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• W2032945178 abstract "The inhibition of cyclin-dependent kinase activity by p27 contributes to regulation of cell cycle progression. Serine 10 is the major phosphorylation site of p27, and its phosphorylation has been shown to affect the stability and nuclear export of p27 at the G0-G1 transition in transfected cultured cells. To investigate the physiological relevance of p27 phosphorylation on Ser10, we generated p27 “knock-in” mice that harbor an S10A mutation in this protein. Mice homozygous for the mutation (p27S10A/S10A mice) were normal in body size, but the abundance of p27 was decreased in many organs, including brain, thymus, spleen, and testis. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27S10A/S10A mice compared with that in wild-type cells, whereas p27 stability in S phase was similar in cells of the two genotypes. The degradation of p27 in cells of the mutant mice at G0 phase was prevented by a proteasome inhibitor. These data indicate that the physiological role of p27 phosphorylation on Ser10 is to stabilize the protein in G0 phase. Unexpectedly, the nuclear export of p27 at the G0-G1 transition occurred normally in p27S10A/S10A mouse embryonic fibroblasts, indicating that phosphorylation of Ser10 is dispensable for this process. The inhibition of cyclin-dependent kinase activity by p27 contributes to regulation of cell cycle progression. Serine 10 is the major phosphorylation site of p27, and its phosphorylation has been shown to affect the stability and nuclear export of p27 at the G0-G1 transition in transfected cultured cells. To investigate the physiological relevance of p27 phosphorylation on Ser10, we generated p27 “knock-in” mice that harbor an S10A mutation in this protein. Mice homozygous for the mutation (p27S10A/S10A mice) were normal in body size, but the abundance of p27 was decreased in many organs, including brain, thymus, spleen, and testis. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27S10A/S10A mice compared with that in wild-type cells, whereas p27 stability in S phase was similar in cells of the two genotypes. The degradation of p27 in cells of the mutant mice at G0 phase was prevented by a proteasome inhibitor. These data indicate that the physiological role of p27 phosphorylation on Ser10 is to stabilize the protein in G0 phase. Unexpectedly, the nuclear export of p27 at the G0-G1 transition occurred normally in p27S10A/S10A mouse embryonic fibroblasts, indicating that phosphorylation of Ser10 is dispensable for this process. Progression of the cell cycle in eukaryotic cells is regulated by a series of protein complexes composed of cyclins and cyclin-dependent kinases (CDKs), 1The abbreviations used are: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; ES, embryonic stem; MEF, mouse embryonic fibroblast; GSK-3β, glycogen synthase kinase-3β; PDBu, phorbol 12,13-dibutyrate; BrdUrd, bromodeoxyuridine. the activity of which is in turn controlled by a group of CDK inhibitors (CKIs) (1Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar, 2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4986) Google Scholar). Among these CKIs, p27 plays a pivotal role in the control of cell proliferation (3Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar, 4Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar). In normal cells, the amount of p27 is high during the G0 phase of the cell cycle, but it decreases rapidly upon the mitogen-induced reentry of cells into G1 phase (5Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar, 6Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar). Forced expression of p27 results in cell cycle arrest in G1 phase (3Polyak K. Lee M.H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar, 4Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar), and, conversely, inhibition of p27 expression by antisense oligonucleotides increases the proportion of cells in S phase (7Coats S. Flanagan W.M. Nourse J. Roberts J.M. Science. 