Title: Ubiquitin modification of cyclin G1 protein stability
Abstract: 5192 Cyclins G1 and G2 are unconventional cyclins correlated with cell cycle arrest that associate with active protein phosphatase 2A (PP2A). Cyclin G-family mRNAs are low in proliferating cells, but significantly upregulated during stress-induced cell cycle exit. Upon stress, the cyclin G1 gene is transactivated by p53, but the G2 gene is a target of the growth inhibitory transcription factor FOXO3a.We reported thatectopic expression of either cyclin G1 or G2 induces cell cycle arrest.Supporting our hypothesis that upregulation of G1 or G2 promotes growth arrest, multiple microarray studies of others have repeatedly correlated their elevation coincident with diverse cell cycle-inhibitory signals. Though cyclins G1 and G2 are close homologs, they differ in their subcellular localization and ability to associate with some binding partners. In a previous collaborative study we determined that cyclin G1 is an unstable protein subjected to proteasomal degradation in the presence of the E3 ubiquitin (Ub) ligase and p53 antagonist Mdm2, and that the level of G1 expression impacts its cell cycle effects. We showed that G1 associates with PP2A, p53, and Mdm2 . In the present study we investigated the regulation of cyclin G1 stability compared to G2, and evaluated the importance of G1 expression to growth inhibitory responses elicited by ectopic expression of the Mdm2 antagonist, p19ARF. Here we show that cyclin G1 is a polyubiquitinated protein that interacts with the 26S proteasome subunit S5a, and is targeted for degradation in the presence of abundant Mdm2. Inhibition of poly-Ub chain formation via ectopic expression of Ub-lysine mutants (K48R) increased the abundance of cyclin G1, and its association with both Mdm2 and PP2A. We used pulldown assays to map the respective regions required for cyclin G1-Mdm2 interactions and hypothesize that, because G1 is reduced in the presence of active Mdm2, it directly contributes to cyclin G1 downregulation. To determine the importance of site-specific ubiquitin modification of cyclin G1 for its stability and associated cell cycle inhibitory activity, we generated several cyclin G1 point mutants. We identified mutants that are stabilized in the presence of Mdm2 and appears to exhibit increased intrinsic cell cycle inhibitory activity. Using shRNA-mediated knockdown of endogenous G1, we have preliminary evidence that the growth-arrest response elicited by ectopic expression of p19ARF is blunted upon cyclin G1 down-regulation. We are evaluating the cellular effects of these G1 mutations and the contribution of endogenous cyclins G1 and G2 to cell cycle arrest. Ub-modification of cell cycle regulatory proteins is central to growth control, and many human cancers exhibit alterations in p53 signaling pathways. Thus determining how cyclin G1 abundance is modulated (in the presence or absence of cyclin G2 expression), and the effect this has on tumor suppressor function is clearly warranted.
Publication Year: 2007
Publication Date: 2007-05-01
Language: en
Type: article
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