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Molecular and Cellular Biology, April 2007, p. 3187-3198, Vol. 27, No. 8
0270-7306/07/$08.00+0     doi:10.1128/MCB.01461-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Glycogen Synthase Kinase 3ß Phosphorylates p21WAF1/CIP1 for Proteasomal Degradation after UV Irradiation{triangledown} ,{dagger}

Ji Young Lee,1,2,3 Su Jin Yu,1,3 Yun Gyu Park,1,3 Joon Kim,2* and Jeongwon Sohn1,3*

Department of Biochemistry, Korea University College of Medicine, Seoul 136-705, South Korea,1 School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea,2 Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705, South Korea3

Received 8 August 2006/ Returned for modification 4 September 2006/ Accepted 23 January 2007


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
UV irradiation has been reported to induce p21WAF1/CIP1 protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3ß (GSK-3ß) is required for its degradation in response to UV irradiation and that GSK-3ß activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3ß activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3ß. ser-114, located within the putative GSK-3ß target sequence, was phosphorylated by GSK-3ß upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3ß is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3ß as the missing link between UV-induced ATR activation and p21 degradation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
p21WAF1 (hereafter referred to as p21), which belongs to the CIP/KIP family of cyclin-dependent kinase inhibitors, is best known as an inhibitor of cell proliferation, although it also plays a role in cell differentiation, senescence, modulation of apoptosis, and activation of cyclin D-Cdk4/Cdk6 (18). The protein is short lived, with a half-life of ~30 min, and its expression is tightly regulated by proteasome-mediated protein degradation. Turnover of p21 protein occurs through both ubiquitination-dependent and -independent proteasome pathways (7, 40, 48). E3-ubiquitin ligases, such as SCFSkp2 and p53RFP, have been implicated in the ubiquitination-dependent pathway of p21 degradation (6, 8, 29). The carboxy-terminal region of p21 directly binds to the C8{alpha} subunit of the 20S proteasome, leading to ubiquitination-independent degradation by the proteasome (12, 48, 54).

Although the function of p21 is critical in DNA damage responses, such as cell cycle arrest, DNA repair, and apoptosis, its expression is differentially regulated by {gamma}- and UV irradiation. {gamma}-Irradiation enhances p21 expression by p53-dependent transcriptional activation, whereas UV irradiation often down-regulates p21 expression by increasing proteasome-mediated protein degradation. Since p21 functions in a variety of different cellular processes, the consequences of changes in p21 regulation after DNA damage are complex. The increased expression of p21 protein induced by DNA damage inhibits cell cycle progression and replication through its interactions with cyclin-Cdk complexes and PCNA (11, 32, 50). At the same time, p21 inhibits DNA repair through its effect on PCNA (14, 43, 44). Recently, p21 was reported to inhibit PCNA ubiquitination relevant to DNA repair (45). However, a positive role for p21 in DNA repair has been found by other groups (28, 41). Similarly, previous reports indicate that p21 plays both anti- and proapoptotic roles (22, 42, 51). Cytoplasmic p21 can interact with ASK1 and procaspase 3 to suppress apoptosis (3, 47). In contrast, overexpression of p21 induces apoptosis in T47D breast carcinoma cells, and retinoic acid-induced p21 forms a complex with cyclin E-Cdk2 to promote apoptosis (5, 42).

The signal that leads to p21 degradation in responses to UV irradiation is poorly understood. Bendjennat et al. (6) reported that ubiquitination by SCFSkp2 was required for the UV-induced degradation of p21 protein. Although the involvement of SCFSkp2 implies phosphorylation of p21, the identity of the responsible kinase was not established. p21 contains many consensus sites for various kinases, and kinases such as Cdk2, Akt (protein kinase B [PKB]), Jun N-terminal protein kinase, p38{alpha}, glycogen synthase kinase 3ß (GSK-3ß), PKA, and PKC have been shown to phosphorylate p21. Phosphorylation of p21 not only regulates the stability of the protein but also affects interaction with its binding partners and modulates its subcellular localization. Protein interactions and the intracellular localization of p21 may influence its stability as well. For example, Cdk binding greatly enhances the proteasomal degradation of p21 (10), whereas cyclin D1 competes with the C8{alpha} subunit of the 20S proteasome complex for binding p21, thereby interfering with its proteasomal degradation (12). A link between intracellular localization and proteolysis has been demonstrated for a related protein, p27KIP1 (13).

GSK-3ß is an evolutionarily conserved and ubiquitously expressed serine/threonine kinase (23) that functions in multiple biological processes, including embryonic development, cell differentiation, apoptosis, cell cycle progression, and insulin response. In contrast to other protein kinases, GSK-3ß is constitutively active in intact cells (23, 46), and its activity can be down-regulated by external stimuli, such as insulin and growth factors. ser-9 phosphorylation mediated by serine/threonine kinases, such as Akt, PKA, and PKC, leads to inactivation of GSK-3ß (15, 20, 21). GSK-3ß suppresses cell proliferation, and it also induces apoptosis under a number of conditions (16, 30). Recent work has shown that GSK-3ß mediates apoptosis by interaction with p53, inhibition of NF-{kappa}B signaling, and prevention of binding of hexokinase II to mitochondria (31, 38, 52).

In the present study, we investigated the signaling intermediates involved in p21 degradation following UV irradiation. Our data demonstrate that ATR stimulates GSK-3ß activity upon UV irradiation and that UV-activated GSK-3ß plays a critical role in p21 protein degradation by phosphorylating p21 at ser-114.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human malignant melanoma cell lines (SK-MEL-1 and SK-MEL-2), a human cervical cancer cell line (HeLa), and a human breast cancer cell line (MCF-7) were purchased from the Korea Cell Line Bank, and a human keratinocyte cell line (HaCaT) was kindly provided by N. E. Fusenig (German Cancer Research Center, Germany). SK-MEL-1 and SK-MEL-2 cells were maintained in RPMI 1640. HeLa, MCF-7, and HaCaT cells were cultured in Dulbecco's modified Eagle's medium. Both media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B.