1996; 272: 877-880Crossref PubMed Scopus (652) Google Scholar). Moreover, we and others have shown that mice with a homozygous deletion of the p27 gene (p27) are larger than normal mice and exhibit both multiple organ hyperplasia and a predisposition to the development of spontaneous and radiation- or chemical-induced tumors (8Fero M.L. Rivkin M. Tasch M. Porter P. Carow C.E. Firpo E. Polyak K. Tsai L.H. Broudy V. Perlmutter R.M. Kaushansky K. Roberts J.M. Cell. 1996; 85: 733-744Abstract Full Text Full Text PDF PubMed Scopus (1343) Google Scholar, 9Kiyokawa H. Kineman R.D. Manova-Todorova K.O. Soares V.C. Hoffman E.S. Ono M. Khanam D. Hayday A.C. Frohman L.A. Koff A. Cell. 1996; 85: 721-732Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar, 10Nakayama K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Nakayama K-I. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar, 11Fero M.L. Randel E. Gurley K.E. Roberts J.M. Kemp C.J. Nature. 1998; 396: 177-180Crossref PubMed Scopus (685) Google Scholar). These observations support the notion that p27 is a key determinant of both body size and organ size as a result of its role in the control of cell proliferation and that the loss of p27 function may lead to carcinogenesis. Indeed, many studies have shown that the expression of p27 is deregulated in various human cancers (12Tsihlias J. Kapusta L. Slingerland J. Annu. Rev. Med. 1999; 50: 401-423Crossref PubMed Scopus (291) Google Scholar). The abundance of p27 is thought to be controlled by multiple mechanisms that operate at the level of the synthesis (transcription and translation) (13Hengst L. Reed S.I. Science. 1996; 271: 1861-1864Crossref PubMed Scopus (823) Google Scholar, 14Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1231) Google Scholar), proteolysis (15Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Sal DelG. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1736) Google Scholar, 16Shirane M. Harumiya Y. Ishida N. Hirai A. Miyamoto C. Hatakeyama S. Nakayama K.I. Kitagawa M. J. Biol. Chem. 1999; 274: 13886-13893Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), and localization of this protein (17Tomoda K. Kubota Y. Kato J. Nature. 1999; 398: 160-165Crossref PubMed Scopus (551) Google Scholar). The proteolysis of p27 is mediated predominantly by the ubiquitin-proteasome pathway (15Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Sal DelG. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1736) Google Scholar). The protein is phosphorylated on Thr187 by the cyclin E-CDK2 complex (18Sheaff R.J. Groudine M. Gordon M. Roberts J.M. Clurman B.E. Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (797) Google Scholar, 19Vlach J. Hennecke S. Amati B. EMBO J. 1997; 16: 5334-5344Crossref PubMed Scopus (609) Google Scholar), and the phosphorylation of this residue is required for binding of p27 to Skp2, an F-box protein that is thought to function as the receptor component of an SCF ubiquitin ligase complex; such binding then results in the ubiquitylation and degradation of p27 (20Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1341) Google Scholar, 21Montagnoli A. Fiore F. Eytan E. Carrano A.C. Draetta G.F. Hershko A. Pagano M. Genes Dev. 1999; 13: 1181-1189Crossref PubMed Scopus (512) Google Scholar, 22Sutterluty H. Chatelain E. Marti A. Wirbelauer C. Senften M. Muller U. Krek W. Nat. Cell Biol. 1999; 1: 207-214Crossref PubMed Scopus (628) Google Scholar, 23Nakayama K. Nagahama H. Minamishima Y.A. Matsumoto M. Nakamichi I. Kitagawa K. Shirane M. Tsunematsu R. Tsukiyama T. Ishida N. Kitagawa M. Nakayama K-I. Hatakeyama S. EMBO J. 2000; 19: 2069-2081Crossref PubMed Scopus (634) Google Scholar, 24Ganoth D. Bornstein G. Ko T.K. Larsen B. Tyers M. Pagano M. Hershko A. Nat. Cell Biol. 2001; 3: 321-324Crossref PubMed Scopus (424) Google Scholar, 25Spruck C. Strohmaier H. Watson M. Smith A.P. Ryan A. Krek T.W. Reed S.I. Mol. Cell. 2001; 7: 639-650Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). We have shown that the degradation of p27 at the G0-G1 transition