The cell-cycle regulatory protein Cks1 is required for SCFSkp2-mediated ubiquitinylation of p27

Abstract

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.

Mutant B-RAF signaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells

“…Phosphorylation at threonine 187 is permissive for p27 Kip1 degradation (Vlach et al, 1997;Carrano et al, 1999;Sutterluty et al, 1999). However, as threonine 187-phosphorylated p27 Kip1 levels increased in parallel with total p27 Kip1 (data not shown), we focused on possible defects in the SCF complex containing Skp2 and its cofactor, Cks1, that recognizes phosphorylated p27 Kip1 and targets it for degradation (Ganoth et al, 2001;Spruck et al, 2001). We demonstrate that B-RAF and cyclin D1 control Skp2 and Cks1 levels in melanoma cells.…”

“…Figure 6a); hence, we investigated the role of Cks1, a cofactor for Skp2 that prevents Skp2 auto-ubiquitylation and degradation (Ganoth et al, 2001;Spruck et al, 2001;Wang et al, 2004). Expression of Cks1 was higher in WM793 and WM115 cells compared to NHEM (Figure 6b).…”

Deregulated degradation of the cdk inhibitor p27 and malignant transformation

“…of purified SCF Skp2 , cyclin E/cdk2, Ubc3, and E1, and the factor competent for this effect was purified and identified as Cks1 [25]. This data allowed for an in vitro ubiquitination assay for p27 using completely purified components.…”

“…2). New evidence pointed to cdk subunit 1 (Cks1) as an integral part of the ubiquitination machinery for p27 [25,26]. Using a biochemical approach, fractions of Hela extract were assayed for their ability to promote p27 ubiquitination in the presence .…”

CRM1/Ran-Mediated Nuclear Export of p27Kip1Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

“…The ubiquitin-dependent proteolysis of p27 (Pagano et al, 1995) is regulated by its phosphorylation at threonine 187 (T187) by cyclin E-Cdk 2 in late G1 and S phase (Sheaff et al, 1997;Vlach et al, 1997;Montagnoli et al, 1999). T187 phosphorylation allows recognition of p27 by its SCF-type E3 ligase, comprised of Skp1, Cul1, and the F-box protein, Skp2 and Roc1 and the Cks1 cofactor Ohta et al, 1999;Sutterluty et al, 1999;Tsvetkov et al, 1999;Ganoth et al, 2001;Spruck et al, 2001). Recent evidence suggests that p27 proteolysis is regulated by at least two distinct mechanisms, with mitogenic signaling conditioning p27 for degradation in early G1 in a manner independent of T187 phosphorylation (Hara et al, 2001;Malek et al, 2001), whereas Skp2-dependent cyclin E-Cdk 2-mediated degradation occurs in S phase after T187 phosphorylation (Malek et al, 2001).…”