Phosphorylation of p27Kip1 at Thr-157 Interferes with Its Association with Importin α during G1 and Prevents Nuclear Re-entry
2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês
10.1074/jbc.m412367200
ISSN1083-351X
AutoresIncheol Shin, Jeremy D. Rotty, Frederick Y. Wu, Carlos L. Arteaga,
Tópico(s)RNA Research and Splicing
ResumoWe have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G1 and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G1 phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin α. A T157A-p27 mutant protein exhibited higher association with importin α than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin α. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G1 phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin α thus preventing its re-entry into the nucleus. We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G1 and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G1 phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin α. A T157A-p27 mutant protein exhibited higher association with importin α than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin α. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G1 phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin α thus preventing its re-entry into the nucleus. Cell cycle progression is dependent on the activity of complexes containing cyclins and cyclin-dependent kinases (Cdk). 1The abbreviations used are: Cdk, cyclin-dependent kinase; NLS, nuclear localization sequence; WT, wild-type; hKIS, human kinase-interacting stathmin; CIAP, calf intestine alkaline phosphates; FBS, fetal bovine serum; LMB, leptomycin B; PBS, phosphate-buffered saline; HA, hemagglutinin.1The abbreviations used are: Cdk, cyclin-dependent kinase; NLS, nuclear localization sequence; WT, wild-type; hKIS, human kinase-interacting stathmin; CIAP, calf intestine alkaline phosphates; FBS, fetal bovine serum; LMB, leptomycin B; PBS, phosphate-buffered saline; HA, hemagglutinin. The Cdk inhibitor p27 is an important regulator of the mammalian cell cycle (1Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5097) Google Scholar). p27 causes G1 arrest by inhibiting the activities of G1 cyclins and Cdks. An increase in p27 levels results in a delay of cell cycle progression, whereas a decrease in p27 promotes arrested cells to resume proliferation. The expression of p27 is regulated both transcriptionally and post-translationally. Akt-mediated phosphorylation and inhibition of Forkhead transcription factors were reported to decrease p27 gene transcription (2Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1216) Google Scholar). Cdk2-mediated phosphorylation of p27 at Thr-187 results in complex formation with ubiquitin ligase SCFSkp2 leading to 26 S proteasome-mediated degradation of p27 in proliferating cells (3Montagnoli A. Fiore F. Eytan E. Carrano A.C. Draetta G.F. Hershko A. Pagano M. Genes Dev. 1999; 13: 1181-1189Crossref PubMed Scopus (508) Google Scholar, 4Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1327) Google Scholar). During G0/G1 phase of the cell cycle, p27 degradation can also occur independently of Thr-187 phosphorylation and binding to SCFSkp2 (5Hara T. Kamura T. Nakayama K. Oshikawa K. Hatakeyama S. Nakayama K. J. Biol. Chem. 2001; 276: 48937-48943Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 6Malek N.P. Sundberg H. McGrew S. Nakayama K. Kyriakides T.R. Roberts J.M. Kyriakidis T.R. Nature. 2001; 413: 323-327Crossref PubMed Scopus (225) Google Scholar). Localization of p27 in the nucleus is required to inhibit Cdk activation by Cdk-activating kinase (7Darbon J.M. Devault A. Taviaux S. Fesquet D. Martinez A.M. Galas S. Cavadore J.C. Doree M. Blanchard J.M. Oncogene. 