is independent of Skp2, however, and occurs in the cytoplasm, whereas the Skp2- and Thr187 phosphorylation-dependent degradation of p27 occurs at S and G2 phases in the nucleus (26Hara T. Kamura T. Nakayama K. Oshikawa K. Hatakeyama S. Nakayama K-I. J. Biol. Chem. 2001; 276: 48937-48943Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). These observations suggest that the nuclear export of p27 may be critical for its down-regulation early during reentry of quiescent cells into the cell cycle. We previously identified Ser10 as the major phosphorylation site of p27, showing that it accounts for ∼70% of the total phosphorylation of this protein; the extent of phosphorylation at this site was 75 times that at Thr187 (27Ishida N. Kitagawa M. Hatakeyama S. Nakayama K.I. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The extent of Ser10 phosphorylation is markedly increased in cells in the G0 phase of the cell cycle compared with that apparent for cells in S or M phase. Mutational analysis suggested that phosphorylation of Ser10, like that of Thr187, contributes to regulation of p27 stability. The p27 protein is translocated from the nucleus to the cytoplasm at the G0-G1 transition of the cell cycle. We and others showed that substitution of Ser10 with Ala (S10A) markedly reduced the extent of p27 export from the nucleus at the G0-G1 transition. Furthermore, phosphorylation on Ser10 is required for the binding of p27 to CRM1, a carrier protein for nuclear export (28Rodier G. Montagnoli A. Di Marcotullio L. Coulombe P. Draetta G.F. Pagano M. Meloche S. EMBO J. 2001; 20: 6672-6682Crossref PubMed Scopus (252) Google Scholar, 29Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). These observations suggest that phosphorylation on Ser10 is essential for nuclear export of p27. Residues 32–45 of p27 constitute a putative nuclear export sequence, the mutation of which reduces both the interaction of p27 with CRM1 as well as the nuclear export and degradation of p27 (30Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar). Human kinase-interacting stathmin has been identified as a nuclear serine-threonine kinase that binds to the COOH-terminal region of p27 and phosphorylates p27 on Ser10 both in vitro and in vivo, thereby promoting its export from the nucleus to the cytoplasm (31Boehm M. Yoshimoto T. Crook M.F. Nallamshetty S. True A. Nabel G.J. Nabel E.G. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (247) Google Scholar). Most studies that have evaluated the effect of Ser10 phosphorylation on p27 function have been based on overexpression of exogenous mutant proteins in cultured cell lines. Under such conditions, however, it is difficult to exclude the possibility that physiologically irrelevant levels of p27 inhibit cell cycle progression, resulting in apparent inhibition of the nuclear export of p27. It is therefore important that the effects of mutant proteins expressed under the control of the endogenous promoter of p27 be examined. To this end, we have adopted a “knock-in” strategy to express the S10A mutant of p27 in mice. We now show that the stability of the mutant protein is markedly reduced in certain cell types of p27S10A/S10A mice at G0 phase. Unexpectedly, the export of p27 from the nucleus at the G0-G1 transition appeared to be unaffected by the S10A mutation. Our data suggest that phosphorylation of Ser10 is important for p27 stability in G0 phase but is not required for nuclear export of this CKI at the G0-G1 transition. Generation of p27S10A/S10A Knock-in Mice—We isolated p27 from a 129/Sv mouse genomic library as described previously (10Nakayama K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Nakayama K-I. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar). The codon for Ser10 in exon 1 was mutated to an alanine codon (GGG AGC → GGC GCC) to create the p27S10A allele. The targeting vector was constructed by insertion of a loxP-flanked PGK-neo-poly(A) cassette into the HindIII site of intron 1 of p27 and ligation of a PGK-TK-poly(A) cassette at the 5′ end of the insert (Fig. 1A). The maintenance, transfection, and selection of mouse embryonic stem (ES) cells were performed as described (10Nakayama K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Nakayama K-I. