Skp2 Regulates G2/M Progression in a p53-dependent Manner

Abstract

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27 Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27 Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27 Kip1 . These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27 Kip1 degradation. INTRODUCTIONUbiquitin-mediated proteolysis of cell cycle regulators is critical for tight control of normal cell proliferation. The rate-limiting step in ubiquitin-dependent degradation is substrate recognition by E3 ubiquitin ligases. Altered expression and/or activity of E3 ubiquitin ligases in cancer cells leads to deregulated proteolysis and aberrant proliferation (Guardavaccaro and Pagano, 2004;Nakayama and Nakayama, 2006). It is critical to understand how E3 ubiquitin ligases contribute to malignant cell proliferation, because they represent a potential new class of therapeutic targets (Nalepa et al., 2006).One major class of E3 ubiquitin ligases that regulates cell cycle progression contains Skp1/Cullin/F-box protein (SCF) complexes (Krek, 1998;Patton et al., 1998). Within these complexes, the cullin protein serves as a scaffold for the E2 ubiquitin-conjugating enzyme and Skp1/F-box protein complex. The F-box protein determines the substrate specificity of the SCF complex; ϳ70 exist in the human genome. The F-box protein S phase kinase-associated protein 2 (Skp2) has received attention as a putative oncogene. Its expression correlates with tumor malignancy in several tumor types, and the SKP2 gene is amplified in small cell lung and biliary tract cancers (reviewed in Guardavaccaro and Pagano, 2004). High expression of Skp2 is sufficient to promote anchorage-independent growth (Carrano et al., 1999), and Skp2 cooperates with mutant Ras to transform rat fibroblasts (Gstaiger et al., 2001). Evidence from transgenic mice models shows that expression of Skp2 in the T-lymphoid lineage cooperates with activate...

Per2 Inhibits K562 Leukemia Cell Growth In Vitro and In Vivo Through Cell Cycle Arrest and Apoptosis Induction

“…Meanwhile, P53 is a regulator of Cdc2-cyclin B1 and can transcriptionally suppress both genes. Moreover, previous studies indicated that circadian regulation of cyclin B1 appears to be indirect and to involve a p53-dependent mechanism, p53 helps to block entry into mitosis and strengthens the G2 arrest [26].…”

Unfolded‐protein response‐associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract

“…When p27 is phosphorylated on Thr 187 , it is degraded by the Ub proteasome system (UPS) via SCF skp2 ‐mediated ubiquitination (28–30). Several lines of evidence suggest that p27 is a common upstream regulator of the Cdk1‐driven cyclic process of mitosis and the unidirectional LFCD process: 1 ) overexpression of mutant Ub in human lens epithelial cells (HLECs) also causes the accumulation of p27, concurrent with G 2 /M phase arrest (12, 31); 2 ) a few reports indicate that p27 has roles in directing the G 2 /M transition during the cell cycle via activation of Cdk1 (32–34); and 3 ) Cdk1 is necessary in directing LFCD (19–22).…”

Induction of G2M Arrest by Flavokawain A, A Kava Chalcone, Increases the Responsiveness of HER2 Overexpressing Breast Cancer Cells to Herceptin

“…for promoting cell cycle progression [43]. Skp2 depletion in melanoma cells and mouse embryonic fibroblasts (MEFs) results in G2M arrest and polyploidy accumulation [44,45]. More importantly, Skp2 deletion in a Skp2 knockout mouse model has been shown by multiple groups to markedly restrict tumorigenesis under different conditions of tumor initiation and promotion, including PTEN, ARF, pRB inactivation as well as HER-2/Neu overexpression [46,47].…”