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Previous Article | Next Article 
Molecular and Cellular Biology, March 2006, p. 2430-2440, Vol. 26, No. 6
0270-7306/06/$08.00+0 doi:10.1128/MCB.26.6.2430-2440.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Emma S. Child,2,
Grzegorz Sarek,1
Papinya Sirimongkolkasem,2
Gordon Peters,3
Päivi M. Ojala,1,
and
David J. Mann2,*
Molecular Cancer Biology Program, Institute of Biomedicine, Biomedicum Helsinki, P.O. Box 63, FIN-00014 University of Helsinki, Finland,1 Biochemistry Building, Division of Cell and Molecular Biology, Faculty of Life Sciences, Imperial College, South Kensington, London SW7 2AZ, United Kingdom,2 London Research Institute, Cancer Research United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom3
Received 19 August 2005/ Returned for modification 23 September 2005/ Accepted 20 December 2005
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ABSTRACT |
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INTRODUCTION |
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Our understanding of the roles of endogenous cell cycle regulatory proteins has been greatly increased through the analysis of the functions of a variety of viral proteins that promote proliferation. The cyclin encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) (K cyclin) is the viral homologue of the mammalian D-type cyclins, and it preferentially interacts with endogenous cdk6 (7, 13). K cyclin/cdk6 complexes have unusual properties, preeminent being their ability to maintain kinase activity in the presence of p21Cip1 and p27Kip1 (37). We have previously shown that K cyclin is able to circumvent the G1 blockades imposed by p21Cip1 and p27Kip1 (37). To overcome the arrest imposed by p27Kip1, K cyclin promotes the cdk6-dependent phosphorylation of p27Kip1 on threonine 187, thereby stimulating p27Kip1 degradation (11, 22). The phosphorylation-resistant T187A mutant of p27Kip1 is able to restrict cell cycle progression even in the presence of K cyclin (22, 37). These data indicate that K cyclin expression provides conditions that are permissive for cyclin/cdk2 activation by eliminating p27Kip1 and that the viral cyclin must facilitate the activation of endogenous cyclin/cdk complexes to enable cell cycle progression (21, 34).
To further dissect the properties of the viral cyclin and shed additional light on the control of cdk inhibitors in vivo, we addressed the mechanism by which K cyclin can bypass the G1 arrest imposed by p21Cip1, given that this cdk inhibitor, like p27Kip1, can inhibit the G1-specific cyclin/cdk holoenzymes. Our results demonstrate that K cyclin/cdk6 can phosphorylate p21Cip1 on serine 130 (S130) in vitro and in vivo and that this phosphorylation is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Also, we show that K cyclin expression enabled S-phase entry under oxidative stress-induced cell cycle arrest resulting from elevated levels of p21Cip1. This was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells and baculovirus.
NIH 3T3-K cells were isolated, cultured, and made quiescent as previously described (37). K cyclin expression was induced with 1 mM IPTG (isopropyl-
-D-thiogalactopyranoside). Culture of U2-OS cells and transfection has been previously described (16, 23). The U2-OS-K cyclin cell line (U2-OS-K) was a kind gift from Heike Laman (Division of Virology, Department of Pathology, University of Cambridge) and Flag-tagged K cyclin expression was induced with 5 mM IPTG. Culture and infection of Sf9 cells with recombinant baculoviruses were performed as previously described (22). The BC-3 cell line (3) was kindly provided by Ethel Cesarman (Cornell Medical College, New York City, NY). BC-3 and JOK-1 hairy cell leukemia cells (a kind gift from Leif Andersson, University of Helsinki, Finland) were cultured in a humidified 5% CO2 atmosphere at 37°C in RPMI 1640 medium supplemented with 15% fetal calf serum (Invitrogen, Carlsbad, CA). For treatment with hydrogen peroxide, NIH 3T3-K or U2-OS-K cells were cultured for 24 h in the absence or presence of IPTG. For BrdU incorporation, hydrogen peroxide was added to 250 µM to NIH 3T3-K cells, and the cells were incubated for 4.5 h. BrdU was added for the last 30 min before the cells were harvested. For the kinase assays, U2-OS-K cyclin cells were treated with 500 µM hydrogen peroxide for 20 h before being harvested.
In vitro kinase assays and phosphopeptide analysis.
