680 Elevated Chemokine mRNA in Rectal Mucosa Following Gastrointestinal Infection and in Irritable Bowel Syndrome

deletion mutations of ING2 vectors were also used.ING2 RNA interference (RNAi) was used to examine the role of the endogenous ING2.Bromodeoxyuridine (BrdUrd) assay was done to see the synergistic effects of ING2 and TGF-β on TGF-β-mediated cell growth regulation.Gal4-Smad2 transcriptional activity with ING2 and rTGF-β1 was examined.Immunoprecipitation (IP) was done by co-expressing ING2, Smad2 and SnoN.Intracellular localization of ING2, Smad2 and SnoN was examined by using immunofluorescence microscopy.Results: 1. ING2 strongly activated PAI-1 promoter synergistically with TGF-β signal about 60 times.The plant homeodomain (PHD) zinc finger of ING2 played crucial roles for this activity.2. ING2 RNAi inhibited the transcriptional activity by TGF-β signal.3. ING2 promoted the ability of rTGF-β1 to inhibit cell growth and ING2 RNAi suppressed this effect.4. ING2 increased TGF-β-induced Gal4-Smad2 transcriptional activity and the PHD of ING2 was required.5. Smad-interacting transcriptional modulator SnoN interacted with ING2 and promoted the assembly of a protein complex containing SnoN, ING2, and Smad2.Knockdown of endogenous SnoN blocked the ability of ING2 to promote TGF-βdependent transcription, and conversely expression of SnoN augmented ING2 enhancement of the TGF-β response.6. ING2, Smad 2 and SnoN coexisted in the nucleus.Conclusions: Tumor suppressor, ING2, collaborates with Smad2 and SnoN to mediate TGF-β-induced Smad-dependent transcription and cellular responses.Down-regulation of ING2 in cancer cells might be one of the mechanisms to evade the tumor suppressive effects of TGF-β. 676The β-Trcp Ubiquitin Ligase Targets PHLPP1 for Ubiquitin-Dependent Degradation Xin Li, Jianyu Liu, Tianyan Gao Hyperactivation of PI3K/Akt signaling is commonly associated with human cancers.PHLPP, a novel family of Ser/Thr protein phosphatases, serves as a negative regulator of Akt by directly dephosphorylating the kinase.Our recent studies have demonstrated that loss of PHLPP expression occurs at high frequency in colorectal cancer, supporting its role as a tumor suppressor.In the present study, we examined the molecular mechanism by which PHLPP1 protein is downregulated in cancer cells.Pulse-chase experiments reveal that the endogenous PHLPP1 is a short-lived protein with half-life around 3 hours.Treating cells with proteasome inhibitors effectively prevents turnover of PHLPP1 protein suggesting that PHLPP1 is degraded via the ubiquitin-proteasome pathway in cells.In searching for the E3 ubiquitin ligase of PHLPP1, we identified a F-box protein, β-Trcp, as the E3 ligase targeting PHLPP1 for degradation.Overexpression of the wild-type, but not a F-box deletion mutant, of β-Trcp results in a decrease of PHLPP1 protein expression and an increase of ubiquitination, whereas knockdown of the endogenous β-Trcp has the opposite effect.Furthermore, we find that the β-Trcp-mediated degradation depends on casein kinase I (CK1) and GSK-3β mediated phosphorylation of PHLPP1, and the activation of PI3K/Akt pathway suppresses the degradation of PHLPP1 by inhibiting the GSK3 activity.More importantly, a phosphorylation mutant of PHLPP1, which resistant to β-Trcp-mediated degradation, consistently expresses at a higher level in colon cancer cells, resulting in a more effective dephosphorylation of Akt and inhibition of cell growth.Taken together, we demonstrate that the degradation of PHLPP1 protein is promoted by β-Trcp-mediated ubiquitination in a phosphorylation dependent manner.Our findings support a negative feedback model in which the amplitude of PI3K/Akt signaling is controlled by Akt-mediated inhibition of PHLPP degradation.