Linkage of replication to start by the Cdk inhibitor Sic1

Science

Volumn 272, Issue 5261, 1996, Pages 560-562

  Schneider, B L ^a   Yang, Q H ^a,b   Futcher, A B ^a  

^a Watson School of Biological Sciences   (United States)

^b STONY BROOK UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN KINASE INHIBITOR;

ARTICLE; CELL DIVISION; DNA REPLICATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN PHOSPHORYLATION; SACCHAROMYCES CEREVISIAE;

EID: 0029923035     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5261.560     Document Type: Article

References (28)

1. K Nasmyth, Curr. Opin. Cell Biol. 5, 166 (1993).
2. E. Schwob, T. Bohm, M. D. Mendenhall, K. Nasmyth, Cell 79, 233 (1994).
3. M D. Mendenhall, Science 259, 216 (1993); T. T. Nugroho and M. D. Mendenhall, Mol. Cell. Biol. 14, 3320 (1994)
4. M D. Mendenhall, Science 259, 216 (1993); T. T. Nugroho and M. D. Mendenhall, Mol. Cell. Biol. 14, 3320 (1994)
5. S. I. Reed, J. A. Hadwiger, A. T. Lörincz, Proc. Natl. Acad. Sci. U.S.A. 82, 4055 (1985); M. D Mendenhall, C A. Jones, S. I Reed, Cell 50, 927 (1987).
6. S. I. Reed, J. A. Hadwiger, A. T. Lörincz, Proc. Natl. Acad. Sci. U.S.A. 82, 4055 (1985); M. D Mendenhall, C A. Jones, S. I Reed, Cell 50, 927 (1987).
7. J. Garrels and B Futcher, unpublished results
8. Two-dimensional gel electrophoresis of Sic1 phosphorylated in vitro by Cdc28 showed 13 labeled charge isoforms, suggesting 13 phosphorylation sites. There are nine Ser-Pro or Thr-Pro sites in Sic1. Even the most highly phosphorylated Sic1 showed only a modest change in mobility in the SDS-PAGE dimension, consistent with the data shown in Figs. 1 and 2
9. Sic1 from cells arrested in various states was treated with phosphatase as in Fig. 1 to show that the mobility shift was due to a change in phosphorylation. Consistent with these results, the mobility of Sic1 is altered in cdc28 mutants (2).
10. C. B. Epstein and F. R Cross, Mol Cell. Biol. 14, 2041 (1994).
11. F. R Cross, personal communication
12. V. Measday, L. Moore, J. Ogas, M Tyers, B Andrews, Science 266, 1391 (1994), C. B. Epstein and F. R. Cross, Genes Dev. 6, 1695 (1992); E. Schwob and K. Nasmyth, ibid. 7, 1160 (1993).
13. V. Measday, L. Moore, J. Ogas, M Tyers, B Andrews, Science 266, 1391 (1994), C. B. Epstein and F. R. Cross, Genes Dev. 6, 1695 (1992); E. Schwob and K. Nasmyth, ibid. 7, 1160 (1993).
14. V. Measday, L. Moore, J. Ogas, M Tyers, B Andrews, Science 266, 1391 (1994), C. B. Epstein and F. R. Cross, Genes Dev. 6, 1695 (1992); E. Schwob and K. Nasmyth, ibid. 7, 1160 (1993).
15. L. Dirick, T. Bohm, K. Nasmyth, EMBO J 14, 4803 (1995), B. L Schneider and A B Futcher, unpublished results.
16. L. Dirick, T. Bohm, K. Nasmyth, EMBO J 14, 4803 (1995), B. L Schneider and A B Futcher, unpublished results.
17. J Yaglom et al , Mol. Cell. Biol 15, 731 (1995).
18. C. J Sherr, Trends Biochem. Sci. 20, 187 (1995).
19. Antibody to rabbit Sic1 (12.5 μl) [J. D Donovan, J. H. Toyn, A. L. Johnson, L. H. Johnston, Genes Dev. 8, 1640 (1994)] was added to a 3-mg cell extract. After incubation for 1 hour at 0°C, protein A beads (30 μl) were added, and the mixture was rocked at 4°C for 1 hour Beads were washed four times with alkaline phosphatase buffer (APB) [50 mM tris-HCl (pH 8), 10 mM dithiothreitol, 0.6 mM dimethylaminopurine, 1 mM phenylmethylsulfonyl fluoride, leupeptin (5 μg/ ml), tosyl-L-phenylalanine-chloromethyl ketone (10 μg/ml), pepstatin (5 μg/ml), and soybean trypsin inhibitor (10 μg/ml)].