UV irradiation. Cells were grown to approximately 70 to 80% confluence in 35-mm dishes. Prior to UV (UVC) irradiation (XL-1500 UV cross-linker; Spectronics), the culture medium was removed to deliver as exact a UV dose as possible and then restored afterwards. A UVC dose which induced death in 50% of the cells after 24 h was used for most experiments.

Antibodies, reagents, and plasmids. Polyclonal antibodies to p21 (SC-397) and GSK-3ß (SC-9166) were purchased from Santa Cruz Biotechnology. Monoclonal antibodies to p53 (Ab-6) and GSK-3ß (610201) were from Oncogene and BD Transduction Laboratories, respectively. Phospho-specific antibodies to GSK-3{alpha}/ß (9331) and Akt (9271) were from Cell Signaling Technology. Horseradish peroxidase-conjugated anti-mouse (NA 931) and anti-rabbit (NA 934) secondary antibodies were purchased from Amersham Pharmacia. Caffeine, wortmannin, staurosporine, DRB (5,6-dichlorobenzamidazole riboside), lithium chloride, acetyl-leu-leu-norleucinal (ALLN), and clasto-lactacystin ß-lactone were purchased from Sigma-Aldrich. Thiadiazolidinone-8 (TDZD-8) and 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate were obtained from Calbiochem. N-acetyl-leu-leu-norleucinal (LLnL) and carbobenzol-L-leucyl-L-leucyl-L-leucinal (MG-132) were from Calbiochem and Alexis, respectively. SB216763 was obtained from Tocris. Recombinant tau and p21 were obtained from Invitrogen and Spring Bioscience, respectively. The pCMV5-FLAG, pCMV5-FLAG-GSK-3ß (wild-type), and pCMV5-FLAG-GSK-3ß (K85A) plasmids were kindly provided by Eui-ju Choi (Korea University, South Korea). The pcDNA3.1-Myc-His-p21 wild-type and S27A, T57A, S114A, and T145A mutant p21 plasmids were kind gifts from S. Dimmeler (University of Frankfurt, Germany).

Reverse transcription-PCR. Total cellular RNA was prepared using TRIzol (GIBCO BRL), and RNA (1 µg) was reverse transcribed using oligo(dT)17 primer (Bioneer) and Moloney murine leukemia virus reverse transcriptase. cDNA synthesized from the total RNA was amplified with primers specific for human p21. The primer sequences of human p21 and ß-actin are as follows: p21 forward primer, 5'-CGAAGTCAGTTCCTTGTGGA-3'; p21 reverse primer, 5'-GGCAGAAGATGTAGAGCGGG-3'; ß-actin forward primer, 5'-CAGAGCAAGAGAGGCATC-3'; ß-actin reverse primer, 5'-CGTAGATGGGCACAGTGT-3'.

Immunoblot analysis. Immunoblot analysis was carried out as previously described (53). Briefly, total cell lysates (30 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked in 5% skim milk for 1 h and incubated sequentially with the primary antibody for 2 h and a horseradish peroxidase-conjugated secondary antibody for 1 h. The protein band of interest was detected using an enhanced chemiluminescence reagent (Amersham Pharmacia).

Immunoprecipitation and in vitro kinase assay. Cells were harvested in lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 10 mM ß-glycerophosphate, 1 mM dithiothreitol, 0.1 mM Na3VO4, 0.2 µg/ml each of leupeptin and aprotinin, 500 µg/ml phenylmethylsulfonyl fluoride). For each sample, total cellular protein (300 µg) was precleared with protein A-Sepharose for 1 h at 4°C and centrifuged at 20,000 x g for 5 min. Anti-GSK-3ß or anti-p21 antibody (2 µg) was added to the supernatant, and the mixture was incubated overnight at 4°C. The immunocomplex was captured by protein A-Sepharose beads (Amersham Pharmacia), centrifuged (4,000 x g, 2 min, 4°C), and washed three times with lysis buffer. For the in vitro kinase assay, immunoprecipitated GSK-3ß was washed three times with kinase buffer (20 mM Tris [pH 7.5], 5 mM MgCl2, 1 mM dithiothreitol) at 4°C and incubated with 1.4 µCi [{gamma}-32P]ATP (Amersham Pharmacia), 2.5 µg recombinant tau (Invitrogen), or 1 µg recombinant p21 (Spring Bioscience) in 20 µl kinase buffer. The kinase reaction was carried out for 30 min at 30°C and stopped by the addition of 2x Laemmli sample buffer. Samples were boiled for 10 min and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After drying, the gel was exposed to an X-ray film (Agfa).

Plasmid transfection. For stable transfection of wild-type and mutant GSK-3ß plasmids, MCF-7 cells (3 x 105/well) were seeded in six-well plates and cultured for 18 h before transfection. MCF-7 cells were used for transfection experiments, since it was difficult to transfect SK-MEL-1 cells. The plasmids pCMV5-FLAG, pCMV5-FLAG-GSK-3ß (the wild type), and pCMV5-FLAG-GSK-3ß (K85A) (2 µg) were transfected into cells using Lipofectamine and Plus reagent (Invitrogen). After 48 h, transfected cells were selected with neomycin (500 µg/ml). For transient transfection of the wild-type and mutant p21 constructs, the pcDNA3.1-Myc-His or pcDNA3.1-Myc-His-p21 wild-type or mutant plasmids (1 µg) were transfected into SK-MEL-2 cells (1 x 105/well) in six-well plates with Fugene 6 (Roche) according to the manufacturer's instructions. Experiments were carried out 24 h after transfection.