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar). The loxP-flanked PGK-neo-poly(A) cassette was deleted in transfected ES cells by infection with an adenovirus encoding Cre recombinase. For detection of mutated ES clones, genomic DNA was digested with BamHI and subjected to Southern hybridization with a 0.9-kb NdeI-EcoRI probe (Fig. 1A). The mutant ES cells were microinjected into C57BL/6 blastocysts, and the resulting male chimeras were mated with female C57BL/6 mice. Heterozygous offspring were intercrossed to produce homozygous mutant animals. All mice were maintained in a specific pathogen-free animal facility. Immunoblot Analysis—Tissues from p27+/+ or p27S10A/S10A mice were homogenized in homogenization buffer (50 mm Tris-HCl (pH 7.5), 0.25 m sucrose, 1 mm EDTA) supplemented with 0.4 mm Na3VO4, 0.4 mm EDTA, 10 mm NaF, and 10 mm sodium pyrophosphate, as well as antipain, pepstatin, chymostatin, leupeptin, and phenylmethylsulfonyl fluoride, each at a concentration of 10 μg/ml. The resulting homogenates as well as cultured primary lymphocytes or mouse embryonic fibroblasts (MEFs) were lysed in radioimmunoprecipitation assay buffer supplemented as for the homogenization buffer. Lysates were incubated on ice for 15 min and then centrifuged at 20,000 × g for 15 min at 4 °C; after determination of its protein concentration with the Bradford assay (Bio-Rad), the resulting supernatant (50 μg of protein) of each lysate was subjected to SDS-PAGE. The separated proteins were transferred to a Hybond P membrane (Amersham Biosciences) and subjected to immunoblot analysis with antibodies (1 μg/ml) to p27 (Transduction Laboratories) or to glycogen synthase kinase-3β (GSK-3β) (Transduction Laboratories). A rabbit polyclonal antibody specific for p27 phosphorylated on Ser10 (Zymed Laboratories Inc.) could not be used because it reacted with p27(S10A) (Supplementary Material, Fig. S1). Immune complexes were detected with appropriate horseradish peroxidase-conjugated secondary antibodies and SuperSignal West Pico chemiluminescence reagents (Pierce). Band intensity was measured with an LAS-1000 chemiluminescence imager (FujiFilm). Cell Culture—Single-cell suspensions of lymphocytes were prepared from the lymph nodes of p27+/+ or p27S10A/S10A mice and cultured (1.0 × 107 cells in 5 ml) in RPMI 1640 medium supplemented with 10% fetal bovine serum; the cells were exposed to 10 nm phorbol 12,13-dibutyrate (PDBu) (Sigma), 300 nm ionomycin (Sigma), 10 μm MG132 (Peptide Institute), cycloheximide (10 μg/ml), or vehicle (dimethyl sulfoxide), as indicated. Primary MEFs were isolated on embryonic day 13.5 and cultured as described (10Nakayama K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Nakayama K-I. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar). For analysis of synchronized cells, MEFs were arrested at G0 phase by serum deprivation (incubation in medium supplemented with 0.1% fetal bovine serum) for 96 h; they were then cultured for the indicated times in medium containing 20% serum. Cell Cycle Analysis by Flow Cytometry—Lymphocytes cultured as described above were exposed to 10 μm bromodeoxyuridine (BrdUrd) (Sigma) for 30 min, harvested, fixed overnight in 70% ethanol at –20 °C, and denatured for 30 min at room temperature in 2 m HCl containing 0.5% Triton X-100. After neutralization with borax buffer (pH 8.5), the cells were subjected to dual color staining with fluorescein isothiocyanate-conjugated antibodies to BrdUrd (Becton Dickinson) and propidium iodide (5 μg/ml). They were then analyzed with a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson). Two-dimensional PAGE—Lymphocytes, MEFs, and tissues were lysed in a solution containing 8 m urea, 2% Triton X-100, 65 mm dithiothreitol, and 0.2% Ampholine (pH 3.5–9.5) (Amersham Biosciences). Lysates containing 50–150 μg of protein were applied to an Immobiline DryStrip (13 cm, pH 4–7) (Amersham Biosciences) and subjected to isoelectric focusing for 12 h at 0 V, 1 h at 500 V, 1 h at 1000 V, and 2 h at 8000 V. The DryStrip was then equilibrated with a solution containing 50 mm Tris-HCl (pH 6.8), 6 m urea, 30% glycerol, 2% SDS, and 65 mm dithiothreitol before the separated proteins were resolved in the second dimension by standard PAGE on an 11% gel and subjected to immunoblot analysis. 