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Vol. 19, Issue 11, 4602-4610, November 2008

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

Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208

Submitted November 12, 2007; Revised June 17, 2008; Accepted August 11, 2008
Monitoring Editor: William P. Tansey

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.

Address correspondence to: Andrew E. Aplin (aplina{at}mail.amc.edu)

Abbreviations used: CDK1, cyclin-dependent kinase 1; Orc1, origin recognition complex-1; qRT-PCR, real-time quantitative reverse transcription-polymerase chain reaction; SCF, Skp1/Cullin/F-box protein; shRNA, short hairpin RNA; siRNA, short-interfering RNA; Skp2, S phase kinase-associated protein 2.