siRNA transfection. MCF-7 or SK-MEL-1 cells (3 x 105 to 5 x 105/well) were seeded in six-well plates and cultured for 18 h before transfection. Green fluorescent protein (GFP), ATM, or ATR small interfering RNA (siRNA; 100 nM each) was transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Inhibition of ATM or ATR protein expression was assessed by immunoblot analysis 48 h after transfection. Experiments were carried out 48 h after siRNA transfection. The coding strand sequence of the ATM siRNA was 5'-CUCUGUUAACCAUUGUAGA-3'. The ATR siRNA sequence was 5'-GAGUAGAAGAUUCAAGCUU-3', the GSK-3ß siRNA sequence 5'-GGACAAGAGAUUUAAGAAU-3', and the GFP siRNA sequence 5'-CCUACGCCACCAAUUUCGU-3'.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of UV irradiation on p21 protein expression. There have been conflicting reports regarding the effect of UV on the regulation of p21 protein expression (2, 6, 33). However, as shown in Fig. 1A, exposure of various human cancer cell lines to UV, including SK-MEL-1 (melanoma), HeLa (cervical cancer), and MCF-7 (breast cancer) as well as an untransformed keratinocyte cell line, HaCaT, resulted in a dose- and time-dependent decline in the p21 protein level. Depending on the cell line, the level of p21 protein was maximally reduced from 1 to 8 h after UV exposure, and expression partially recovered thereafter (Fig. 1A). Although p53 is well known to activate p21 transcription following DNA damage (19), expression of p21 protein was down-regulated regardless of p53 status (for MCF-7 and HeLa, wild-type p53; for SK-MEL-1 and HaCaT, mutated p53). Furthermore, the steady-state mRNA levels of p21 in both MCF-7 and SK-MEL-1 cells were not affected by UV exposure, although the p53 protein level in MCF-7 cells increased (Fig. 1B). Expression of p21 mRNA was up-regulated in MCF-7 cells after {gamma}-irradiation (see Fig. S1 in the supplemental material).


Figure 1
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  		FIG. 1. UV irradiation reduces p21 protein expression. (A) UV reduces p21 protein expression in a time- and dose-dependent manner in various cell lines. Upper panels: time-dependent analysis. SK-MEL-1, HeLa, MCF-7, and HaCaT cells were irradiated with 100, 30, 20, and 25 J/m2 UV, respectively, and incubated for the indicated time periods before cell lysates were prepared for immunoblot analysis. The UV doses used here represent those that induce 50% lethality at 24 h. Lower panels: dose-dependent analysis. Cell lysates were prepared 2 h after UV irradiation, and p21 protein levels were determined by immunoblot analysis. ß-Actin and {alpha}-tubulin served as loading controls. (B) Expression of p53 protein and p21 mRNA after UV irradiation. SK-MEL-1 and MCF-7 cells were irradiated with UV at 100 and 20 J/m2, respectively. Upper panels: reverse transcription-PCR (RT-PCR) analysis of p21 mRNA expression. The band intensities were measured by densitometry, and the levels of p21 mRNA relative to those of the respective ß-actins are indicated. Lower panels: immunoblot analysis of p53 protein expression.

 
To determine whether decreased p21 protein expression was due to reduced stability, its half-life in UV-irradiated SK-MEL-1 and MCF-7 cells was measured. Cells were treated with cycloheximide (CHX) to prevent new protein synthesis, and p21 protein levels were analyzed at different time points after UV irradiation. As shown in Fig. 2A and B, the half-life of p21 protein in SK-MEL-1 cells was reduced from 40 min before UV irradiation to 17 min after UV irradiation and from 2.0 h to 1.3 h in MCF-7 cells. The longer half-life of p21 in MCF-7 cells is probably due to a defect in protein degradation (37). Furthermore, enhanced proteasomal degradation was found to be responsible for the down-regulation of p21 protein, since LLnL and other proteasome inhibitors effectively blocked the reduction of p21 protein expression (Fig. 2C). These results demonstrate that UV irradiation enhances the proteasomal degradation of p21 protein, independent of p53 status.


Figure 2
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  		FIG. 2. UV irradiation reduces p21 protein stability by increasing proteasome-mediated degradation. (A) SK-MEL-1 and MCF-7 cells were treated with CHX (30 µg/ml) alone or CHX and UV (100 and 20 J/m2, respectively). p21 protein levels at the indicated time points were assessed by immunoblot analysis. (B) The half-life of p21 protein was determined by densitometric quantitation of three independent immunoblots. (C) SK-MEL-1 cells were pretreated for 1 h with LLnL (10 µM) or dimethyl sulfoxide (left panel) or with various proteasome inhibitors, ALLN (50 µM; Al), LLnL (Ln; 10 µM), MG-132 (MG; 50 µM), clasto-lactacystin ß-lactone (Lac; 2.5 µM), or dimethyl sulfoxide (C) (right panel), before UV irradiation (100 J/m2). Protein levels of p21 were determined by immunoblot analysis 2 h after UV irradiation. ß-Actin served as a loading control.

 
Requirement of ATR and GSK-3ß for p21 protein degradation after UV irradiation. To investigate the signaling molecule(s) mediating UV-induced p21 protein degradation, the effects of various kinase inhibitors, such as caffeine (an ATM/ATR inhibitor), lithium chloride (a GSK-3ß inhibitor), staurosporine (a PKC inhibitor), wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor), DRB (a casein kinase II inhibitor), and 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (an Akt inhibitor), were assessed. Among these, only caffeine and lithium chloride prevented the UV-induced degradation of p21 protein, implying that ATM/ATR and GSK-3ß are involved in p21 degradation (Fig. 3A). When cells were treated with LiCl, staurosporin, or wortmannin, the p21 protein level decreased even without UV exposure, presumably because they affect p21 expression at or before translation. In these cases, CHX was included to eliminate the effect of the inhibitor alone on p21 expression.


