Translational control of p27Kip1 accumulation during the cell cycle

Science

Volumn 271, Issue 5257, 1996, Pages 1861-1864

  Hengst, Ludger ^a   Reed, Steven I ^a  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EUKARYOTA;

CELL CYCLE PROTEIN; CYCLIN DEPENDENT KINASE; CYCLIN DEPENDENT KINASE INHIBITOR 1B; ENZYME INHIBITOR; MESSENGER RNA; MEVINOLIN; MICROTUBULE ASSOCIATED PROTEIN; TUMOR SUPPRESSOR PROTEIN;

AMINO ACID SEQUENCE; ARTICLE; BIOSYNTHESIS; CELL CULTURE; CELL CYCLE; CELL CYCLE G1 PHASE; CELL CYCLE S PHASE; CELL LINE; DRUG ANTAGONISM; GENETICS; HALF LIFE TIME; HELA CELL; HUMAN; METABOLISM; MOLECULAR GENETICS; PROTEIN SYNTHESIS; CELL GROWTH; CONTROLLED STUDY; ENZYME ACTIVITY; ENZYME INDUCTION; FIBROBLAST; HUMAN CELL; PRIORITY JOURNAL; RNA TRANSLATION; TRANSLATION REGULATION;

AMINO ACID SEQUENCE; CELL CYCLE; CELL CYCLE PROTEINS; CELL LINE; CYCLIN-DEPENDENT KINASE INHIBITOR P27; CYCLIN-DEPENDENT KINASES; ENZYME INHIBITORS; G1 PHASE; HALF-LIFE; HELA CELLS; HUMANS; LOVASTATIN; MICROTUBULE-ASSOCIATED PROTEINS; MOLECULAR SEQUENCE DATA; PROTEIN BIOSYNTHESIS; RNA, MESSENGER; S PHASE; TUMOR CELLS, CULTURED; TUMOR SUPPRESSOR PROTEINS;

EID: 0029670477     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5257.1861     Document Type: Article

References (40)

