F -box proteins and co-factors of SCF E3 ubiquitin ligases in melanoma
Aberrant cell cycle control is a hallmark of cancer cells. Alterations in E3 ubiquitin ligase-mediated proteasomal activity contribute to malignant cell proliferation. As potential therapeutic targets, it is critical to understand how ubiquitin ligases contribute to malignant cell proliferation. F-box proteins, sometimes with the aid of a co-factor, are the substrate-recognizing component of the Skp1/Cul1/F-box (SCF)-E3 ubiquitin ligase complexes. Cks1 and αB-crystallin are the co-factors for the F-box proteins Skp2 and Fbx4, respectively. SCFSkp2/Cks1 targets p27Kip1 and cyclin E for proteasomal degradation, whereas SCFFbx4/αB-crystallin targets cyclin D1 and Pin2. In this study, we examined the expression and role of SCFSkp2/Cks1 and SCFFbx4/αB-crystallin in melanoma and melanocytes. We show that SCFSkp2/Cks1 and SCFFbx4/αB-crystallin are differentially regulated in melanoma. The expression of Skp2 and Cks1 are up-regulated while αB-crystallin is down-regulated in melanoma cells. Importantly, the expression of both Cks1 and αB-crystallin is regulated by the mutant B-RAF signaling pathway. The up-regulated Skp2 and Cks1 are required for melanoma cell proliferation, as depletion of either resulted in G2 arrest and endoreplication. Surprisingly, accumulated p27 Kip1, a Skp2 degradation target, is dispensable for Skp2 knockdown-induced G2 arrest and endoreplication. The G2 arrest resulting from Skp2 depletion is mediated by the down-regulation of G2/M regulators, including cyclin A, CDK1, and cyclin B. Expression of nuclear localized cyclin B1 prevented G2 arrest after Skp2 knockdown. Furthermore, the G2 arrest and down-regulation of G2/M regulatory genes were strongly dependent on p53, which frequently remains wild-type in melanoma. In melanoma cell lines harboring mutant p53, Skp2 depletion did not induce G2 arrest despite upregulation of p27 Kip1. These results suggest that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells. The examination of the αB-crystallin co-factor revealed its role in regulating cyclin D1 turnover in melanocytic cells. Knockdown of αB-crystallin in melanocytes decreases the cyclin D1 turnover rate. Expression of αB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. These data indicate that SCFSkp2/Cks1 and SCFFbx4/αB-crystallin are differentially regulated by mutant B-RAF signaling and contribute to melanocyte and melanoma cell cycle control.
Hu, Rong, "F -box proteins and co-factors of SCF E3 ubiquitin ligases in melanoma" (2009). ETD Collection for Thomas Jefferson University. AAI3390427.