Sf9 cells were infected with the appropriate recombinant baculoviruses and lysed 72 h later by swelling in K buffer (approximately 2.5 x 106 cells/ml; K buffer is 25 mM HEPES [pH 7.9], 5 mM MgCl, 0.1% 2-mercaptoethanol, and 0.1 mM EDTA) for 10 min on ice. Cell debris was removed by centrifugation. Kinase assays were performed in K buffer containing 0.5 to 1 µg of the appropriate substrate, 100 µM ATP, and 2.5 µCi [
-32P]ATP at 30°C for 30 min. Reactions were then terminated by the addition of an equal volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and resolved by SDS-PAGE. Primary effusion lymphoma (PEL) cells, transfected cells, or U2-OS-K cells were lysed into the ELB lysis buffer (150 mM NaCl, 50 mM HEPES [pH 7.4], 0.1% Igepal, 5 mM EDTA, 2 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride, leupeptin [2 µg/ml], pepstatin [2 µg/ml], and aprotinin [1.5 µg/ml]) supplemented with 25 mM ß-glycerophosphate. For measurement of in vitro kinase activity towards pRb, histone H1, endogenous p21Cip1, or glutathione S-transferase (GST)-p21Cip1 lysates were incubated 2 h at 4°C with anti-K cyclin, anti-CDK2, or anti-Myc antibody. Immunocomplexes were coupled to protein A-Sepharose beads for an additional 1 h at 4°C and washed three times with lysis buffer, followed by one wash with kinase buffer (20 mM Tris [pH 7.5], 50 mM KCl, 7.5 mM MgCl2, 1 mM DTT, 25 mM ß-glycerophosphate, leupeptin [2 µg/ml], pepstatin [2 µg/ml], and aprotinin [1.5 µg/ml]). Immunodepletion was performed with three rounds of immunoprecipitation with either anti-cdk6, anti-cdk4, anti-cdk2, or anti-p21Cip1 antibody; the depleted lysates were immunoprecipitated with anti-K cyclin antibody. For blocking treatments, 1 µg of K cyclin antibody was pretreated with 10 µg of GST-K cyclin at room temperature for 2 h prior to addition to the lysate. Kinase reactions were performed in the presence of 2 µCi of [-32P]ATP for 15 min at 30°C using 0.5 µg of wild-type or S130A mutant GST-p21Cip1 or 2 µg GST-pRb and histone H1 as a substrate or without any exogenous substrates. Phosphorylated proteins were analyzed by SDS-PAGE and autoradiography.
For the phosphopeptide analysis, in vitro kinase assays were performed as described above except that 20 µCi [-32P]ATP and GST-p21Cip1 were used. The phosphorylated GST-p21Cip1 products were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Radioactive bands were identified by autoradiography, excised, and digested with trypsin. Peptides were resolved in two dimensions on thin-layer cellulose chromatography plates using a Hunter Thin Layer Peptide Mapping Electrophoresis system as previously described (22). The first dimension was resolved by electrophoresis in pH 1.9 buffer (25 ml formic acid, 78 ml acetic acid, and 897 ml distilled H2O) for 50 min at 1,000 V, followed by chromatography with 3:10:12:15 acetic acid:pyridine:water:butan-1-ol for 8 h for the second dimension. Plates were dried and subjected to autoradiography. Phosphoamino acid analysis was performed as previously described (25).
Cell extractions and immunological methods. For immunoblotting, cells were washed twice with phosphate-buffered saline (PBS), lysed in situ in SDS-PAGE sample buffer, and collected by scraping. Samples were then boiled and subjected to SDS-PAGE using 10% polyacrylamide gels to resolve cyclins and cdk's and 12.5% polyacrylamide gels for p21Cip1. Immunoblotting was performed as described previously (9). Blots were analyzed by autoradiography or by data capture via a Fuji LAS-3000 and quantification with Fujifilm Science Lab 2003 ImageQuant 4.21 software. Phosphatase treatment was performed by incubation of the cell extract with calf intestinal alkaline phosphatase (Roche) at 37°C for 30 min prior to immunoblotting.
For immunoprecipitations, NIH 3T3-K cells were washed twice with PBS, lysed in situ in IP-K buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween-20, 50 mM NaF, 0.1 mM Na2VO3, and 1 mM DTT) and collected by scraping, followed by five passes through a 21-gauge needle. Samples were incubated for 30 min at 4°C before centrifugation to remove cell debris. Immunoprecipitations were performed at 4°C for 2 h using protein A- or protein G-Sepharose beads and the appropriate antibody. The immunoprecipitates were washed three times in IP-K buffer prior to resuspension in SDS-PAGE loading buffer and immunoblot analysis or kinase assay (9).