32P and 35S Labeling of p27 in Vivo—Freshly isolated lymphocytes were incubated for 2 h in phosphate-free RPMI 1640 medium supplemented with dialyzed fetal bovine serum and then metabolically labeled with [32P]orthophosphate (Amersham Biosciences) at a concentration of 1 mCi/ml for 8 h at 37 °C in the same medium with or without 10 nm PDBu and 300 nm ionomycin. Alternatively, the cells were incubated for 8 h with or without PDBu and ionomycin and labeled with [35S]methionine and [35S]cysteine (Amersham Biosciences) at a concentration of 80 mCi/ml for the final 1 h. After extensive washing with radioisotope-free medium, the cells were lysed and subjected to immunoprecipitation with antibodies to p27. The resulting precipitates were fractionated by SDS-PAGE and subjected to autoradiography with a BAS-2000 image analyzer (FujiFilm). Immunofluorescence Analysis of p27 Expression—MEFs were grown on glass coverslips and subjected to immunofluorescence staining as described previously (32Hatakeyama S. Kitagawa M. Nakayama K. Shirane M. Matsumoto M. Hattori K. Higashi H. Nakano H. Okumura K. Onoe K. Good R.A. Nakayama K.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3859-3863Crossref PubMed Scopus (182) Google Scholar). Endogenous p27 was stained with a monoclonal antibody to p27 (Transduction Laboratories), and immune complexes were detected with Alexa488-conjugated goat antibodies to mouse immunoglobulin G (Molecular Probes, Inc., Eugene, OR). Nuclei were stained with Hoechst 33258 dye. Lymphocytes were plated on glass coverslips precoated with poly-l-lysine (200 μg/ml) and were then subjected to immunofluorescence staining as described previously (32Hatakeyama S. Kitagawa M. Nakayama K. Shirane M. Matsumoto M. Hattori K. Higashi H. Nakano H. Okumura K. Onoe K. Good R.A. Nakayama K.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3859-3863Crossref PubMed Scopus (182) Google Scholar). Endogenous p27 was stained with polyclonal antibodies to p27 (C-19 and N-20 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; Ab-2 from Neomarkers), and immune complexes were detected with Alexa488-conjugated goat antibodies to rabbit immunoglobulin G (Molecular Probes). Cell nuclei were stained with propidium iodide. Confocal fluorescence images were obtained with a Radiance2000 microscope (Bio-Rad). Introduction of an S10A Mutation into Mouse p27—We designed a targeting construct to replace the wild-type p27 allele with an allele in which the codon for Ser10 was changed to an alanine codon (Fig. 1A). The linearized targeting vector was introduced into mouse ES cells by electroporation, and transfectants selected on the basis of their ability to grow in the presence of both G418 and gancyclovir were screened for homologous recombination events by the polymerase chain reaction and Southern blot analysis (data not shown). The loxP-flanked neomycin resistance cassette was then deleted by infection of selected ES cells with an adenovirus vector encoding Cre recombinase, after which the mutant p27 allele was amplified by the polymerase chain reaction and confirmed by DNA sequencing (data not shown). The targeted ES cells were then injected into C57BL/6 blastocysts, and chimeric male mice that transmitted the mutant allele in the germ line were obtained. Heterozygotes (p27+/S10A) were bred to produce p27S10A/S10A mice, which were identified by Southern blot analysis of tail DNA (Fig. 1B). Phenotype of p27S10A/S10A Knock-in Mice—Heterozygote matings yielded wild-type (p27+/+), heterozygous (p27+/S10A), and homozygous mutant (p27S10A/S10A) offspring in a ratio similar to that expected for Mendelian inheritance, indicative of no substantial embryonic lethality of the mutant allele. We have previously shown that p27–/– mice are larger than normal mice and exhibit multiple organ hyperplasia (10Nakayama K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Nakayama K-I. