as compared to those expressing the WT protein or the Ala-HSP27 mutant that form both small and large oligomers (Figure 7A
). These latter results further indicated that small oligomers of HSP27 might be the form of the protein involved in p27Kip1 ubiquitination and degradation and thereby in cell cycle progression from G1 to S-phase.
|
To demonstrate that HSP27 overexpression was the responsible for the detected effect in the repartition of cells in the different phases of the cell cycle, we performed loss of function experiments by RNA interference-mediated depletion of HSP27 in HSP27-overexpressing cells. HSP27 stably transfected Hela cells were transiently transfected with a HSP27 siRNA or, as a control, a siRNA targeting an irrelevant gene (luciferase). HSP27-siRNA specifically abrogated the degradation of p27Kip1 (Fig. 10
A) and the accumulation of cells in the S-phase of the cell cycle (Fig. 10B
) observed in HSP27 overexpressing cells after a 48h serum depletion. Similar observations were made in the rescue experiment performed by overexpressing p27Kip1 in HSP27-transfected cells (Fig. 10A
). In these conditions, serum depletion did not anymore induce an accumulation of cells in the S-phase of the cell cycle (Fig. 10B
). Thus, the effects of HSP27 overexpression on cell cycle progression are mediated by induction of p27Kip1 degradation.
|
DISCUSSION
The present study indicates that HSP27 interacts with p27Kip1 and, in some stressful conditions such as prolonged serum depletion and exposure to GTN, enhances the proteolysis of the Cdk inhibitor through the ubiquitin-proteasome pathway to promote progression from G0/G1 to S-phase of the cell cycle. HSP27-mediated p27Kip1 proteolysis occurs in a Thr187 phosphorylation- and Skp2-independent fashion and does not affect the protein when phosphorylated on Ser-10.
It has been recently shown that the main function of stress proteins was to prevent accumulation of denaturated and/or aggregated proteins, as an increase in ubiquitin-dependent degradation of proteins could prevent heat shock toxicity as efficiently as heat shock proteins (30)
. To promote the degradation of proteins, HSP70 and HSP90 require cochaperones such as CHIP, an ubiquitin ligase protein, and Bag1, an ubiquitin-like protein. Their function in protein degradation is directly related to their ATP-dependent chaperone activity and they do not demonstrate any direct ubiquitin-binding ability. In contrast to HSP70 and HSP90, HSP27 exerts its chaperone functions in the form of large oligomers, at least in cultured cells, and functions in the absence of ATP. In the present study, we show that HSP27 induces p27kip1 ubiquitination and degradation under the form of small oligomers, which suggests that this role of HSP27 may not be directly related to its chaperone function. One important property of HSP27 that differs from larger stress proteins such as HSP70 and HSP90 is its ubiquitin-binding ability. The small stress protein demonstrates a high affinity for long chains of ubiquitin (31)
. This effect accounts for the ability of the protein to promote I-B degradation by the 26S proteasome (7)
. HSP27 promotes ubiquitination of a number of other cellular proteins, including p27Kip1. How the protein targets for HSP27-mediated ubiquitination are selected remains to be determined.
It has been well established that the concentration of p27Kip1, which is elevated in quiescent cells and decreases on entry into the cell cycle, is controlled predominantly by the rate of p27Kip1 degradation (32
, 33)
. The first identified mechanism for p27Kip1 degradation involves its phosphorylation on Thr187 by the cyclin E/Cdk2 complex and its subsequent interaction with Skp2 through Cks1, leading to p27Kip1 ubiquitination by SCFSkp2 and proteasomal degradation (8
, 9
, 11
, 12
, 34)
. Analysis of Skp2 protein expression (17)
and subcellular location (18
19
20)
in the different phases of the cell cycle, as well as examination of the consequences of skp2 gene disruption in mouse cells (17)
, have indicated that Skp2 was not involved in the degradation of p27Kip1 at G1-phase. Rather, Skp2 functions to lower the expression of p27Kip1 during S- and G2-phases. A KPC-dependent pathway was shown recently to account for the cytosolic degradation of p27Kip1 in G1-phase. KPC consists of the RING-finger domain containing protein KPC1 and the ubiquitin-like (UBL) domain and ubiquitin-associated (UBA) domain containing protein KPC2. Interestingly, this pathway requires the nuclear export of p27Kip1 by CRM1 and Jab1 (21)
. Phosphorylation on Ser-10, which increases p27Kip1 stability, could be a determinant of its Jab-1-mediated nuclear export (17
, 35)
. The HSP27-mediated proteasomal degradation of p27Kip1 does not affect the protein when phosphorylated on either Ser-10 or Thr187, which suggests an additional mechanism. Another protein, known as SAG (sensitive-to-apoptosis gene), which is a component of an E3 ubiquitin ligase complex (SAG/ROC2/Rbx2/Hrt2) and regulates culin functions, also prevents p27Kip1 accumulation induced by serum starvation by increasing its proteasomal degradation (36)
. Whether SAG cooperates with HSP27 in this function will require further investigation.