1994; 9: 3127-3138PubMed Google Scholar). The binding to and inhibition of Cdks in the nucleus by p27 is probably impaired (8Orend G. Hunter T. Ruoslahti E. Oncogene. 1998; 16: 2575-2583Crossref PubMed Scopus (119) Google Scholar) in many human tumor cells where cytosolic redistribution of p27 has been observed (9Ciaparrone M. Yamamoto H. Yao Y. Sgambato A. Cattoretti G. Tomita N. Monden T. Rotterdam H. Weinstein I.B. Cancer Res. 1998; 58: 114-122PubMed Google Scholar, 10Singh S.P. Lipman J. Goldman H. Ellis Jr., F.H. Aizenman L. Cangi M.G. Signoretti S. Chiaur D.S. Pagano M. Loda M. Cancer Res. 1998; 58: 1730-1735PubMed Google Scholar).More recent evidence suggests that phosphorylation of p27 is an important determinant of its subcellular localization. Akt-mediated phosphorylation of p27 at Thr-157 results in retention of p27 in the cytosol (11Liang J. Zubovitz J. Petrocelli T. Kotchetkov R. Connor M.K. Han K. Lee J.H. Ciarallo S. Catzavelos C. Beniston R. Franssen E. Slingerland J.M. Nat. Med. 2002; 8: 1153-1160Crossref PubMed Scopus (802) Google Scholar, 12Shin I. Yakes F.M. Rojo F. Shin N.Y. Bakin A.V. Baselga J. Arteaga C.L. Nat. Med. 2002; 8: 1145-1152Crossref PubMed Scopus (680) Google Scholar, 13Viglietto G. Motti M.L. Bruni P. Melillo R.M. D'Alessio A. Califano D. Vinci F. Chiappetta G. Tsichlis P. Bellacosa A. Fusco A. Santoro M. Nat. Med. 2002; 8: 1136-1144Crossref PubMed Scopus (602) Google Scholar). The nuclear localization sequence (NLS) of p27 contains an Akt consensus motif RXRXXT157D. Expression of constitutively active mutants of Akt results in cytosolic localization of wild-type (WT) p27. However, a mutant p27 protein in which Thr-157 has been replaced with Ala shows nuclear localization regardless of high cellular Akt activity (11Liang J. Zubovitz J. Petrocelli T. Kotchetkov R. Connor M.K. Han K. Lee J.H. Ciarallo S. Catzavelos C. Beniston R. Franssen E. Slingerland J.M. Nat. Med. 2002; 8: 1153-1160Crossref PubMed Scopus (802) Google Scholar, 12Shin I. Yakes F.M. Rojo F. Shin N.Y. Bakin A.V. Baselga J. Arteaga C.L. Nat. Med. 2002; 8: 1145-1152Crossref PubMed Scopus (680) Google Scholar, 13Viglietto G. Motti M.L. Bruni P. Melillo R.M. D'Alessio A. Califano D. Vinci F. Chiappetta G. Tsichlis P. Bellacosa A. Fusco A. Santoro M. Nat. Med. 2002; 8: 1136-1144Crossref PubMed Scopus (602) Google Scholar). Furthermore, high expression of active (phosphorylated) Akt in primary human breast cancers statistically correlates with localization of p27 in tumor cytosol (12Shin I. Yakes F.M. Rojo F. Shin N.Y. Bakin A.V. Baselga J. Arteaga C.L. Nat. Med. 2002; 8: 1145-1152Crossref PubMed Scopus (680) Google Scholar). Ser-10 is another major phosphorylation site in p27 (14Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), and this modification is necessary for the nuclear export of the protein mediated by the exportin (15Ishida 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 (237) Google Scholar, 16Rodier G. Montagnoli A. Di Marcotullio L. Coulombe P. Draetta G.F. Pagano M. Meloche S. EMBO J. 2001; 20: 6672-6682Crossref PubMed Scopus (250) Google Scholar). The binding of p27 to CRM1 requires Ser-10 phosphorylation, as an S10A p27 mutant shows reduced association with CRM1, whereas phospho-mimicking p27 mutants in which Ser-10 has been substituted with aspartic acid (S10D) or glutamic acid (S10E) show a markedly enhanced interaction with CRM1 (15Ishida 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 (237) Google Scholar, 17Connor 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 (158) Google Scholar). In addition to point mutations in Ser-10, mutations in basic residues within the nuclear export sequence also impair p27 nuclear export, suggesting that export is mediated by the interaction of CRM1 with the atypical nuclear export sequence in p27 (17Connor 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 (158) Google Scholar). The kinase responsible for Ser-10 phosphorylation is human kinase-interacting stathmin (hKIS) (18Boehm M. Yoshimoto T. Crook M.F. Nallamshetty S. True A. Nabel G.J. Nabel E.G. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (241) Google Scholar). The forced expression of hKIS reverses the cell cycle arrest induced by WT-p27 overexpression but not by S10A-p27, indicating that hKIS modulates cell cycle progression through Ser-10 phosphorylation and nuclear export (18Boehm M. Yoshimoto T. Crook M.F. Nallamshetty S. True A. Nabel G.J. Nabel E.G. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (241) Google Scholar).The macromolecular cargo transport between the nucleus and the cytosol is mediated by nuclear pore complexes. Mammalian nuclei typically contain several thousand nuclear pore complexes (19Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar) each composed of proteins termed nucleoporins, which form a complex of around <125 MDa (20Doye V. Hurt E. Curr. Opin. Cell Biol. 1997; 9: 401-411Crossref PubMed Scopus (213) Google Scholar). Soluble transport receptors (karyopherins) that bind either the NLS or nuclear export sequence in cargo proteins can be classified into exportins and importins. The most widely characterized exportin is CRM1, which binds basic amino acid residues in the nuclear export sequence. Importin α recognizes NLS, and the association of the importin α-NLS complex with the nuclear pore complex is mediated by importin β (19Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar, 21Gorlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (409) Google Scholar). The C terminus of p27 contains a classical bipartite NLS consisting of an N-terminal cluster of three basic residues and a C-terminal cluster of two basic residues separated from each other by 10 amino acids (22Gorlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1062) Google Scholar). This NLS is thought to be responsible for p27 import into the nucleus (23Zeng Y. Hirano K. Hirano M. Nishimura J. Kanaide H. Biochem. Biophys. Res. Commun. 2000; 274: 37-42Crossref PubMed Scopus (52) Google Scholar). The binding of NLS to importin α might be mediated by the ionic interaction between basic amino acid residues in the NLS and acidic residues in importin α (19Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar).We report herein that Akt-mediated phosphorylation of p27 at Thr-157 occurs in the cytosol during the G1 phase of the cell cycle. Blockade of nuclear export with the CRM1 inhibitor leptomycin B eliminated detectable levels of cytosolic Thr-157 P-p27, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation in the cytosol. A T157A p27 mutant protein exhibited high association with importin α and exclusive nuclear localization, whereas p27 phosphorylation at this site prevented the association with importin α. The binding with importin α was dependent on Thr-157 dephosphorylation, because treatment with calf intestine alkaline phosphates (CIAP) restored the association of both transfected and endogenous p27 with importin α, although there was no effect of CIAP on the association between T157A-p27 and importin α. Co-expression of a vector encoding active Akt abrogated the association of WT-p27 with importin α. These results along with previous reports (15Ishida 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 (237) Google Scholar, 16Rodier G. Montagnoli A. Di Marcotullio L. Coulombe P. Draetta G.F. Pagano M. Meloche S. EMBO J. 2001; 20: 6672-6682Crossref PubMed Scopus (250) Google Scholar, 17Connor 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 (158) Google Scholar, 18Boehm M. Yoshimoto T. Crook M.F. Nallamshetty S. True A. Nabel G.J. Nabel E.G. