Ubiquitination of p21^Cip1/WAF1 by SCF^Skp2: Substrate Requirement and Ubiquitination Site Selection

Abstract

Multiple proteolytic pathways are involved in the degradation of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1). Timed destruction of p21(Cip1/WAF1) plays a critical role in cell-cycle progression and cellular response to DNA damage. The SCF(Skp2) complex (consisting of Rbx1, Cul1, Skp1, and Skp2) is one of the E3 ubiquitin ligases involved in ubiquitination of p21(Cip1/WAF1). Little is known about how SCF(Skp2) recruits its substrates and selects particular acceptor lysine residues for ubiquitination. In this study, we investigated the requirements for SCF(Skp2) recognition of p21(Cip1/WAF1) and lysine residues that are ubiquitinated in vitro and inside cells. We demonstrate that ubiquitination of p21(Cip1/WAF1) requires a functional interaction between p21(Cip1/WAF1) and the cyclin E-Cdk2 complex. Mutation of both the cyclin E recruitment motif (RXL) and the Cdk2-binding motif (FNF) at the N terminus of p21(Cip1/WAF1) abolishes its ubiquitination by SCF(Skp2), while mutation of either motif alone has minimal effects, suggesting either contact is sufficient for substrate recruitment. Thus, SCF(Skp2) appears to recognize a trimeric complex consisting of cyclin E-Cdk2-p21(Cip1/WAF1). Furthermore, we show that p21(Cip1/WAF1) can be ubiquitinated at four distinct lysine residues located in the carboxyl-terminal region but not two other lysine residues in the N-terminal region. Any one of these four lysine residues can be targeted for ubiquitination in the absence of the others in vitro, and three of these four lysine residues are also ubiquitinated in vivo, suggesting that there is limited specificity in the selection of ubiquitination sites. Interestingly, mutation of the carboxyl-terminal proline to lysine enables ubiquitin conjugation at the carboxyl terminus of the substrate both in vitro and in vivo. Thus, our results highlight a unique property of the ubiquitination enzymatic reaction in that substrate ubiquitination site selection can be remarkably diverse and occur in distinct spatial areas.

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex

“…7B). Consistent with the role of Skp2 in promoting p21 degradation in unperturbed cells (Yu et al 1998;Bornstein et al 2003;Sarmento et al 2005;Wang et al 2005), si-RNA against Skp2 elevated the basal p21 protein levels during the G1 and S phases of the cell cycle (Fig. 7B, cf.…”

“…Therefore, the CRL4 Cdt2 ubiquitin ligase is redundant with SCF Skp2 in promoting the destruction of p21 in S-phase cells in a similar fashion to that observed for the replication factor Cdt1. Interestingly, whereas the CRL4 Cdt2 -dependent ubiquitylation of p21 depends on p21 interaction with PCNA and is stimulated by GSK3␤-dependent phosphorylation on Ser114, SCF Skp2 -dependent ubiquitylation of p21 is promoted by p21 binding to and phosphorylation by Cdk1/2 on Ser130 (Bornstein et al 2003;Wang et al 2005;Zhu et al 2005). Thus, it is possible that CRL4 Cdt2 preferentially frees PCNA from p21, while SCF Skp2 preferentially targets p21 in complex with Cdk1/2 (see model in Fig.…”

“…Several studies have shown how p21 is degraded during an unperturbed cell cycle: At the G1/S transition and during the S phase of the cell cycle, the SCF Skp2 ubiquitin ligase targets p21 for degradation (Yu et al 1998;Bornstein et al 2003;Sarmento et al 2005;Wang et al 2005), while the CDK inhibitor is ubiquitylated via the APC/C Cdc20 ubiquitin ligase during G2/M (Amador et al 2007). Our understanding of p21 degradation following UV irradiation, on the other hand, remains rather confusing.…”

p53- and p21-dependent premature APC/C–Cdh1 activation in G2 is part of the long-term response to genotoxic stress

“…First, the APC/C-Cdh1 has a much broader substrate specificity, including many crucial activators of cell division and DNA replication that are not targeted by APC/C-Cdc20 (Peters, 2006;Wa¨sch et al, 2010). Second, because p21 is a substrate of the APC/CCdc20 in mitosis (Amador et al, 2007) and of SCF Skp2 during interphase (Yu et al, 1998;Bornstein et al, 2003;Wang et al, 2005), APC/C-Cdh1-dependent degradation of Cdc20 (Pfleger and Kirschner, 2000) and Skp2 (Bashir et al, 2004;Wei et al, 2004;Liu et al, 2007) might reinforce the p53-dependent induction of p21 expression after DNA damage. Given that p21 on the one hand leads to activation of APC/C-Cdh1 (this study) and on the other hand inhibits CDK-dependent phosphorylation, which otherwise would protect Skp2 from APC/C-Cdh1 targeting (Rodier et al, 2008), the existence of a positive feedback loop between p21 expression and APC/C-Cdh1 activation appears to be an attractive option and might be complemented and further enforced by p53/p21-dependent repression of Cdc20 transcription (Kidokoro et al, 2008).…”

NUB1, an interferon-inducible protein, mediates anti-proliferative actions and apoptosis in renal cell carcinoma cells through cell-cycle regulation

“…Hence, identifying the molecules critical for immunotherapy with IFN-a is still crucial to developing treatments for metastatic RCC. NEDD8, one of the ubiquitin-like proteins, reportedly forms conjugates with cullin family proteins and thereby activates an Skp1-Cullin-F-box (SCF) ubiquitin protein ligase complex that catalyses the ubiquitination of many cellcycle regulators, for example, cyclin E, p21, p73 and p27 (Singer et al, 1999;Podust et al, 2000;Wang et al, 2005;Watson et al, 2006). NEDD8 ultimate buster 1 (NUB1) is a NEDD8-interacting protein composed of 601 amino-acid residues with a calculated molecular mass of 69.1 kDa.…”