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar). In contrast, the body size (Fig. 1C) and organ size (data not shown) of p27S10A/S10A mice appeared normal. The increase in body weight with age also did not differ markedly among p27+/+, p27+/S10A, and p27S10A/S10A mice (Fig. 1D). This difference in phenotype between p27–/– and p27S10A/S10A mice suggests that the S10A mutant of p27 is not functionally null. Consistent with this notion, we previously showed that mutation of Ser10 in p27 does not affect the CDK-inhibitory function of this protein (27Ishida N. Kitagawa M. Hatakeyama S. Nakayama K.I. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). We next examined the abundance of p27 in various organs by immunoblot analysis. The amount of p27(S10A) was reduced in the brain, thymus, spleen, and testis of p27S10A/S10A mice compared with that of the wild-type protein in p27+/+ animals, whereas the expression of p27 in liver, heart, lung, and skeletal muscle was not affected by the mutation (Fig. 2, A and B). For all organs examined, the abundance of p27 mRNA was similar in p27S10A/S10A and p27+/+ mice (data not shown). Two-dimensional PAGE revealed that the proportion of p27 molecules that were phosphorylated in various organs of wild-type mice varied from 30 to 70% (Fig. 2C) and was not correlated with the extent of p27 down-regulation apparent in the corresponding organs of p27S10A/S10A mice. No signal corresponding to phosphorylated p27 was detected in thymus or lung of p27S10A/S10A mice (Fig. 2C), indicating that the signal observed in wild-type animals was attributable to phosphorylation of p27 on Ser10. These data suggest that phosphorylation of Ser10 affects the abundance of p27 in some, but not all, organs. Cell Cycle-dependent Phosphorylation of p27 on Ser10 in Primary Lymphocytes—We have previously shown that p27 is phosphorylated on Ser10 at G0-G1 phase of the cell cycle in cultured HeLa and NIH 3T3 cells, whereas the extent of this phosphorylation is markedly reduced in cells in S and M phases (27Ishida N. Kitagawa M. Hatakeyama S. Nakayama K.I. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 29Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). We examined the phosphorylation status of Ser10 of p27 in primary lymphocytes isolated from wild-type mice. Almost all mature lymphocytes are in the G0 phase in the absence of mitogenic stimulation (Fig. 3A). Stimulation of the cells with the combination of PDBu and ionomycin for 12 h (G1 phase) or 24 h (S phase) resulted in a substantial reduction in the abundance of p27 (Fig. 3B). We evaluated the relative amounts of phosphorylated and nonphosphorylated forms of p27 in lymphocytes and MEFs by two-dimensional PAGE (Fig. 3, C and D). Similar to our previous observations with HeLa and NIH 3T3 cells (27Ishida N. Kitagawa M. Hatakeyama S. Nakayama K.I. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 29Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), about half of p27 molecules were phosphorylated in wild-type lymphocytes at G0 phase, whereas no such phosphorylation was detected in p27S10A/S10A lymphocytes (Fig. 3C). These data suggest that most phosphorylation of p27 at G0 phase is attributable to Ser10. Only ∼8% of p27 molecules were phosphorylated in wild-type lymphocytes at S phase, suggesting that the phosphorylated molecules present in G0 phase had been dephosphorylated or selectively degraded. We obtained similar results with MEFs (Fig. 3D). The detection of marked phosphorylation of p27 on Ser10 at G0 phase does not necessarily indicate that p27 undergoes phosphorylation during G0 phase. It might become phosphorylated before the entry