BrdU incorporation was determined as previously described (9) 24 h after addition of the BrdU. Cells were cotransfected with histone H2B-green fluorescent protein (GFP) fusion construct, and at least 100 GFP-positive cells were scored for BrdU incorporation. For half-life determinations, NIH 3T3-K cells were metabolically labeled for 120 min with approximately 100-µCi/ml [35S]methionine and cysteine (27) in the absence and presence of K cyclin expression, and p21Cip1 was immunoprecipitated as described above. For immunohistochemical analysis, transfected cells were fixed with 3.5% (wt/vol) paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 min. Immunofluorescence labeling was performed as previously described (16).
Nuclear-cytoplasmic fractionation. For nuclear-cytoplasmic extractions, NIH 3T3-K cells were trypsinized, washed in PBS, and resuspended in buffer A (10 mM HEPES [pH 7.9]-10 mM KCl-1.5 mM MgCl2-0.34 M sucrose-10% glycerol-1 mM DTT). Triton X-100 was added to 0.1%, and the sample was incubated on ice for 5 min. Nuclei were pelleted by centrifugation at 3,500 rpm for 4 min at 4°C and washed once in buffer A. The cytoplasmic supernatant solution was clarified by centrifugation at 14,000 rpm for 10 min at 4°C. For PEL cell fractionation, BC-3 cells were resuspended in hypotonic lysis buffer (20 mM Tris [pH 7.5], 10 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 0.1% Triton X-100, 10 mM MnCl2, 20% glycerol, 1 mM DTT, 25 mM ß-glycerophosphate, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 1.5 µg/ml aprotinin) to a concentration of 5 x 107 cells/ml. Samples were incubated on ice for 5 min with gentle mixing. Nuclei were pelleted by being spun at 800 rpm for 5 min at 4°C. The supernatant (cytosol) was then carefully removed from the pellet, which was washed with PBS. The cytosolic extract was further clarified by centrifugation at 14,000 rpm for 15 min at 4°C. The clarified supernatant was collected and represents the cytosolic fraction. The nuclear pellet was resuspended in hypotonic lysis buffer plus 0.5 M NaCl and vortexed twice for 10 s. Nuclei were spun at 14,000 rpm for 15 min. The supernatant (nuclei) was collected and represents the nuclear fraction.
Gel filtration chromatography. Cell lysates prepared in ELB lysis buffer were passed through a 0.22-µm-pore-size MILLEX-GS filter (Millipore) and fractionated on a Superdex 200 HR column with a fast-performance liquid chromatography system (Pharmacia Biotech, Uppsala, Sweden). Samples were loaded onto the column and separated in gel filtration buffer (50 mM HEPES [pH 7.5], 150 mM NaCl) at a flow rate of 0.3 ml/min. Column calibration was performed under the same conditions with blue dextran (2,000 kDa) thyroglobulin (669 kDa), apoferritin (443 kDa), ß-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa). For each fractionation, 30 fractions of 0.5 ml each were collected. A total of 50 µl of each fraction was used for immunoblotting, and 450 µl of each fraction was used for immunoprecipitation experiments.
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RESULTS |
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To determine if this phosphorylation event promoted by K cyclin and causing retarded mobility of p21Cip1 on SDS-PAGE was related to the S130 phosphorylation observed in vitro (Fig. 1), we raised an antibody to the phosphopeptide GEQAEGpSPGGPGDS, where pS represents phosphoserine 130. In vitro characterization indicated that this antiserum reacted only with serine 130-phosphorylated p21Cip1 (Fig. 2D). Moreover, immunoprecipitation with the phosphospecific serum from lysates of NIH 3T3-K cells expressing the viral cyclin specifically isolated the slower-migrating form of p21Cip1, while an antibody against a p21Cip1 carboxy-terminal peptide immunoprecipitated both forms of the cdk inhibitor (Fig. 2E). Taken together, these data demonstrate that K cyclin can promote the phosphorylation of p21Cip1 on S130 in vitro and in vivo and that this phosphorylation leads to reduced mobility of p21Cip1 on SDS-PAGE. We note that Kim et al. (18) demonstrated that p38 kinase can phosphorylate p21Cip1on S130 in vivo. In our hands, the p38 inhibitor SB203580 had little effect on the presence of the S130 phosphoform of p21Cip1 in our asynchronous cultures (data not shown).