Figure 3
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  		FIG. 3. The UV-induced degradation of p21 protein is prevented by inhibition of ATR and GSK-3ß. (A) SK-MEL-1 cells were pretreated for 1 h with caffeine (5 mM), LiCl (100 mM), staurosporine (STP; 1 nM), wortmannin (Wort; 50 nM), DRB (35 to 70 µM), 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (ICIO; 20 µM), or dimethyl sulfoxide and irradiated with UV (100 J/m2). CHX (30 µg/ml) was added to cells at the time of UV irradiation. After 1 h of incubation, cells were harvested for immunoblot analysis of p21 protein. ß-Actin served as a loading control. (B) MCF-7 cells were transfected with siRNA specific to GFP, ATR, or ATM (100 nM). After 48 h, the protein levels of ATR and ATM were analyzed by immunoblotting. The band intensities were measured by densitometry, and the levels of p21 protein expression relative to those of the respective {alpha}-tubulins are indicated. (C) MCF-7 cells were transfected with siRNA specific to ATR, ATM, or GFP as a control. After 48 h, cells were irradiated with UV (20 J/m2), and p21 protein levels were analyzed by immunoblotting. (D) TDZD-8 also blocks degradation of p21 protein induced by UV. Cells were pretreated with various doses of TDZD-8 alone (upper panels) or treated with both TDZD-8 (120 µM for SK-MEL-1 and 60 µM for MCF-7) and CHX (30 µg/ml) (lower panels) for 1 h. Cells were then irradiated with UV, and p21 protein levels were analyzed 1 (SK-MEL-1) or 3 (MCF-7) h afterwards. ß-Actin and {alpha}-tubulin served as loading controls. (E) SK-MEL-1 cells were transfected with siRNA (100 nM) specific to GSK-3ß or GFP as a control. Left panel: at 48 h after siRNA transfection, cells were irradiated with UV, and 1 h later, the p21 protein expression was assessed by an immunoblot analysis. Right panel: the level of GSK-3ß protein was measured by an immunoblot analysis 48 h after siRNA transfection.

 
The involvement of ATM/ATR in p21 protein degradation in response to UV was previously reported (6), but the downstream molecule(s) involved in the UV-induced destabilization of p21 protein was not described. Therefore, we further investigated the roles of ATM/ATR and GSK-3ß as well as a causal relationship between their activation. First, ATM or ATR siRNA was introduced to MCF-7 cells, and their effect on p21 degradation was examined. Introduction of ATM or ATR siRNA significantly reduced the respective protein and mRNA expressions (Fig. 3B; see also Fig. S2 in the supplemental material). As shown in Fig. 3C, only the ATR siRNA prevented p21 protein degradation, suggesting that ATR but not ATM is required for p21 degradation by UV irradiation. Since the Chk1 and Chk2 protein kinases are activated by ATR and ATM, respectively, the abilities of the Chk1 and Chk2 inhibitors {SB218078 and 2-[4-(4-chlorophenoxy)phenyl]-1H- benzimidazole-5-caboxamide, respectively} to block UV-induced p21 protein destabilization were assessed. However, these agents had no effect (data not shown).

Although inhibition by lithium chloride suggested that GSK-3ß plays a role in UV-induced p21 destabilization (Fig. 3A), there have been no reports that GSK-3ß is involved in stress-induced p21 regulation. To confirm the role of GSK-3ß in p21 degradation by UV irradiation, two other GSK-3ß inhibitors, TDZD-8 (a non-ATP-competitive inhibitor) and SB216763 (an ATP-competitive inhibitor), as well as siRNA for GSK-3ß were used. TDZD-8 specifically inhibits GSK-3ß activity with little effect on other kinases (27). Figure 3D shows that TDZD-8 prevented UV-induced p21 protein degradation in a dose-dependent manner, and complete inhibition was observed with doses of 60 and 120 µM in MCF-7 and SK-MEL-1 cells, respectively. Pretreatment of the cells with SB216763 or introduction of GSK-3ß siRNA also blocked p21 degradation (Fig. 3E; see also Fig. S3 in the supplemental material). Furthermore, another line of evidence for the requirement of GSK-3ß in the UV-induced degradation of p21 protein was obtained using the catalytically inactive mutant (K85A) of GSK-3ß. The mutant GSK-3ß gene was stably transfected into MCF-7 cells, and the p21 protein level following UV irradiation was analyzed. Ectopic expression of the mutant GSK-3ß was confirmed by immunoblot analysis (Fig. 4A). As shown in Fig. 4B, cells expressing the K85A mutant GSK-3ß (clones K85A-1 and K85A-2) failed to reduce p21 protein expression following UV irradiation. These results demonstrate that GSK-3ß activity is indeed necessary for UV-induced p21 protein degradation.


Figure 4
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  		FIG. 4. Overexpression of the catalytically inactive GSK-3ß mutant stabilizes the p21 protein. MCF-7 cells were stably transfected with an empty vector (pCMV5-FLAG) or wild-type GSK-3ß or K85A mutant GSK-3ß cDNA. (A) GSK-3ß expression in transfected MCF-7 cells was analyzed by immunoblotting. Two independent clones of MCF-7 cells transfected with the K85A mutant GSK-3ß gene are shown in lanes 3 and 4. (B) Stable clones were treated with CHX (30 µg/ml) and UV (20 J/m2) or CHX alone. Cells were harvested 2 h after UV irradiation, and p21 protein levels were analyzed by immunoblotting. The relative p21 protein expression in lane 3 of each blot, compared to that in lane 2, was calculated based on densitometric measurement of the bands and is indicated as a percentage. *, endogenous GSK-3ß.