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22. To purify the inhibitor protein from Iovastatin-arrested HeLa cells, we separated extracts obtained from 25 liters of arrested culture by size chromatography on a Superdex 200 column (Pharmacia). Fractions containing inhibitory activity (150 to 250 kD) were collected, and the proteins of these fractions were denatured at 100°C for 5 min The heatstable proteins were separated by Superdex 200 chromatography, and the proteins of fractions containing inhibitory activity were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane. The resulting band at 28 kD was digested by trypsin and peptides were microsequenced The following peptide sequences were obtained: NLF-GPDHEEL, NDFQNHKP, YEWQEVEK, LPEFYYRP, and RPQFR (27) The first two peptides exhibit more than 50% sequence identity with the sequence of the inhibitor p21 and were therefore used to design degenerate oligonucleotides to perform a PCR reaction that resulted in the cloning of an incomplete p27 cDNA Using this cDNA fragment as probe, we cloned the complete p27cDNA sequence.
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28. Abbreviations for the amino acid residues are as follows. C, Cys; D, Asp; E, Glu; F, Plie; G, Gly; H, His; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; T, Thr; V, Val, W, Tip; and Y, Tyr
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30. 7 cells/10 ml) and radioactivity (1.5 mCl/10 ml) were used, and cells were starved for 60 min in medium lacking methionine and cysteine before labeling
31. 5 cells/ml in DMEM supplemented with newborn calf serum (10%). Asynchronous growing cells were arrested in G1 by treatment with 66 μM lovastatin for 33 hours as described [K Keyomarsi, L Sandoval, V. Band, A. B Pardee, Cancer Res 51, 3602 (1991)]. For fluorescence-activated cell sorting (FACS) analyses, 5-ml samples were labeled for 15 min with bromodeoxyundine (BrdU; 30 μg/ml). The distribution of the cells in the cell cycle was determined with monoclonal antibodies to BrdU (anti-BrdU) and staining with propidium iodide (Becton Dickinson) according to the manufacturer's directions Proteins and RNA were isolated with the Tri Reagent system (Molecular Research Center). Immunoblots of cyclins, Cdks. and p27 were performed with polyclonal (anti-p27 and anti-cyclin A) or monoclonal antibodies (anti-cyclin E and anti-PSTAIRE) as described (22). Antibodies to p27 were generated against a peptide sequence (CRNLFGPVDHEEL-TRDLE) (27) from the p27 protein, and antibodies affinity-punfied against the immunogenic peptide were used Polyclonal antibodies to p21 were generated with the full-length protein as antigen. RNA was separated through 1% agarose gels, transferred to Nytran plus membrane (Schleicher & Schuell), and hybridized at 65°C in 1 M NaCl, 10% dextran sulfate, 1% SDS, and salmon testis DNA (100 μg/ml).
32. Asynchronously growing human newborn foreskin fibroblasts (HS68) cells were diluted 1:3, seeded in DMEM supplemented with fetal bovine serum (FBS; 10%) at 20% confluency, and grown into contact inhibition. For FACS analyses, cells on one plate were labeled for 30 min with BrdU (30 μg/ml).
33. -7 M final concentration) and indomethacin (Biomol, 18 75 μg/ml final concentration).
34. 5 cells/ml in DMEM supplemented with newborn calf serum (10%) Cells were synchronized by a thymidine and nocodazole block release protocol with 2 mM thymidine for 19 hours, released for 3 hours, and treated with nocodazole (75 ng/ml) for 12 hours. Samples of the synchronized cells were harvested every hour after release from the nocodazole block, and the percentage of cells in different phases of the cell cycle was determined by FACS analysis as described (13)
35. HS68 human diploid fibroblasts were grown to confluency and incubated for three additional days in DMEM growth medium, supplemented with 10% heat-inactivated FBS. Cells were trypsinized and seeded in a dilution of 1:5. Samples were taken at 3-hour intervals after release from the contact inhibition For FACS analyses, samples were labeled for 15 min with BrdU (30 μg/ml).
36. 35S label (ICN) per sample (10 ml), cells were labeled for 30 min and incubated for 0, 15, 30, 60, or 120 min in the presence of methionine (100 mg/liter) and cysteine (150 mg/liter). Cells were collected by centrifugation, washed in PBS, and lysed in RIPA buffer (in PBS: 1% NP-40, 0.5% sodium deoxycholate, 0 1% SDS, PMSF (0.1 mg/ml), aprotinin (30 μl/ml; Sigma), and 0.1 mM sodium orthovanadate). Protein concentrations were determined by absorbance at 280 nm, and samples were adjusted for equal protein amounts. Proteins were denatured by boiling at 100°C for 5 min. p27 was precipitated with a polyclonal antibody to p27 (C19) (23). Immune complexes were collected on protein A-Sepharose beads and washed seven times in RIPA buffer and once in RIPA buffer containing methionine and cysteine (each at 1 mg/ml). The immunoprecipitates were separated by SDS-PAGE and used for autofluorography.
37. HS68 human diploid fibroblasts were grown to contact inhibition and used after 6 days of density-mediated growth arrest. Asynchronous cells were grown to <30% confluency on plates. Labeling was done as described (23), Labeled cells were washed with PBS, scraped in RIPA buffer, extracts were normalized for equal counts per minute (cpm), and p27 immunoprecipitated as described (23)
38. HS68 human diploid fibroblasts were labeled 4 days after density-mediated growth arrest. Asynchronous cells were grown to <30% confluency on plates Pulse-chase labeling was done as described (23). The labeled cells were washed with PBS, trypsinized, and washed several times with ice-cold PBS. Cells were lysed in RIPA buffer, the extracts were normalized for equal cpm, and p27 immunoprecipitated as described (23).
39. 35S label in 10 ml of DMEM without methionine and cysteine (ICN) (per plate). Cells were washed in ice-cold PBS and scraped in PBS containing 1% NP-40 and PMSF (0 1 mg/ml), aprotinin (30 μl/ml; Sigma), and 0.1 mM sodium orthovanadate. The extracts were normalized for equal cpm and boiled for 10 min The heat-stable supernatant containing almost all p27 protein was adjusted to 0.5% sodium deoxycholate and 0.1% SDS The p27 protein was precipitated with affinity-purified antibody generated against a p27 peptide sequence (24). The second antibody cross-reacts with the p21 protein, Immunoprecipitates were washed once in RIPA and three times in PBS containing 1% NP-40, separated by SDS-PAGE, and analyzed by autofluorography.
40. We thank A W. Alberts for lovastatin; T. Hunter, R. Poon, and H Toyoshima for communicating results before publication, and S Haase, F Melchior, B Niculescu, and K. Sato for critical reading of the manuscript Supported by fellowships from the Leukemia Society of America (to L.H ) and by U.S. Public Health Service grant GM46006 and U.S. Army grant DAMD17-94-J4208 (to S.I.R.).