In the absence of stress, interaction of HSP27 with p27Kip1 does not result in its degradation. In response to stress, HSP27 shifts to small oligomers and p27Kip1 is ubiquitinated and degraded. HSP27 favors p27Kip1 ubiquitination rather than prevents deubiquitination since, when the proteasome enzymatic function is inhibited, the ubiquitinated protein similarly accumulates in control, HSP27 sense-, antisense-, and siRNA-transfected cells (data not shown). In our experimental conditions, we detected only a mono-ubiquitination of p27Kip1, while polyubiquitination is usually required for the protein targeting to the proteasome and degradation. HSP27 could induce p27Kip1 mono-ubiquitination with other finalities than targeting to the proteasome such as facilitation of the nuclear export of the protein (37)
, which, in turn, facilitates its proteosomal degradation in the cytosol. However, as HSP27 is located mainly in the cytosol, a more likely possibility is that mono-ubiquitination of p27Kip1 is the first step of a polyubiquitination process that either cannot be detected in our experimental conditions or requires additional proteins. For example, MDM2 was shown to trigger only mono-ubiquitination of p53 in vitro while inducing polyubiquitination and degradation of the protein in vivo, which was related to insufficient amounts of p300, a subtype 4 ubiquitin ligase protein necessary for extension of ubiquitin chains on mono-ubiquitinated p53 (38)
. Whatever the type of p27Kip1 ubiquitination mediated by HSP27, this event is an initial step toward the proteasomal degradation of the protein.
The F-box protein Skp2, which together with Skp1, Cul1, and Roc1/Rbx1 forms the SCFSkp2 E3 ubiquitin ligase complex, can interact with HSP27 protein in the absence of other proteins of the SCF complex. The Skp2/HSP27 interaction appears to antagonize the ability of HSP27 to promote protein ubiquitination and degradation in vitro and in vivo. The retinoblastoma protein Rb was recently shown to interact also with Skp2, which prevents its interaction with p27Kip1 (39)
; whereas another tumor suppressor protein, tuberin, binds to p27Kip1, sequesters it from Skp2 and prevents its degradation (40)
. In both cases, cell-cycle progression is impaired. Under stress conditions, overexpressed HSP27 could sequester Skp2 from the SCFSkp2 ubiquitin ligase complex while favoring the degradation of p27Kip1 through a Skp2-independent pathway. An interaction between an F-box protein and a small heat shock protein has already been described, i.e., the small heat shock protein B-crystallin could interact with the F-box-containing protein FBX4 to favor ubiquitination of a still-unidentified protein. Interestingly, this interaction depends on the serine phosphorylation status of B-crystallin and the cell cycle (41)
. Phosphorylation of HSP27 could also play a role in regulating its interaction with Skp2 and its ability to promote protein ubiquitination and degradation as this event induces the formation of small oligomers, which appear to be the active form of the stress protein for these functions.
A major expectation of our observations was that overexpressed HSP27 would promote cell-cycle progression by increasing p27Kip1 degradation. The HSP27-mediated, Skp2-independent degradation of p27Kip1 at the G0/G1 transition, identified in tumor cells under certain stress conditions, could account for a previously suggested role for HSP27 in cell proliferation, e.g., overexpression of HSP27 was shown to enhance the anchorage-dependent and -independent growth of breast cancer cells (42)
. In addition, HSP27 has been reported to accumulate in tumor cells grown at confluence (43
, 44)
. The accumulated protein could facilitate the re-entry of the quiescent cells into the cell cycle. Therefore, part of the protective effect of this late stress responsive protein would be to restart cell proliferation.
Mouse models have provided evidence that p27 gene is a haplosufficient tumor-suppressor gene (45)
. Mice lacking p27Kip1 develop spontaneous pituitary tumors (46
47
48)
and become more susceptible to tumor formation in multiple tissues when challenged with carcinogens (49)
. Deregulation of p27Kip1 is observed in human tumors, due to posttranslational mechanisms that include enhanced proteolysis, and low concentration of p27Kip1 is an independent negative prognostic marker in many tumor types (50)
. This suggests that proteins involved in p27Kip1 degradation may have oncogenic properties. HSP27 has been observed to be highly expressed in transformed cells compared with its normal counterparts, and some clinical studies have associated an increased concentration of HSP27 in tumors with poor outcome (51
52
53
54)
. The present study suggests that HSP27 overexpression could prevent p27Kip1 from arresting cell proliferation in response to stresses, which, depending on the cell context, may favor cell death by apoptosis or prevent DNA damage repair and favor transformation. This latter effect may be enforced by the ability of HSP27 to prevent cell death; HSP27 is a powerful protective protein that increases cancer cell resistance to chemotherapeutic agents. In addition, HSP27 expression is often constitutively high in human cancer cells (4
, 26
, 55)
. High concentration of Skp2 was correlated with low levels of p27Kip1 in some high-grade lymphomas (56)
. Whether high expression concentration of HSP27 correlates with low expression of p27Kip1 in human tumors has now to be determined. Such a correlation would suggest that overexpressed HSP27 may be a potential target for tumor cell eradication.
ACKNOWLEDGMENTS
This work was supported by grants from the Ligue Nationale Contre le Cancer and its committees in the Nièvre and Côte d’Or departments. A.P. is a recipient of a doctoral fellowship from l’Association pour la Recherche contre le Cancer; M.B., from the Ministère de l’Education; E.C., a postdoctoral fellowship from Sidaction; and C.D., a postdoctoral fellowship from Fondation pour la Recherche Médicale. We thank A. Bouchot, N. Sassi, and S. Cathelin for technical assistance and M. Townsend for English corrections.
FOOTNOTES
1 These authors contributed equally to this work. 
Received for publication April 19, 2005. Accepted for publication February 10, 2006.
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). Cells were either left untreated (NT), treated during 24 h with GTN (500 µM), or serum depleted for 48 h (SD) in the presence or absence of the proteasome inhibitor MG132 (MG, 20 µM). C) Suc-LLVY-AMC cleavage was measured in lysates from control (