K cyclin associates with and promotes phosphorylation of p21Cip1 in both transfected and PEL-derived cells.
We have previously shown that K cyclin associates with p27Kip1 in vivo in transfected cells and PEL-derived cells (16). To assess the potential physical interactions between K cyclin and p21Cip1, we expressed HA-tagged versions of wild-type and phosphosite mutants of p21Cip1 in the presence of Myc-tagged K cyclin in U2-OS cells. Cell lysates from transfections were immunoprecipitated with anti-Myc antibody and either subjected to an in vitro kinase assay without adding any exogenous substrates or immunoblotted for p21Cip1. As shown in Fig. 3A, K cyclin associated with both wild-type p21Cip1 and the phosphosite mutants of p21Cip1. The coimmunoprecipitated wild-type p21Cip1 displayed two immunoreactive species, in contrast to the S130A mutant p21Cip1, which migrated as a single species. Mutation of the site of Akt phosphorylation (T145A) in p21Cip1 (31, 42) did not prevent either the association of K cyclin and p21Cip1 or the appearance of the retarded mobility form of p21Cip1. The double mutant S130A/T145A migrated in a manner indistinguishable from the S130A mutant protein. A similar mobility shift of p21Cip1 was also seen when total p21Cip1 was immunoblotted from the total lysates. Addition of [-32P]ATP to the K cyclin immunoprecipitates resulted in the phosphorylation of coimmunoprecipitated p21Cip1 only when the S130 site was present (Fig. 3A, top). Next, we analyzed whether p21Cip1 is associated with K cyclin in BC-3 PELs, which are latently infected with KSHV and express latent viral proteins like K cyclin. BC-3 cell lysates were immunoprecipitated with either anti-K cyclin or anti-p21Cip1 antibodies, and coimmunoprecipitated proteins were analyzed by immunoblotting. As shown in Fig. 3B, p21Cip1 and K cyclin were in complex in BC-3 cells, confirming our results from transfected cells and in agreement with recently published data (39). JOK-1 is a KSHV-negative leukemia cell line, which served as a negative control.
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K cyclin-mediated phosphorylation of p21Cip1 on S130 has little effect on p21Cip1 protein stability or localization but appears to abolish cdk2 binding. We next began to dissect the physiological consequences of p21Cip1 S130 phosphorylation. Phosphorylation of cdk inhibitors has been shown to modulate their activity via two major mechanisms: protein stability and subcellular localization. We assessed the effects of S130 phosphorylation on each of these parameters. NIH 3T3-K cells were metabolically labeled with [35S]methionine and cysteine for 120 min prior to chasing in the presence of excess unlabeled amino acids in the absence or presence of K cyclin and the abundance of p21Cip1 assessed over time by immunoprecipitation (Fig. 5A and B). Quantitation of p21Cip1 in immunoprecipitates following resolution by SDS-PAGE indicated that the turnover of p21Cip1 was not appreciably altered by K cyclin expression. Similar results were obtained using the translational inhibitor cycloheximide (not shown). Thus, K cyclin expression had little effect on p21Cip1 turnover.
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-tubulin) and nuclear (E2F-1) markers (Fig. 5C). To study the distribution of p21Cip1 in a more physiological setting, the BC-3 PEL cells were fractionated into nuclear and cytoplasmic samples and immunoblotted with p21Cip1 antibody (Fig. 5D). The purity of the nuclear and cytoplasmic fractions was verified using markers for the nucleus (Sp1) and cytosol (ß-tubulin). Again, p21Cip1 was detected in both compartments. The slower-migrating form of p21Cip1 corresponding to the phosphorylated p21Cip1 was also present in both compartments, supporting our fractionation data from NIH 3T3 cells expressing K cyclin. These biochemical data were further verified by immunofluorescence studies in which K cyclin-induced phosphorylation of p21Cip1 on S130 had no discernible effect on the nuclear retention of p21Cip1. However, K cyclin expression caused a slight increase in the proportion of p21Cip1 in the cytoplasm, although this was independent of S130 phosphorylation (data not shown). To address the function of S130 phosphorylated p21Cip1 as a cdk inhibitor directly, we assessed the proportion of each p21Cip1 species in complex with different cyclin/cdk's in vivo. We immunoprecipitated cdk2 or cyclin D1 from NIH 3T3-K cells and immunoblotted the precipitates for p21Cip1 (Fig. 5E). Cyclin D1 immunoprecipitates contained both mobility forms of p21Cip1 while cdk2 immunoprecipitates displayed only the faster-migrating form of p21Cip1 lacking S130 phosphorylation. Total cell lysates clearly demonstrated the presence of p21Cip1, displaying its typical dual mobility. In reciprocal experiments, the antiserum directed against the S130 phosphoform of p21Cip1 did not coimmunoprecipitate cdk2 but did associate with cyclin D1; both of these cell cycle regulators were found in immune complexes and captured with antibodies directed against total cellular p21Cip1 (Fig. 5F). Thus, in vivo, S130-phosphorylated p21Cip1 appears specifically unable to bind cyclin/cdk2.
