Mitotic and G 2 Checkpoint Control: Regulation of 14-3-3 Protein Binding by Phosphorylation of Cdc25C on Serine-216-Reference-Cited by-同舟云学术

Mitotic and G 2 Checkpoint Control: Regulation of 14-3-3 Protein Binding by Phosphorylation of Cdc25C on Serine-216

Published:1997-09-05 Issue:5331 Volume:277 Page:1501-1505
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Peng Cheng-Yuan¹²³⁴,Graves Paul R.¹²³⁴,Thoma Richard S.¹²³⁴,Wu Zhiqi¹²³⁴,Shaw Andrey S.¹²³⁴,Piwnica-Worms Helen¹²³⁴

Affiliation:

1. C.-Y. Peng, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA, and Committee on Virology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

2. P. R. Graves, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

3. R. S. Thoma, Z. Wu, H. Piwnica-Worms, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA, and Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

4. A. S. Shaw, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

Abstract

Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G 2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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3. Myc-epitope–tagged Cdc25C and Cdc25(S216A) were subcloned into pUHD10-3 (25). Plasmids were cotransfected with pBabe a plasmid encoding a puromycin resistance gene into the HeLa tTA cell line (23). Clones resistant to G418 (geneticin Gibco) and puromycin were screened for inducible expression of Myc-tagged Cdc25C or Cdc25(S216A). Clones were expanded in Dulbecco's minimum essential medium (DMEM) containing G418 (400 μg/ml) puromycin (1 μg/ml) and tetracycline (2 μg/ml). Cells were trypsinized and washed four times with warm DMEM lacking tetracycline to induce protein expression. Upon replating cells were grown in DMEM containing G418 and puromycin. Indirect immunofluorescence indicated that 85 to 90% of cells were induced to express Cdc25C and Cdc25(S216A) at levels 10- to 50-fold as high as endogenous Cdc25C (4). In some cases cells were subjected to a double-thymidine block (7) or were incubated during the last 8 to 16 hours of induction with nocodazole (0.15 μg/ml) (Calbiochem) followed by mechanical agitation. Cells were lysed in mammalian cell lysis buffer [50 mM tris (pH 8.0) 2 mM dithiothreitol (DTT) 5 mM EDTA 0.5% NP-40 100 mM NaCl 1 μM microcystin 1 mM sodium orthovanadate 2 mM phenylmethylsulfonyl fluoride (PMSF) aprotinin (0.15 U/ml) 20 μM leupeptin and 20 μM pepstatin]. Antibodies used for Cdc25C detection included a monoclonal antibody to the Myc epitope (9E10 myc-agarose Santa Cruz Biotechnology) a monoclonal antibody generated to Cdc25C (174E10-3) and an affinity-purified rabbit polyclonal antibody to glutathione-S-transferase (GST). 14-3-3 proteins were detected with antibody to 14-3-3 β (K-19 Santa Cruz) which is broadly reactive with members of the 14-3-3 family of proteins . Bound primary antibodies were detected with horseradish peroxidase–conjugated anti-rabbit or anti-mouse secondary antibodies (Cappel) and an ECL detection system (Amersham).

4. C.-Y. Peng and H. Piwnica-Worms unpublished results.

5. Cells were incubated for 4 hours in phosphate-free media supplemented with 32 P-labeled inorganic phosphate (4 mCi/ml) 2 mM glutamine and 1.5% dialyzed calf serum. Cells were lysed in 1 ml of mammalian cell lysis buffer and Cdc25C was immunoprecipitated with antibody 174E10-3. Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) transferred to nitrocellulose and visualized by autoradiography. The nitrocellulose containing radiolabeled protein was excised blocked with 0.5% polyvinylpyrrolidone (PVP-40) in 100 mM acetic acid for 30 min at 37°C washed six times with water and digested with TPCK trypsin (Worthington) at a final concentration of 30 mg/ml in 0.1 M NH 4 HCO 3 (pH 8.0). Proteolysis and two-dimensional phosphopeptide mapping were performed as described (24).

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