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (241) Google Scholar) suggest that Akt-mediated phosphorylation of p27 at Thr-157 prevents nuclear re-entry by inhibiting the association of p27 with importin α.MATERIALS AND METHODSCell Culture and Vectors—BT-474, 293T, NIH3T3 (mouse), and HeLa (human) cells were all from the American Type Culture Collection (ATCC; Manassas, VA). BT-474 cells were maintained in improved minimal essential medium, 10% FBS (Invitrogen). 293T, NIH3T3, and HeLa cells were passaged in Dulbecco's modified Eagle's medium, 10% FBS. The WT and S10A human p27 genes each in a pET vector were provided by Dr. Michele Pagano (New York University). They were subcloned into the BamH1 and XhoI sites of the FLAG-tagged vector pCMV-Tag2B (Stratagene). The T157A mutant was described previously (12Shin I. Yakes F.M. Rojo F. Shin N.Y. Bakin A.V. Baselga J. Arteaga C.L. Nat. Med. 2002; 8: 1145-1152Crossref PubMed Scopus (680) Google Scholar). pCDNA-HA-AktDD was a gift from Dr. Jim Woodgett (University of Toronto). In this mutant, Thr-308 and Ser-473 have been substituted with aspartic acid, resulting in a constitutively active Akt kinase (24Hutchinson J. Jin J. Cardiff R.D. Woodgett J.R. Muller W.J. Mol. Cell. Biol. 2001; 21: 2203-2212Crossref PubMed Scopus (249) Google Scholar). The transfection of plasmids into cell lines was performed using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. CIAP was purchased from Promega. Propidium iodide, polyethylene glycol 8000, leptomycin B (LMB), cycloheximide, and Hoechst 33342 were from Sigma. The protease inhibitor mixture was from Roche Applied Science. Erlotinib (OSI-774, Tarceva) was provided by Dr. Mark Sliwkowski (Genentech, South San Francisco, CA).Cell Fractionation, Immunoprecipitation, and Immunoblot Analysis—Subcellular fractionation was performed as described (25Lenferink A.E. Busse D. Flanagan W.M. Yakes F.M. Arteaga C.L. Cancer Res. 2001; 61: 6583-6591PubMed Google Scholar). Briefly, cell pellets from a monolayer culture were incubated in a hypotonic buffer (10 mm HEPES (pH 7.2), 10 mm KCl, 1.5 mm MgCl2, 0.1 mm EGTA, 20 mm NaF, 100 μm Na3VO4, and protease inhibitor mixture) for 30 min at 4 °C on a rocking platform. Cells were homogenized (Dounce, 30 strokes), and their nuclei were pelleted by centrifugation (10 min, 3500 rpm). The supernatant was saved as the cytosolic fraction, and nuclear pellets were incubated in nuclear lysis buffer (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, and 1% Triton X-100) for 1 min in a sonicating water bath. Prior to immunoblotting, cells were washed twice with ice-cold PBS, scraped in EBC lysis buffer (50 mm Tris-HCl (pH 8.0), 120 mm NaCl, 0.5% Nonidet P-40, 100 mm NaF, 200 μm Na3VO4, and protease inhibitor mixture), and incubated for 20 min at 4 °C while rocking. Lysates were cleared by centrifugation (10 min at 12,000 rpm, 4 °C). Fifty μg of total protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Immunoblot analysis was performed as described previously (25Lenferink A.E. Busse D. Flanagan W.M. Yakes F.M. Arteaga C.L. Cancer Res. 2001; 61: 6583-6591PubMed Google Scholar) using the primary antibodies described below and horseradish peroxidase-linked IgG (Amersham Biosciences) as the secondary antibodies. Immunoreactive bands were visualized by chemiluminescence (Roche Applied Science). Antibodies used were as follows: monoclonal against importin α (karyopherin α/Rch-1, BD Transduction Laboratories, Franklin Lakes, NJ), FLAG-M2 monoclonal (Sigma), p27, hemagglutinin (HA), tubulin 14-3-3 (H-8), and c-jun (Santa Cruz Biotechnology, Santa Cruz, CA); total Akt, P-S473 Akt, and P-T308 Akt (Cell Signaling, Beverly MA);Texas