Phosphorylation of p21Cip1 on S130 is required for G1 release. We next addressed whether the phosphorylation of p21Cip1 on S130 was important for K cyclin-mediated bypass of a p21Cip1-imposed G1 arrest by measuring S-phase entry by BrdU incorporation (Fig. 6A). Wild-type and S130A mutant p21Cip1 were expressed at similar levels (Fig. 6B) and efficiently prevented S-phase entry (Fig. 6A). Coexpression of K cyclin readily circumvented the cell cycle arrest imposed by wild-type p21Cip1 but was ineffective against the S130A mutant p21Cip1 (Fig. 6A and C). Similar results were obtained by flow cytometry (data not shown). Importantly, the increase in S phase enforced by K cyclin in the presence of wild-type p21Cip1 was accompanied by retardation of the migration of ectopic p21Cip1 on SDS-PAGE (Fig. 6B). The S130A mutation of p21Cip1, like the wild-type protein, was ineffective in the inhibition of K cyclin/cdk6 kinase activity but was as efficacious as the wild protein in inhibiting cyclin D2/cdk6 or cyclin E/cdk2 (Fig. 6D and data not shown), demonstrating that the S130A mutation did not impair p21Cip1 inhibitory function.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whereas the mechanism of p27Kip1 inactivation occurs through phosphorylation-promoted degradation, this did not appear to be the primary cause of p21Cip1 inactivation. Previous studies have produced conflicting data on the effects of modification of this residue of p21Cip1 on inhibitor stability. Kim et al. (18) observed a stabilization of p21Cip1 correlating with S130 phosphorylation by p38 kinase, whereas Bornstein et al. (6) reported that the ability of active cyclin E/cdk2 to promote p21Cip1 ubiquitylation in vitro was partially dependent on the presence of serine at position 130 of p21Cip1; that is, the S130A mutant was stabilized. Ubiquitylation of p21Cip1 has been intimately linked to turnover of this cdk inhibitor (for a review, see reference 5), although recent studies have questioned this view (8). Whatever the mechanism of p21Cip1 proteolysis, our data indicate that protein degradation is not a major cause for K cyclin-mediated release of a p21Cip1-induced G1 arrest.
p21Cip1 inactivation through phosphorylation has also been described by enforcing its cytoplasmic retention: phosphorylation of p21Cip1 in the carboxy-terminal region on residue T145 by Akt led to relocalization of p21Cip1 to the cytoplasm (42), thereby restricting inhibition of nuclear cyclins/cdk's. Our subcellular fractionation and immunohistochemical studies indicated that such a mechanism is not involved in the inactivation of p21Cip1 by K cyclin described here (Fig. 5 and data not shown). Another potential mechanism of inactivation is p21Cip1 sequestration in protein/protein complexes. D-type cyclins are known to regulate cdk inhibitor function through sequestration (28). K cyclin is most similar in amino acid sequence to the D-type cyclins (21). Here, we demonstrate association between K cyclin and p21Cip1 in vivo (Fig. 3 and 4), although only a small fraction of the p21Cip1 cofractionates with K cyclin (Fig. 4A), making it unlikely that sequestration can explain the phosphorylation-dependent inactivation of p21Cip1.