 
Activation of GSK-3ß by UV irradiation. The results shown above strongly demonstrate the critical role of GSK-3ß in the UV-induced degradation of p21. Therefore, we investigated whether UV stimulates GSK-3ß kinase activity. Since the presence of serum in the culture medium could interfere with GSK-3ß activity, a serum-free condition was employed to assess GSK-3ß activity. As shown in Fig. 5A, the serum-free culture condition did not affect the UV-induced degradation of p21 protein or inhibition of degradation by TDZD-8. An in vitro kinase assay using recombinant tau protein as a substrate showed that the kinase activity of GSK-3ß increased in a time-dependent manner. Maximum activation (2.2-fold) was observed at 30 min, and activity declined to the basal level at 60 min (Fig. 5B, left panel). In the control reactions in which GSK-3ß immunoprecipitation was performed with normal serum instead of anti-GSK-3ß antibody or the recombinant tau substrate was not included, phosphorylated tau bands were not observed (Fig. 5B, right panel). Figure 5C shows that TDZD-8 suppressed the UV-induced activation of GSK-3ß. Since ser-9 phosphorylation of GSK-3ß is known to inhibit kinase activity, we next examined whether the ser-9 residue of GSK-3ß underwent changes upon UV stimulation. The data shown in Fig. 5D demonstrate that ser-9 phosphorylation of GSK-3ß decreased from 15 to 30 min after UV irradiation, indicating GSK-3ß activation. ser-9 phosphorylation of GSK-3ß was recovered to a control level at 60 min. These data strongly indicate that the activity of GSK-3ß increases transiently after UV irradiation.


Figure 5
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  		FIG. 5. GSK-3ß is activated by UV irradiation. (A) The serum-free condition employed in an in vitro kinase assay does not affect UV-induced p21 degradation or inhibition of degradation by TDZD-8. SK-MEL-1 cells were pretreated with 60 µM TDZD-8 for 1 h prior to UV exposure (100 J/m2). Cells were harvested after 2 h, and the levels of p21 protein were assessed by immunoblot analysis. (B) Left panel: SK-MEL-1 cells were cultured in a serum-free medium for 4 h and irradiated with UV (100 J/m2). GSK-3ß was immunoprecipitated with a monoclonal anti-GSK-3ß antibody from the whole-cell lysate. The kinase activity of GSK-3ß was measured using recombinant tau protein as a substrate as described in Materials and Methods. GSK-3ß was immunoblotted as an input control. Right panel: the in vitro kinase activity of GSK-3ß at 30 min after UV irradiation was measured as described for the left panel. (C) TDZD-8 inhibits UV-induced GSK-3ß activity. SK-MEL-1 cells were cultured in a serum-free medium for 4 h and treated with 60 µM TDZD-8 for 1 h, followed by UV irradiation (100 J/m2). After 30 min of incubation, cells were harvested and GSK-3ß was immunoprecipitated. An in vitro kinase assay for GSK-3ß was performed as described for panel B. (D) The inhibitory ser-9 phosphorylation of GSK-3ß is abolished by UV irradiation. SK-MEL-1 cells were cultured in a serum-free medium for 4 h, UV irradiated (100 J/m2), and harvested at the indicated time points. ser-9 phosphorylation of GSK-3ß was analyzed by immunoblotting using an antibody specific for GSK-3ß phosphorylated at ser-9. Total GSK-3ß served as a loading control.

 
GSK-3ß is a downstream effector in the UV-induced ATR signaling pathway. Since ATR activation is required for the UV-induced destabilization of p21 protein, we investigated whether GSK-3ß was activated through the ATR signaling pathway. As shown in Fig. 6A, treatment with both caffeine and TDZD-8 at suboptimal doses did not exert an additive effect on the recovery of p21 protein expression, suggesting that ATR and GSK-3ß participate in the same signaling pathway. In addition, an in vitro kinase assay showed that caffeine prevented the increase of GSK-3ß activity after UV irradiation (Fig. 6B), indicating that GSK-3ß activation is ATM/ATR dependent. We then abrogated the expression of ATM or ATR using siRNA and assessed GSK-3ß activity before and after UV irradiation. In an in vitro kinase assay, UV-induced GSK-3ß activation was inhibited by ATR siRNA, further demonstrating that ATR is required for UV-induced GSK-3ß activation (Fig. 6C). Introduction of ATM or GFP siRNA did not affect GSK-3ß activation by UV. Altogether, these results demonstrate that GSK-3ß is activated through the UV-triggered ATR signaling pathway.


Figure 6
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  		FIG. 6. GSK-3ß activation by UV irradiation occurs downstream of ATR activation. (A) Inhibitors of ATM/ATR and GSK-3ß do not exert an additive effect on the UV-induced degradation of p21. SK-MEL-1 cells were pretreated with caffeine (3 mM), TDZD-8 (40 µM), or both for 1 h and irradiated with UV (100 J/m2). Two hours after UV exposure, p21 protein levels were determined by immunoblot analysis. (B) Caffeine inhibits UV-induced GSK-3ß activation in an in vitro kinase assay. SK-MEL-1 cells were cultured in a serum-free medium for 4 h, pretreated with caffeine (3 mM) for 1 h, and irradiated with 100 J/m2 of UV. After 30 min, cells were harvested and GSK-3ß was immunoprecipitated. The in vitro kinase assay was performed as described in Materials and Methods. One-tenth of the immunoprecipitated GSK-3ß was immunoblotted as an input control. (C) ATR siRNA inhibits UV-induced GSK-3ß activation. SK-MEL-1 cells were transfected with GFP, ATM, or ATR siRNA and irradiated with UV (100 J/m2) 48 h later. The in vitro kinase assay was performed as described for panel B.

 
GSK-3ß directly phosphorylates p21 protein at ser-114. To determine whether GSK-3ß directly phosphorylates p21 protein after UV irradiation, an in vitro kinase assay with GSK-3ß was performed using recombinant or immunoprecipitated p21 protein as a substrate. GSK-3ß immunoprecipitated from SK-MEL-1 cells 30 min after UV irradiation was indeed found to phosphorylate both recombinant p21 protein and p21 immunoprecipitated from SK-MEL-2 cells overexpressing the protein (Fig. 7). The p21 protein overexpressed in SK-MEL-2 cells appeared to be phosphorylated better than recombinant p21 by GSK-3ß. Increased phosphorylation of immunoprecipitated p21 protein by UV-activated GSK-3ß was observed from 15 to 45 min, with peak phosphorylation at 30 min (Fig. 7B). It was noted that immunoprecipitated p21 protein was phosphorylated at a low level by control unstimulated GSK-3ß, indicating the possibility that unstimulated GSK-3ß phosphorylated p21 at a low level, but UV further increased p21 phosphorylation by GSK-3ß. On this line, thr-57 of p21 was shown to be phosphorylated by GSK-3ß under resting-state conditions (35).


