The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27.
The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. 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 complex. 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.
Unit of Biochemistry, B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096, Israel.
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Ganoth
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Bornstein
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Larsen
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Tyers
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Pagano
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Hershko
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
C093090:CKS1B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; D042846:CDC2-CDC28 Kinases; D018844:Cyclin-Dependent Kinases; D008025:Ligases