Our most striking observations concerning the mechanism of p21Cip1 inactivation through S130 phosphorylation is the lack of association of the S130 phosphoform of p21Cip1 with cyclin/cdk2 (Fig. 5), resulting in restoration of cdk2 kinase activity in the presence of p21Cip1 (Fig. 7). The mechanism through which S130 phosphorylation of p21Cip1 negates its cdk-binding function is unclear at present. Preliminary experiments indicate that in vitro-phosphorylated p21Cip1 can still interact with cyclin/cdk2 complexes (E. S. Child and D. J. Mann, unpublished observations), ruling out the possibility that an autonomous structural change due to S130 phosphorylation is responsible for the lack of interaction with cyclin/cdk2 seen in vivo. A number of accessory proteins are known to be recruited to p21Cip1, and some of these have been reported to modulate its cdk inhibitory activity: TOK-1 interacts with p21Cip1 close to the PCNA-binding region and enhances the inhibitory action of p21Cip1 towards cdk2 (26), while SET also binds to the p21Cip1 carboxy-terminal region and modulates the inhibitory action of p21Cip1 towards cyclin A/cdk2 but not cyclin E/cdk2 complexes (12). Although neither of these modulatory proteins would seem to account for the loss of cdk2 binding observed here, phosphorylation-dependent recruitment of a regulatory protein remains an attractive hypothesis to explain our data.
Inactivation of p21Cip1 by phosphorylation may play an important role in hyperproliferative disorders promoted by viral infection and in tumorigenesis. KSHV is the causative agent of Kaposi's sarcoma and is associated with a number of other lymphoproliferative diseases (for a review, see reference 40). Expression of the KSHV-encoded K cyclin may contribute to these proliferative disorders. K cyclin expression leads to the functional inactivation of the two major antiproliferative Cip/Kip proteins: p27Kip1, through phosphorylation-triggered proteolysis (11, 22) and p21Cip1, apparently through phosphorylation-mediated functional inactivation, as described here. Thus, a cell expressing K cyclin will essentially be devoid of inhibitory p21Cip1 and p27Kip1 proteins, favoring activation of endogenous cyclin/cdk2 kinases and, hence, cell cycle progression. Although it is uncertain whether the viral cyclin contributes to the oncogenic properties of KSHV, it is strikingly evident that K cyclin is fully equipped to undermine the normal proliferative control pathways.
The inactivation of p21Cip1 described here through the use of the viral cyclin reveals another level of p21Cip1 molecular biology that is likely to be of importance in vivo. Our data indicate the existence of a p21Cip1 S130 kinase in vivo in the absence of K cyclin, although the expression of the viral cyclin greatly enhanced this phosphorylation (Fig. 2). Through the use of the viral cyclin, we have demonstrated a role for this phosphorylation event in the inactivation of the cell cycle arrest function of p21Cip1. It is noteworthy that some tumor tissues express high levels of p21Cip1 but proliferate rapidly (19, 24, 35, 41), raising the intriguing possibility that the endogenous p21Cip1 is functionally inactivated, perhaps through S130 phosphorylation.
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ACKNOWLEDGMENTS |
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This work was funded by Cancer Research UK (D.J.M. and G.P.) and the BBSRC (D.J.M.); by grants from the Academy of Finland, Finnish Cancer Foundations, and Sigrid Juselius Foundation (P.M.O.); and by the University of Helsinki Graduate School in Biotechnology and Molecular Biology (A.J.).
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FOOTNOTES |
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These authors made equal contributions to this work.
These authors made equal contributions to this work.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Aprelikova, O., Y. Xiong, and E. T. Liu. 1995. Both p16 and p21 families of cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin-dependent kinases by the CDK-activating kinase. J. Biol. Chem. 270:18195-18197.
3. Arvanitakis, L., E. A. Mesri, R. G. Nador, J. W. Said, A. S. Asch, D. M. Knowles, and E. Cesarman. 1996. Establishment and characterization of a primary effusion (body cavity-based) lymphoma cell line (BC-3) harboring Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus. Blood 88:2648-2654.
4. Barnouin, K., M. L. Dubuisson, E. S. Child, S. Fernandez de Mattos, J. Glassford, R. H. Medema, D. J. Mann, and E. W.-F. Lam. 2002. H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1 expression. J. Biol. Chem. 277:13761-13770.
5. Bloom, J., and M. Pagano. 2004. To be or not to be ubiquitinated? Cell Cycle 3:138-140.[Medline]
6. Bornstein, G., J. Bloom, D. Sitry-Shevah, K. Nakayama, M. Pagano, and A. Hershko. 2003. Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase. J. Biol. Chem. 278:25752-25757.
7. Chang, Y., P. S. Moore, S. J. Talbot, C. H. Boshoff, T. Zarkowska, K. Godden, H. Paterson, R. A. Weiss, and S. Mittnacht. 1996. Cyclin encoded by KS herpesvirus. Nature 382:410.[CrossRef][Medline]
8. Chen, W. J., and J. K. Lin. 2004. Induction of G1 arrest and apoptosis in human Jurkat T cells by pentagalloylglucose through inhibiting proteasome activity and elevating p27Kip1, p21Cip1/WAF1, and Bax proteins. J. Biol. Chem. 279:13496-13505.