Figure 7
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  		FIG. 7. UV-activated GSK-3ß phosphorylates p21 protein in vitro. SK-MEL-1 cells were cultured under a serum-free condition for 4 h prior to UV irradiation (100 J/m2), and GSK-3ß was immunoprecipitated at the indicated time points after UV irradiation. An in vitro kinase assay of GSK-3ß was performed using recombinant (A) or immunoprecipitated (B) p21 protein as a substrate. Immunoprecipitated p21 protein was obtained from SK-MEL-2 cells which were transfected with wild-type p21 cDNA. One-tenth volumes of the immunoprecipitated GSK-3ß and p21 proteins were immunoblotted as input controls. *, nonspecific bands.

 
In order to identify the specific p21 residue phosphorylated by GSK-3ß following UV irradiation, mutant (S27A, T57A, S114A, T145A, T57A/S114A, and T57A/T145A) p21 proteins deficient in potential phosphoacceptor residues were overexpressed in SK-MEL-2 cells that expressed very low levels of endogenous p21. ser-27 and ser-114 are located within the matching consensus sequence for GSK-3ß, (S/T)XXXP(S/T). In addition, thr-57 was shown to be phosphorylated by GSK-3ß under resting-state conditions, although it is not in the GSK-3ß consensus sequence (35). Therefore, these three residues are the primary candidates phosphorylated by GSK-3ß. thr-145 is phosphorylated by Akt and PKA (36, 39, 55), and it was included as a control. The T57A/S114A and T57A/T145A double mutants were employed to assess UV-induced phosphorylation of p21 without possible phosphorylation at thr-57 by the resting GSK-3ß (35). Mutant p21 proteins were immunoprecipitated and used as substrates for the in vitro GSK-3ß kinase assay. As shown in Fig. 8A and B, the wild-type p21 as well as the T145A, T57A, and T57A/T145A double mutants was phosphorylated by UV-activated GSK-3ß. However, UV-induced phosphorylation was not observed for the S114A and T57A/S114A mutants, demonstrating that ser-114 is the specific site phosphorylated by GSK-3ß upon UV irradiation. Phosphorylation of p21 proteins containing T57A mutations (T57A, T57A/S114A, and T57A/T145A) by unstimulated GSK-3ß was not detected. This finding explains the phosphorylation of p21 by resting GSK-3ß as shown in Fig. 7B and is consistent with a report by Rossig et al. (35) that GSK-3ß phosphorylated thr-57 of p21 in the resting state.


Figure 8
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  		FIG. 8. GSK-3ß phosphorylates ser-114 of p21 following UV irradiation, triggering p21 degradation. (A) The ser-114 residue is phosphorylated by UV-activated GSK-3ß. SK-MEL-2 cells were transiently transfected for 24 h with plasmids encoding wild-type or mutant p21 or an empty vector (pcDNA3.1). Mutant and wild-type (wt) p21 proteins were immunoprecipitated and used as substrates for the in vitro GSK-3ß kinase assay. GSK-3ß was immunoprecipitated from SK-MEL-1 cells 30 min after UV irradiation (100 J/m2). The in vitro kinase assay was performed as described in Materials and Methods. One-tenth volumes of the immunoprecipitated GSK-3ß and p21 proteins were immunoblotted as input controls. (B) Relative band intensity of phosphorylated p21 was determined by densitometric analysis from three independent experiments. The relative levels of p21 phosphorylation are plotted. (C) Phosphorylation at ser-114 is required for the UV-induced degradation of p21 protein. SK-MEL-2 or MCF-7 cells were transiently transfected with wild-type or mutant p21 plasmids or an empty vector (pcDNA3.1). p21 protein levels were assessed by immunoblot analysis 24 h after UV irradiation (20 or 10 J/m2, respectively). (D) The UV-induced destabilization of wild-type and T145A and T57A mutant p21 proteins is reversed by TDZD-8. SK-MEL-2 cells were transiently transfected with wild-type or mutant p21 plasmids. After 24 h, cells were pretreated with TDZD-8 (40 µM) for 1 h and UV irradiated (20 J/m2). Protein levels of p21 were analyzed by immunoblotting after 24 h. (E) The expression levels of p21 and {alpha}-tubulin proteins in panel D were determined by densitometric analysis. The average ratios of p21 to {alpha}-tubulin expression for three independent experiments are plotted.

 
Next, the levels of p21 mutant proteins after UV irradiation were examined by immunoblot analysis to confirm the importance of ser-114 phosphorylation. As shown in Fig. 8C, degradation of p21 protein by UV irradiation was largely blocked by S114A mutation, whereas mutation of other residues had no significant effect. To further demonstrate that ser-114 phosphorylation by GSK-3ß is critical for the UV-induced destabilization of p21, SK-MEL-2 cells overexpressing mutant or wild-type p21 were pretreated with TDZD-8, and p21 protein levels were analyzed after UV irradiation. If ser-114 phosphorylation were directly involved in UV-induced p21 degradation, pretreatment with TDZD-8 would not result in a substantial increase in the p21 protein level in SK-MEL-2 cells which expressed only the p21 S114A mutant. As expected, TDZD-8 pretreatment did not affect the protein level of the p21 S114A mutant (Fig. 8D and E). In contrast, expression of the p21 T145A and T57A mutants as well as of wild-type p21, which was reduced by UV irradiation, was restored by treatment with TDZD-8. These data strongly suggest that GSK-3ß phosphorylates p21 protein at ser-114 after UV irradiation, thereby triggering the degradation of p21.