Red-conjugated antibody against rabbit IgG and Oregon Green-conjugated antibody against mouse IgG (Molecular Probes, Eugene, OR). A phospho-specific Thr-157 p27 antibody was a gift from Dr. Giuseppe Viglietto (Centro di Endocrinologia et Oncologia Sperimentale, Naples, Italy) (13Viglietto G. Motti M.L. Bruni P. Melillo R.M. D'Alessio A. Califano D. Vinci F. Chiappetta G. Tsichlis P. Bellacosa A. Fusco A. Santoro M. Nat. Med. 2002; 8: 1136-1144Crossref PubMed Scopus (602) Google Scholar).Immunoprecipitations were carried out by incubating 0.5–1 mg of total cell lysates with primary antibody at 4 °C overnight. Protein A-Sepharose (Sigma, 1:1 slush in PBS) was then added for 2 h at 4 °C while rocking. The precipitates were washed four times with ice-cold PBS, resuspended in 6× Laemmli sample buffer, and resolved by SDS-PAGE followed by immunoblot analysis. For studies involving phosphatase treatment, cells were lysed in CIAP buffer (20 mm Tris (pH 8), 150 mm NaCl, 1 mm MgCl2, 1 mm dithiothreitol, 0.5% Triton X-100, and protease inhibitor mixture). The lysates were treated with or without 200 units of CIAP at 37 °C for 1 h after which phosphatase inhibitors (Na3VO4 (1 mm) and NaF (50 mm)) were added.Cell Cycle Analysis—293T and BT-474 cells were synchronized in G1 phase of the cell cycle by serum starvation for 48 h. BT-474 cells were also treated with the erbB tyrosine kinase inhibitor erlotinib (3 μm) (26Hidalgo M. Siu L.L. Nemunaitis J. Rizzo J. Hammond L.A. Takimoto C. Eckhardt S.G. Tolcher A. Britten C.D. Denis L. Ferrante K. Von Hoff D.D. Silberman S. Rowinsky E.K. J Clin. Oncol. 2001; 19: 3267-3279Crossref PubMed Scopus (959) Google Scholar, 27Hernan R. Fasheh R. Calabrese C. Frank A.J. Maclean K.H. Allard D. Barraclough R. Gilbertson R.J. Cancer Res. 2003; 63: 140-148PubMed Google Scholar) during this time. At 48 h, medium was removed and replenished with fresh medium containing 10% FBS. At variable times after the addition of serum, cells were trypsinized, fixed with methanol, and their nuclei were labeled with propidium iodide as described (25Lenferink A.E. Busse D. Flanagan W.M. Yakes F.M. Arteaga C.L. Cancer Res. 2001; 61: 6583-6591PubMed Google Scholar). 2 × 104 propidium iodide-positive nuclei were gated and analyzed in a FACS/Calibur Flow Cytometer (Becton Dickinson).Immunofluorescence Staining—293T cells were seeded onto coverslips in 12-well plates at the density of 105 cells/well. On the next day, cells were transfected with 1 μg of FLAG-p27 with or without 1 μg HA-AktDD. Eighteen hours later, the cells were serum-starved for 48 h. FBS (10%) was added for 6–24 h, the time at which the monolayers were washed with PBS, fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 (15 min), and blocked for nonspecific binding in PBS, 3% milk for 30 min. Subcellular localization of FLAG-p27 was monitored using a FLAG antibody (1:500) diluted in PBS, 1% milk. Expression of HA-AktDD was monitored with a polyclonal HA antibody (1:250). Endogenous Thr(P)-157-p27 in BT-474 cells was stained with a Thr(P)-157 p27 antibody (1:250) described above. Incubation with primary antibodies was for 1 h. After three washes with PBS, samples were treated with Texas Red-conjugated anti-rabbit IgG (1:250) or Oregon Green-conjugated anti-mouse IgG (1:250) for 30 min. For DNA staining, samples were incubated with Hoechst 33342 dye (1 μg/ml, 10 min) after incubation with secondary antibodies. Immunofluorescence was monitored with a cooled digital CCD camera (Princeton Instruments, Monmouth Junction, NJ) on a Zeiss Axiophot upright microscope.Heterokaryon Shuttling Assay—An interspecies heterokaryon shuttling assay was performed as described (28Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (450) Google Scholar) with some