20. Extract (50 μg) was loaded per lane on a 16 cm by 18 cm by 0.75 mm gel and run for 20 hours at 100 V.
21. Proteins were transferred to nitrocellulose for 30 min at 10 V Blots were blocked by using non-fat milk (5%) in tns-buffered saline [TBS; 140 mM NaCl, 2.5 mM KCl, 25 mM tris-HCl (pH 7.4)] for 1 hour Blots were incubated overnight with a 1.100 dilution of rabbit antibody to Sic1 (14) Blots were washed four times in TBS, then incubated with a 1:2000 dilution of alkaline phosphatase-conjugated goat antibody to rabbit immunoglobulin G (Pierce) for 1.5 hours, washed, and finally incubated at room temperature with 10 ml of NBT-BCIP (nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolphosphate ρ-toluidine) (Gibco-BRL) for 5 to 10 min β-Tubulin was used as a loading control.
22. M. Tyers, G. Tokiwa, B. Futcher, EMBO J. 12, 1955 (1993).
23. Beads (30 μl) carrying immunoprecipitated Sic1 (14) were divided into three portions (10 μl), and these were treated with APB (10 μl) (14), APB (8 μl) plus calf intestinal phosphatase (CIP) (2 μl, 2 U) (Boehringer), or APB (7 μl), CIP (2 μl), and B-glycerolphosphate (1 μl of 1 M).
24. +]) [B. J. Thomas and R Rothstein, Cell 56, 619 (1989)], #31 (MATα cdc34-2 cln1::HIS3 cln2::TRP1 ura3::GAL-CLN3 leu2 ura3), BS100 (MATαcln1::LEU2-GAL-CLN1-HA3 cln2::TRP1 cln3::HIS3 leu2 his3 ura3 ade2 trp1), BS147 (MATa cln1 cln2 cln3 sic1., TRP1 [pGAL-CLN3 CEN UR43] ura3 leu2 trp1 his2 ade1], BS152 (cln1 cln2 cln3 sic1::TRP1) (derived from BS147 by plasmid loss), and BS178 [(MATa cln1::LEU2-GAL-CLN1-HA3 cln2 cln3 sic1::TRP1 ura3 trp1 his(2 or 3)]. The sic1::TRP1 allele was from M. Tyers; the parent of BS147 was from F. Cross.
25. 7 cells per milliliter The galactose medium was YEP (1% yeast extract, 1% peptone) with 1% raffinose and 1% galactose, the glucose medium was YEP with 1% raffinose and 2% glucose Before shifting from one medium to another, cells were first washed twice with the new medium that had been prewarmed to the target temperature. Sic1 was detected as described (15, 16) Representative samples were treated with phosphatase as shown (Fig. 1) to demonstrate that the mobility shift was due to phosphorylation.
26. 7 cells per milliliter with 2% filter-sterilized sucrose (W303 and BS193) or 1% filter-sterilized sucrose plus 1% galactose (BS147). Cells were centrifuged, sonicated, and elutriated in medium at 30°C
27. A computer curve-fitting algorithm estimated the number of cells with DNA content of 1N, 2N, or between 1N and 2N.
28. We thank L. Johnston for antibody to Sic1; M. Mendenhall, M. Tyers (who independently found suppression of the cln1 cln2 cln3 mutant by sic1), and F. Cross for strains, reagents, helpful discussions, and communication of unpublished results, and M. Cleary and M. Luke for reading the manuscript. Supported by NIH grant GM 39978 and U S Army Breast Cancer grant DAMD17-94-J-4050 to A B.F