of cells into G0 phase. To test this hypothesis, we labeled lymphocytes freshly isolated from wild-type or p27S10A/S10A mice with [32P]orthophosphate for 8 h in the absence or presence of mitogenic stimulation (Fig. 3E). In the absence of stimulation (G0 phase), neither wild-type nor p27S10A/S10A cells incorporated [32P]orthophosphate into p27. In contrast, p27 present in both types of cells was labeled with [32P]orthophosphate in response to mitogenic stimulation, although the extent of labeling was greater for wild-type cells than for p27S10A/S10A cells. To exclude the possibility that the resting lymphocytes were metabolicallly inactive, the cells from wild-type mice were labeled with [35S]methionine and [35S]cysteine. Lymphocytes in G0 or G1 phases incorporated the radiolabeled amino acids to similar extents. These data suggest that p27 undergoes phosphorylation on Ser10 not during G0 phase but during the cell cycle before entry of cells into G0 phase. Destabilization of p27 by the S10A Mutation Specifically at G0 Phase—The predominance of the Ser10-phosphorylated form of p27 in G0 phase suggested that phosphorylation of this residue might be responsible for the stabilization of p27 apparent in quiescent cells. To test this hypothesis, we measured, as described previously (33Malek N.P. Sundberg H. McGrew S. Nakayama K. Kyriakidis T.R. Roberts J.M. Nature. 2001; 413: 323-327Crossref PubMed Scopus (228) Google Scholar), the half-life of p27 in lymphocytes isolated from wild-type or p27S10A/S10A mice. In G0 phase, whereas wild-type p27 was stable, p27(S10A) was markedly unstable (Fig. 4A). In contrast, wild-type p27 and p27(S10A) were degraded at similar rates in S-phase cells (Fig. 4B). Treatment of p27S10A/S10A lymphocytes with the proteasome inhibitor MG132 largely prevented the degradation of p27(S10A) at G0 phase (Fig. 4C), suggesting that this proteolysis is mediated by the ubiquitin-proteasome system. These data indicate that phosphorylation of Ser10 contributes to the stabilization of p27 apparent during G0 phase. Degradation of p27 at G0-G1 Independent of Ser10Phosphorylation—p27 is rapidly degraded by the ubiquitin-proteasome pathway at the G0-G1 transition. This process is largely independent both of SCFSkp2 and of phosphorylation of p27 on Thr187 (26Hara T. Kamura T. Nakayama K. Oshikawa K. Hatakeyama S. Nakayama K-I. J. Biol. Chem. 2001; 276: 48937-48943Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 33Malek N.P. Sundberg H. McGrew S. Nakayama K. Kyriakidis T.R. Roberts J.M. Nature. 2001; 413: 323-327Crossref PubMed Scopus (228) Google Scholar). We examined whether the degradation of p27 at the G0-G1 transition is dependent on phosphorylation of Ser10. Both wild-type and p27S10A/S10A lymphocytes exhibited similar kinetics of p27 down-regulation at the G0-G1 transition, although the original abundance of p27(S10A) was lower than that of wild-type p27 at G0 phase (Fig. 5A). The timing of entry of cells into S phase after mitogenic stimulation also did not differ substantially between wild-type and p27S10A/S10A lymphocytes (Fig. 5B). These data suggest that the phosphorylation of p27 on Ser10 is not required for the degradation of p27 at the G0-G1 transition. Nuclear Export of p27 in p27S10A/S10A Cells—We and others previously showed that the S10A mutation reduced the efficiency of nuclear export of p27 (28Rodier G. Montagnoli A. Di Marcotullio L. Coulombe P. Draetta G.F. Pagano M. Meloche S. EMBO J. 2001; 20: 6672-6682Crossref PubMed Scopus (252) Google Scholar, 29Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 30Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar). Given that these previous experiments were performed with overexpressed recombinant proteins in cultured cells, ho" @default.
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• W2032945178 title "Role of Serine 10 Phosphorylation in p27 Stabilization Revealed by Analysis of p27 Knock-in Mice Harboring a Serine 10 Mutation" @default.
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