modifications. Briefly, NIH3T3 cells were seeded at a density of 3 × 105 cells/well in a 6-well plate. On the next day, cells were transfected with 2 μg of FLAG-p27 with FuGENE 6 for 48 h. HeLa cells were seeded at 1 × 106 cells/well on top of the NIH3T3 cells. Cell fusions were induced by the addition of 50% polyethylene glycol 8000 for 2 min. To block de novo protein synthesis, 50 μg/ml of cycloheximide was added for 5 min before the addition of polyethylene glycol. After a 1-h incubation in Dulbecco's modified Eagle's medium containing cycloheximide, the samples were fixed in 4% formaldehyde (10 min). In some cases, LMB was added to the medium at 10 ng/ml immediately after seeding HeLa cells on top of NIH3T3 cells.RESULTSAkt Phosphorylates Cytosolic p27 on Thr-157 during G1 Phase—We first analyzed the spatial and temporal regulation of Akt-mediated phosphorylation of p27 in G1-arrested and cycling 293T cells (Fig. 1). Serum starvation for 48 h increased the 293T G1 fraction from 40 to 70%. Akt was predominantly localized in the cytosol throughout the cell cycle. At short intervals (5–30 min) after the addition of serum to G1-arrested cells, total Akt and Ser-473 P-Akt were detectable in 293T nuclear fractions. Ser-473 P-Akt was undetectable in serum-starved cells but was maximal 3–6 h after serum addition. p27 was found both in cytosol and the nucleus in asynchronous cultures, whereas serum-starved quiescent cells exhibited high p27 levels only in the nucleus. p27 translocated to the cytosol 1 h after serum addition reaching its maximal level in this compartment 6 h later in mid G1. Three to six hours after serum addition and when (cytosolic) P-Akt levels were maximal, levels of Thr(P)-157 p27 were also maximal. Phosphorylation of p27 in Thr-157 was only detectable in cytosolic fractions of 293T cells. Twenty-four hours after serum stimulation when cells have entered S phase, p27 was degraded consistent with reports of proteolytic degradation of p27 in cycling cells (29Sutterluty H. Chatelain E. Marti A. Wirbelauer C. Senften M. Muller U. Krek W. Nat. Cell Biol. 1999; 1: 207-214Crossref PubMed Scopus (627) Google Scholar).Similar results were obtained with erbB2-overexpressing BT-474 human breast cancer cells induced into quiescence by serum starvation and treatment with the erbB tyrosine kinase inhibitor erlotinib. P-Akt and Thr(P)-157 p27 were detectable in asynchronous cell cultures but undetectable in quiescent cells (Fig. 2A). Upon serum addition and release from G1, P-Akt and Thr(P)-157 p27 content as monitored by immunohistochemistry increased with similar kinetics achieving a maximum in mid-to-late G1 phase of the cell cycle. Immunofluorescence staining of synchronized cultures showed minimal P-Akt and Thr(P)-157 p27 staining of quiescent BT-474 cells and a time-dependent increase upon serum addition at 5 h (Fig. 2B). These data suggest that temporal and spatial phosphorylation of p27 in Thr-157 correlate with the cellular distribution of Akt and its activation.Fig. 2Time course of Akt and p27 phosphorylation in BT-474 cells.A, BT-474 cells were synchronized by serum starvation and incubation with 3 μm erlotinib for 48 h. At this time, improved minimal essential medium, 10% FBS was added. Cells were then harvested at the indicated times and subjected to flow cytometry as indicated under "Materials and Methods." The percentage of cells in G1,S,andG2M phases of the cell cycle at different times following serum addition are indicated on top. Cell lysates (30 μg/lane) were subjected to immunoblot analyses with the antibodies indicated on the right of each panel. B, simultaneously, BT-474 cells on coverslips were induced to undergo cell cycle arrest (as in Fig. 2). At 48 h, 10% FBS was added to induce the progression into S phase. At the indicated times following serum addition, the cells were fixed and stained with P-Akt and Thr(P)-157 p27 antibodies as well as Hoechst nuclear dye.