The cell-cycle regulatory protein Cks1 is required for SCFSkp2-mediated ubiquitinylation of p27 (original) (raw)

Nature Cell Biology volume 3, pages 321–324 (2001)Cite this article

Abstract

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway1, allowing CDK activity to drive cells into S phase2. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5–8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex9. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate.

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Acknowledgements

We thank C. Segal for technical assistance, S. Reed for sharing results before publication, Z-P. Pan, J. Pines, C. Pickart, S. Reed and E. Yeh for reagents, R. Piva and A. Carrano for their contribution to this work, and J. Bloom and L. Yamasaki for critical reading of the manuscript. M.P. also thanks L. Yamasaki and L. Bragi for their continuous support. This work was supported by grants from the Israel Science Foundation and the Human Frontier Science Program Organization (HFSPO; to A.H.), by an Irma T. Hirschl Scholarship and grants from the HFSPO and the NIH (to M.P), and by grants from the National Cancer Institute of Canada (to M.T). Part of this work was done during the stay of A.H. at New York Univ. Sch. Med. (on sabbatical leave), where he was supported by a UICC Yamagiwa–Yoshida Memorial International Cancer Study Grant.

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Authors and Affiliations

  1. Unit of Biochemistry, B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096, Israel
    Dvora Ganoth, Gil Bornstein & Avram Hershko
  2. Department of Pathology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, 10016, USA
    Tun K. Ko & Michele Pagano
  3. Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, M56 1X5, Canada
    Brett Larsen & Mike Tyers

Authors

  1. Dvora Ganoth
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  2. Gil Bornstein
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  3. Tun K. Ko
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  4. Brett Larsen
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  5. Mike Tyers
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  6. Michele Pagano
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  7. Avram Hershko
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Corresponding authors

Correspondence toMichele Pagano or Avram Hershko.

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Figure S1. The heat-stable factor is sensitive to trypsin action. Heat-treated fraction 1 (∼0.1 mg ml−1 ) was incubated at 37 °C for 60 min with 50 mM Tris–HCl pH 8.0, in the absence (lane 1) or presence (lane 2) of 0.6 mg ml−1 of TPCK-treated trypsin (Sigma T8642). Trypsin activity was terminated by addition of 2 mg ml−1 soybean trypsin inhibitor (STI). In lane 3, STI was added 5 min before a similar incu-bation with trypsin. Samples corresponding to ∼50 ng of heat-treated fraction 1 were then assayed for stimulation of p27–ubiquitin ligation. (PDF 193 kb)

Figure S2. The heat-stable factor is not Nedd8 and is required after modification of Cul-1 by Nedd8. a, Purified Nedd8 does not replace the factor in stimulation of p27–ubiquitin ligation. Where indicated, ∼50 ng of heat-treated fraction 1 or 100 ng of purified recombinant human Nedd8 was added to the p27–MeUb ligation assay. b, Ligation of Nedd8 to Cul-1. Cul−1–ROC1 (3 μl) was incubated with 10 μg Nedd8 and 20 μl of purified Nedd8-conjugating enzymes in a 100-μl reaction mixture containing Tris pH 7.6, MgCl2, ATP, phosphocreatine, creatine phosphokinase, DTT, glycerol and STI at concentrations similar to those used for the p27–ubiquitin ligation assay. A control preparation of Cul1–ROC1 was incubated under similar conditions, but without Nedd8-conjugating enzymes. After incubation at 30°C for 2 h, samples of control (lane 1) or Nedd8-modified (lane 2) preparations were separated on an 8% SDS–polyacrylamide gel and immunoblotted with an anti-Cul-1 antibody (Zymed). c, SCF Skp2 complex containing Nedd8-modified Cul-1 still requires the factor from fraction 1 for p27–ubiquitin ligation. p27–MeUb ligation was assayed as in ‚Experimental procedures’, except that 35S-labelled p27 was replaced with bacterially expressed purified p27 (20 ng), and Cul-1–ROC1 was replaced with 2 μl of unmodified or Nedd8-modified Cul-1–ROC1 (see above). After incubation 30°C for 60 min, samples were separated on a 12.5% SDS–polyacrylamide gel, transferred to nitrocellulose and blotted with an anti-p27 monoclonal antibody (Transduction Laboratories). Asterisk shows a crossreacting protein.

Figure S3. Purification of the factor required for p27–ubiquitin ligation and its identification as Cks1. a, Final step of purification by gel-filtration chromatography. The peak activity from the MonoS step (see Methods) was applied to a Superdex 75 HR 10/30 column (Pharmacia) equilibrated with 20 mM Tris–HCl pH 7.2, 150 mM NaCl, 1 mM DTT and 01% Brij-35. Samples (0.5 ml) were collected at a flow rate of 0.4 ml min−1. Column fractions were concentrated to 50 μl by centrifuge ultrafiltration (Centricon-10, Amicon). Samples (0.004 μl) of column fractions were assayed for their ability to stimulate p27–ubiquitin ligation. Results were quantified by phosphorimager analysis and are expressed as the percentage of [35S]p27 converted to ubiquitin conjugates. Arrows indicate the elution position of marker proteins of the indicated Mr values × 103. b, Silver staining of samples (2.5 μl) from the indicated fractions of the Superdex 75 column, resolved on a 16% SDS–poly-acrylamide gel.

Figure S4. All bacterially expressed Cks/Suc1 proteins stimulate multiphosphorylation of the Cdc27 subunit of cyclosome/APC. Cyclosomes from S-phase HeLa cells were partially purified[13] and were incubated with 500 units of Suc1-free Cdk1–cyclin B[8] as described[13]. Where indicated, 10 ng μl−1 of the corresponding Cks/Suc1 protein was added. Samples were subjected to immunoblotting with a monoclonal antibody against human Cdc27 (Transduction Laboratories).

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Ganoth, D., Bornstein, G., Ko, T. et al. The cell-cycle regulatory protein Cks1 is required for SCFSkp2-mediated ubiquitinylation of p27.Nat Cell Biol 3, 321–324 (2001). https://doi.org/10.1038/35060126

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