9. Child, E. S., and D. J. Mann. 2001. Novel properties of the cyclin encoded by human herpesvirus 8 that facilitate exit from quiescence. Oncogene 20:3311-3322.[CrossRef][Medline]
10. el-Deiry, W. S., T. Tokino, V. E. Velculescu, D. B. Levy, R. Parsons, J. M. Trent, D. Lin, W. E. Mercer, K. W. Kinzler, and B. Vogelstein. 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75:817-825.[CrossRef][Medline]
11. Ellis, M., Y. P. Chew, L. Fallis, S. Freddersdorf, C. Boshoff, R. A. Weiss, X. Lu, and S. Mittnacht. 1999. Degradation of p27(Kip) cdk inhibitor triggered by Kaposi's sarcoma virus cyclin-cdk6 complex. EMBO J. 18:644-653.[CrossRef][Medline]
12. Estanyol, J. M., M. Jaumot, O. Casanovas, A. Rodriguez-Vilarrupla, N. Agell, and O. Bachs. 1999. The protein SET regulates the inhibitory effect of p21Cip1 on cyclin E-cyclin-dependent kinase 2 activity. J. Biol. Chem. 274:33161-33165.
13. Godden-Kent, D., S. J. Talbot, C. Boshoff, Y. Chang, P. Moore, R. A. Weiss, and S. Mittnacht. 1997. The cyclin encoded by Kaposi's sarcoma-associated herpesvirus stimulates cdk6 to phosphorylate the retinoblastoma protein and histone H1. J. Virol. 71:4193-4198.[Abstract]
14. Guan, K. L., and J. E. Dixon. 1991. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192:262-267.[CrossRef][Medline]
15. Harper, J. W., G. R. Adami, N. Wei, K. Keyomarsi, and S. J. Elledge. 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805-816.[CrossRef][Medline]
16. Jarviluoma, A., S. Koopal, S. Rasanen, T. P. Makela, and P. M. Ojala. 2004. KSHV viral cyclin binds to p27KIP1 in primary effusion lymphomas. Blood 104:3349-3354.
17. Kato, J. Y., M. Matsuoka, D. K. Strom, and C. J. Sherr. 1994. Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase. Mol. Cell. Biol. 4:2713-2721.
18. Kim, G.-Y., S. E. Mercer, D. Z. Ewton, Z. Yan, K. Jin, and E. Friedman. 2002. The stress-activated protein kinases p38 and JNK1 stabilize p21Cip1 by phosphorylation. J. Biol. Chem. 277:29792-29802.
19. Krolewski, B., and J. B. Little. 2000. Overexpression of p21 protein in radiation-transformed mouse 10T(1/2) cell clones. Mol. Carcinog. 27:141-148.[CrossRef][Medline]
20. LaBaer, J., M. D. Garrett, L. F. Stevenson, J. M. Slingerland, C. Sandhu, H. S. Chou, A. Fattaey, and E. Harlow. 1997. New functional activities for the p21 family of CDK inhibitors. Genes Dev. 11:847-862.
21. Laman, H., D. J. Mann, and N. C. Jones. 2000. Viral-encoded cyclins. Curr. Opin. Genet. Dev. 10:70-74.[CrossRef][Medline]
22. Mann, D. J., E. S. Child, C. Swanton, H. Laman, and N. Jones. 1999. Modulation of p27(Kip1) levels by the cyclin encoded by Kaposi's sarcoma-associated herpesvirus. EMBO J. 18:654-663.[CrossRef][Medline]
23. Mann, D. J., and N. C. Jones. 1996. E2F-1 but not E2F-4 can overcome p16-induced G1 cell-cycle arrest. Curr. Biol. 6:474-483.[CrossRef][Medline]
24. McKenzie, P. P., M. K. Danks, R. W. Kriwacki, and L. C. Harris. 2003. P21Waf1/Cip1 dysfunction in neuroblastoma: a novel mechanism of attenuating G0-G1 cell cycle arrest. Cancer Res. 63:3840-3844.
25. Neufeld, E., H. J. Goren, and D. Boland. 1989. Thin-layer chromatography can resolve phosphotyrosine, phosphoserine, and phosphothreonine in a protein hydrolyzate. Anal. Biochem. 177:138-143.[CrossRef][Medline]
26. Ono, T., H. Kitaura, H. Ugai, T. Murata, K. K. Yokoyama, S. M. Iguchi-Ariga, and H. Ariga. 2000. TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase. J. Biol. Chem. 275:31145-31154.