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ser-10 Phosphorylation Is Required for p27 Cytosolic Localization and Its Phosphorylation at Thr-157—To further document that phosphorylation of p27 in Thr-157 occurs in the cytosol, FLAG-WT-p27, FLAG-T157A-p27, and FLAG-S10A-p27 were expressed in 293T cells and examined during cell cycle progression. WT-p27 localized in both cytosol and nuclei of asynchronous 293T cells. In quiescent cells WT-p27 was almost exclusively localized in the nucleus with cytosolic FLAG-WT-p27 levels markedly increasing 6 h after the addition of serum (Fig. 3A, lane 3). Co-transfection of activated Akt markedly increased FLAG-WT-p27 cytosolic levels throughout the cell cycle (Fig. 3A, lanes 5–8). Both FLAG-T157A-p27 and FLAG-S10A-p27 were exclusively nuclear in 293T cells in the presence or absence of transfected active Akt, suggesting that phosphorylation at Ser-10 is also required for cytosolic localization of p27 and phosphorylation at Thr-157. Consistent with this possibility, Thr-157 phosphorylation was detectable in transfected wild-type p27 but it was almost undetectable in pull downs of the S10A p27 mutant (Fig. 3B). Finally, treatment of BT-474 cells with leptomycin B, an inhibitor of CRM1-dependent nuclear export, abrogated cellular levels of Thr(P)-157 p27 (Fig. 3C), implying that nuclear export of p27 is required for Thr-157 phosphorylation.Fig. 3Phosphorylation of p27 on Thr-157 occurs in the cytosol.A, 293T cells were transfected with FLAG-WT-p27, FLAG-T157A-p27, or FLAG-S10A-p27 either with or without HA-AktDD. Sixteen hours after transfection, the cells were serum-starved (ss) for 48 h. To release cells into S phase, synchronized cells were treated with 10% FBS and harvested 6 or 24 h after as indicated. Both cytosolic and nuclear fractions were subjected to FLAG immunoblot analysis. Tubulin and c-jun (cytosolic and nuclear markers, respectively) immunoblots confirmed there was no significant cross-contamination between cytosolic and nuclear fractions. B, 293T cells were transfected with FLAG-tagged WT-, T157A- or S10A-p27 for 48 h. FLAG-p27 was precipitated from 500 μg of whole cell lysates and subjected to Thr(P)-157 p27 and FLAG immunoblot analyses. C, BT-474 cells on coverslips were treated with LMB at a concentration of 10 ng/ml for 2.5 h, fixed, and stained with a Thr(P)-157 p27 antibody or Hoechst dye.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The results shown in Fig. 3A were confirmed by immunolocalization studies. 293T cells were transfected with FLAG-WT-p27, FLAG-T157A-p27, and FLAG-S10A-p27, each with or without constitutively active HA-AktDD and subjected to staining with FLAG antibodies. FLAG staining was observed in the cytosol and nuclei of asynchronous 293T cells transfected with FLAG-WT-p27. Serum starvation resulted in nuclear localization of WT-p27, which translocated to the cytosol 6 h after the addition of serum (Fig. 4A). At 24 h, there was a 70% reduction in FLAG-positive cells (data not shown) consistent with p27 degradation during S phase. Expression of HA-AktDD resulted in cytosolic localization of WT-p27 in serum-starved cells and in cycling cells at 6- and 24-h post-serum addition. Staining with an HA antibody indicated that the active Akt mutant was predominantly localized in the cytosol. This result agrees with the localization of endogenous activated Akt in 293T cell fractions (shown in Fig. 1A). Both T157A-p27 and S10A-p27 were exclusively nuclear throughout the cell cycle (Fig. 4,
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