UV-induced degradation of p21 protein is ubiquitination independent. Both ubiquitination-dependent and -independent pathways for p21 protein degradation have been reported. Therefore, we determined whether UV-induced p21 degradation involved ubiquitination and ser-114 phosphorylation after UV irradiation was required for p21 ubiquitination. Thus, p21 protein was immunoprecipitated from SK-MEL-1 cells which were pretreated with a proteasome inhibitor, MG-132, and irradiated with UV. Then, the level of p21 ubiquitination was analyzed by immunoblotting of the immunoprecipitated p21 with antiubiquitin antibody. As shown in Fig. 9A, pretreatment of the cells with MG-132 resulted in accumulation of ubiquitinated p21. However, UV irradiation did not result in a further increase of the level of ubiquitinated p21 (Fig. 9B), suggesting that UV-induced degradation of p21 protein was ubiquitination independent. Next, to determine whether ser-114 phosphorylation after UV irradiation affected the ubiquitination of p21, the ubiquitination level of S114A mutant p21 was analyzed with or without UV irradiation. SK-MEL-2 cells were transfected with wild-type or mutant (S114A or T145A) p21, and the ubiquitination levels of the p21 proteins were analyzed as described above. The T145A mutant was used as a mutant p21 control. Consistent with the notion that UV-induced p21 protein degradation was ubiquitination independent, neither S114A nor T145A mutation affected the level of p21 ubiquitination after UV irradiation (Fig. 9C). These results indicate that p21 protein degradation after UV irradiation occurs through a ubiquitination-independent pathway.


Figure 9
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  		FIG. 9. UV-induced p21 protein degradation occurs though a ubiquitination-independent pathway. (A) SK-MEL-1 cells were treated with MG-132 for 4 h. p21 was immunoprecipitated, and the level of p21 ubiquitination was analyzed by immunoblotting using antiubiquitin antibody. Immunoprecipitated p21 protein was immunoblotted as an input control. (B) SK-MEL-1 cells were treated with MG-132 (50 µM) for 1 h, followed by UV irradiation (100 J/m2). After 4 h of culture, ubiquitination was analyzed as described for panel A. NC indicates a control where immunoprecipitation was carried out with normal serum. (C) SK-MEL-2 cells were transfected with wild-type (WT) or S114A or T145A mutant p21 cDNA. After 24 h, the cells were treated with MG-132 (2.5 µM) for 1 h before UV exposure (10 J/m2). p21 ubiquitination was analyzed 6 h after UV irradiation as described above. * and ** indicate immunoglobulin heavy chain bands.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we explored the signaling mechanism leading to p21 protein degradation after UV irradiation. A schematic model based upon our results is presented in Fig. 10: ATR activated by UV irradiation enhances the activity of GSK-3ß, which then phosphorylates p21 protein at ser-114, leading to the proteasome-mediated degradation of p21.


Figure 10
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  		FIG. 10. Schematic model. UV induces ser-114 phosphorylation of p21 by GSK-3ß, which triggers the proteasome-mediated degradation of p21 protein. GSK-3ß activity is increased by UV irradiation via the ATR signaling pathway, and it phosphorylates p21 protein at ser-114. After phosphorylation, p21 protein is degraded by the proteasome.

 
Here, we demonstrate that GSK-3ß is activated by ATR and that it mediates UV-induced p21 protein degradation. GSK-3ß was not previously shown to be regulated by ATM/ATR, either directly or indirectly. In this study, we provide evidence that GSK-3ß is a downstream effector kinase of ATR in the signaling pathway leading to p21 protein degradation following UV irradiation. First, in an in vitro kinase assay, GSK-3ß was activated by UV, and activation was blocked by caffeine or ATR siRNA, demonstrating that GSK-3ß activation is ATR dependent. Although GSK-3ß is a constitutively active enzyme and primarily subject to negative regulation, the increased activity of GSK-3ß under certain circumstances has been reported (9, 34). Second, GSK-3ß activity is required for the UV-induced destabilization of p21 protein. Inhibition of GSK-3ß by the chemical inhibitors LiCl, TDZD-8, and SB216763, introduction of siRNA, or overexpression of the catalytically inactive GSK-3ß mutant prevented a reduction of the p21 protein level after UV irradiation. Furthermore, treatment with both caffeine and TDZD-8 at suboptimal doses did not exhibit an additive effect on the recovery of p21 protein expression, indicating that GSK-3ß and ATR participate in the same signaling pathway. Interestingly, Chk1, one of the major substrates of ATR, as well as Chk2, did not appear to be involved in GSK-3ß activation or p21 protein degradation by UV irradiation, since chemical inhibitors of Chk1 and Chk2 had no effect on the UV-induced destabilization of p21 (data not shown).

ATM and ATR, the PI3K-related kinases, are critical for transmitting radiation-induced DNA damage signals (4). ATR is activated by various forms of DNA damage, including bulky DNA adducts formed by UV radiation and stalled replication forks. DNA double-strand breaks that are not associated with the replication machinery primarily induce the rapid activation of ATM. Activation of ATM and ATR results in the phosphorylation of a number of downstream proteins involved in checkpoint controls, DNA repair, or apoptosis. For checkpoint controls, ATM activates Chk2, whereas ATR predominantly up-regulates Chk1 activity (1). Bendjennat et al. (6) earlier showed that ATR mediates the UV-induced down-regulation of p21 protein expression. However, they were not able to identify the downstream effector molecule(s) of ATR involved in the destabilization of p21 protein. In this study, we initially obtained a clue that GSK-3ß is involved in UV-induced degradation of p21 by using lithium chloride, a chemical inhibitor of GSK-3ß. Bendjennat et al. (6) failed to prevent UV-induced p21 degradation by lithium chloride, because the cell types were different or the dose that they employed (30 mM) was not sufficient. We found no significant effect of lithium chloride at low doses, but increasing the dose over 50 mM resulted in dose-dependent protection of p21 from UV-induced destabilization. The finding that GSK-3ß is activated by UV irradiation may provide a clue for understanding some important aspects of a cellular response to UV, since GSK-3ß plays a role in multiple biological processes, such as cell cycle progression and apoptosis.