27. Parry, D., S. Bates, D. J. Mann, and G. Peters. 1995. Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product. EMBO J. 14:503-511.[Medline]
28. Perez-Roger, I., S. H. Kim, B. Griffiths, A. Sewing, and H. Land. 1999. Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1). EMBO J. 18:5310-5320.[CrossRef][Medline]
29. Platt, G. M., E. Cannell, M. E. Cuomo, S. Singh, and S. Mittnacht. 2000. Detection of the human herpesvirus 8-encoded cyclin protein in primary effusion lymphoma-derived cell lines. Virology 272:257-266.[CrossRef][Medline]
30. Polyak, K., M. H. Lee, H. Erdjument-Bromage, A. Koff, J. M. Roberts, P. Tempst, and J. Massague. 1994. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78:59-66.[CrossRef][Medline]
31. Rossig, L., A. S. Jadidi, C. Urbich, C. Badorff, A. M. Zeiher, and S. Dimmeler. 2001. Akt-dependent phosphorylation of p21Cip1 regulates PCNA binding and proliferation of endothelial cells. Mol. Cell. Biol. 21:5644-5657.
32. Russo, A. A., P. D. Jeffrey, A. K. Patten, J. Massague, and N. P. Pavletich. 1996. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382:325-331.[CrossRef][Medline]
33. Sarek, G., A. Järviluoma, and P. M. Ojala. 2006. KSHV viral cyclin inactivates p27KIP1 through Ser10 and Thr187 phosphorylation in proliferating primary effusion lymphomas. Blood 107:725-732.
34. Sherr, C. J., and J. M. Roberts. 1999. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13:1501-1512.
35. Skomedal, H., G. B. Kristensen, A. K. Lie, and R. Holm. 1999. Aberrant expression of the cell cycle associated proteins TP53, MDM2, p21, p27, cdk4, cyclin D1, RB, and EGFR in cervical carcinomas. Gynecol. Oncol. 73:223-228.[CrossRef][Medline]
36. Soos, T. J., H. Kiyokawa, J. S. Yan, M. S. Rubin, A. Giordano, A. DeBlasio, S. Bottega, B. Wong, J. Mendelsohn, and A. Koff. 1996. Formation of p27-CDK complexes during the human mitotic cell cycle. Cell Growth Differ. 7:135-146.[Abstract]
37. Swanton, C., D. J. Mann, B. Fleckenstein, F. Neipel, G. Peters, and N. Jones. 1997. Herpes viral cyclin/Cdk6 complexes evade inhibition by CDK inhibitor proteins. Nature 390:184-187.[CrossRef][Medline]
38. van den Heuvel, S., and E. Harlow. 1993. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262:2050-2054.
39. Van Dross, R., S. Yao, S. Asad, G. Westlake, D. J. Mays, L. Barquero, S. Duell, J. A. Pietenpol, and P. J. Browning. 2005. Constitutively active K-cyclin/cdk6 kinase in Kaposi sarcoma-associated herpesvirus-infected cells. J. Natl. Cancer Inst. 97:656-666.
40. Viejo-Borbolla, A., and T. F. Schulz. 2003. Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8): key aspects of epidemiology and pathogenesis. AIDS Rev. 5:222-229.[Medline]
41. Wong, S. C., J. K. Chan, K. C. Lee, and W. L. Hsiao. 2001. Differential expression of p16/p21/p27 and cyclin D1/D3, and their relationships to cell proliferation, apoptosis, and tumour progression in invasive ductal carcinoma of the breast. J. Pathol. 194:35-42.[CrossRef][Medline]
42. Zhou, B. P., Y. Liao, W. Xia, B. Spohn, M. H. Lee, and M. C. Hung. 2001. Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat. Cell Biol. 3:245-252.[CrossRef][Medline]
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16 h. Cells were then treated with vehicle or hydrogen peroxide for 4.5 h before lysis and immunoblotting for the indicated proteins. (B) Cells shown in panel A were treated with BrdU for the final 30 min of hydrogen peroxide treatment, and indirect immunofluorescence was used to detect S-phase progression. Data represent the mean ± standard error of at least four separate experiments with at least 100 cells scored per experiment. (C) U2-OS-K cells were cultured in the absence (–) or presence (+) of IPTG to induce K cyclin expression for