The present study also demonstrates that p21 is directly phosphorylated by UV-activated GSK-3ß. GSK-3ß, immunoprecipitated from UV-irradiated cells, phosphorylated p21 protein which had been overexpressed in SK-MEL-2 cells as well as recombinant p21 in an in vitro kinase assay. ser-114 of p21 was identified as the residue phosphorylated by GSK-3ß after UV irradiation. Phosphorylation of p21 at ser-114 has not been reported thus far. An in vitro kinase assay using phosphoacceptor mutants of p21 showed that phosphorylation of the p21 S114A mutant by UV-activated GSK-3ß did not increase, in contrast to what was observed for wild-type p21 or other (T57A and T145A) mutants. In addition, the p21 S114A mutant was significantly protected from UV-induced degradation, and TDZD-8 was not able to increase the level of this protein after UV irradiation. Taken together, these results clearly demonstrate that ser-114 phosphorylation by GSK-3ß is critical for p21 protein degradation by UV. GSK-3ß immunoprecipitated from unstimulated cells phosphorylated p21 at thr-57, as the p21 T57A mutant did not exhibit phosphorylation by the control GSK-3ß (Fig. 7A). However, the protein level of the p21 T57A mutant was reduced as efficiently as that of wild-type p21 after UV irradiation, indicating that phosphorylation at thr-57 is not involved in UV-induced p21 degradation. As previously reported (35), GSK-3ß may regulate the basal turnover of p21 protein by thr-57 phosphorylation.

Phosphorylation has been shown to affect p21 stability in opposing ways. Phosphorylation of the ser-130 residue by Cdk2 during cell cycle progression (10, 56) seems to trigger ubiquitination by SCFSkp2 and subsequent degradation of p21 protein by the proteasome (8). However, phosphorylation of the same residue, ser-130, by Jun N-terminal protein kinase 1 and p38{alpha}, which is activated by transforming growth factor ß1, has been shown to stabilize p21 protein (24). Akt phosphorylates p21 at thr-145 and ser-146 within the PCNA binding site: ser-146 phosphorylation by Akt significantly increases the p21 protein level (26), and thr-145 phosphorylation by Akt does not affect the stability of p21 while it prevents PCNA binding and promotes the nuclear export of p21 (55). On the other hand, thr-57 phosphorylation mediated by GSK-3ß is required for the basal turnover of p21, and the PI3K/Akt-dependent inhibition of GSK-3ß in response to serum stimulation dramatically increases the half-life of p21 protein (35). thr-57 is also phosphorylated by Cdk2 at the G2/M boundary, which promotes association with cyclin B1 and enhances Cdk1 activity but also destabilizes p21 (17).

There have been contradictory reports regarding the effect of UV on the regulation of p21 protein expression (2, 6, 33). In this study, UV was found to induce degradation of p21 protein, independent of p53 status, in various human cancer cell lines as well as in an untransformed human keratinocyte cell line. It has been suggested that only low doses of UV (<40 J/m2) induce p21 protein degradation, leading to enhanced DNA repair (6). However, our data do not support this contention, since destabilization of p21 protein is dose dependent, up to 100 to ~150 J/m2. Although the reason for this difference is not understood, a reduction of p21 protein expression at high doses of UV has been observed by other groups as well (25, 45). Despite ample evidence that p21 inhibits DNA repair, it is not completely clear whether a decrease in p21 expression caused by UV irradiation contributes primarily to DNA repair: while an elevation of p21 is expected to increase apoptosis if p21 suppresses DNA repair, p21 protein in UV-irradiated cells appears to enhance cell survival. Ectopic expression of p21 protein reduced apoptosis following UV irradiation (data not shown), and repression of p21 protein expression following UV-induced DNA damage changed the cell fate from cell cycle arrest to apoptosis (49).

We found an evidence to indicate that UV-induced p21 protein degradation occurred though a ubiquitination-independent proteasome pathway: ubiquitinated p21 did not accumulate after UV irradiation in cells which were pretreated with a proteasome inhibitor, indicating that p21 degradation after UV irradiation occurs without ubiquitination. Therefore, ser-114 phosphorylation by GSK-3ß in UV-induced p21 destabilization does not appear to induce ubiquitination of p21, and the question of how ser-114 phosphorylation by GSK-3ß contributes to the p21 protein degradation remains to be elucidated.

A number of questions regarding the physiological implication of p21 regulation after UV irradiation and the effect of p21 phosphorylation at ser-114 on its subcellular localization or interaction with other proteins remain open. Also of importance is the mechanism by which ATR activates GSK-3ß. While many questions have been raised, the present study provides important novel insights into the molecular events triggered by UV exposure: GSK-3ß is activated in an ATR-dependent manner, and UV-activated GSK-3ß phosphorylates p21 protein at ser-114, which leads to the down-regulation of p21 protein expression.


   ACKNOWLEDGMENTS
 
This work was supported by Proteomics grant FPR05C2-390 and a grant from the Medical Research Center for Environmental Toxicogenomics and Proteomics funded by the Korea Science and Engineering Foundation.

We thank Bon-Hong Min and Kyung Mi Lee for critical reading of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address for Jeongwon Sohn: Korea University College of Medicine, 126-1 Anam-Dong 5-Ga, Sungbuk-Gu, Seoul 136-705, South Korea. Phone: 82-2-920-6192. Fax: 82-2-922-6702. E-mail: biojs{at}korea.ac.kr. Mailing address for Joon Kim: School of Life Sciences and Biotechnology, Korea University, 126-1 Anam-Dong 5-Ga, Sungbuk-Gu, Seoul 136-701, South Korea. Phone: 82-2-3290-3442. Fax: 82-2-3290-3442. E-mail: joonkim{at}korea.ac.kr Back

{triangledown} Published ahead of print on 5 February 2007. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


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Molecular and Cellular Biology, April 2007, p. 3187-3198, Vol. 27, No. 8
0270-7306/07/$08.00+0     doi:10.1128/MCB.01461-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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