WWP2 isoforms differentially modulate the signaling pathway in SW620 cells

Abstract

WWP2 (WW domain-containing protein 2) is a NEDD4-like E3 ubiquitin-protein ligase whose activity has been indicated in the ubiquitination of PTEN and elevating the activity of phosphatidyl inositol 3-kinase (PI3K) Although it has been indicated in cell proliferation in cancer cells the underlying molecular mechanism is still vague Eps8 (Epidermal growth factor receptor kinase substrate 8) functions in the EGFR-mediated cell proliferation and is a common substrate of both EGFR and Src Colorectal cancer cells SW620 have a significant amount of WWP2 expression whereas Eps8 affects cell growth There are three WWP2 isoforms which are full-length WWP2 (WWP2-FL or V1) C-terminal isoform (WWP2-C or V2) and N-terminal isoform (WWP2-N or V3) WWP2 isoforms have previously been shown to regulate TGFβ/Smads signaling activity linked to epithelial mesenchymal transition (EMT) However the distinct role of the three WWP2 isoforms involving in the activation of Eps8/Src and PI3K and cell proliferation is still unknown To clarify the effect of the three WWP2 isoforms on cell proliferation we generated siWWP2-G knockdown cells which targets WWP2-V1 and WWP2-V2 and siWWP2-H knockdown cells which targets WWP2-V1 and WWP2-V3 Decreased levels of AKT AKT pi- S473 Src Src pi-Y416 FAK FAK pi-Y861 K-Ras and cell proliferation/migration were observed in siWWP2-H knockdown cells indicating WWP2-V1 or WWP2-V3 or both of them plays an important role in cell proliferation To clarify the distinct effect of WWP2-V1 and WWP2-V3 we transfected V1 resistance construct into siWWP2-H type HeLa knockdown cells Interestingly cell proliferation was further decreased when V1 resistance construct was transfected into siWWP2-H HeLa knockdown cells while no significant difference on cell proliferation was shown in vector-transfected siWWP2-H HeLa cells Taken together our results highlight the importance of WWP2 isoforms particularly WWP2-V3 involving in the Src/FAK and PI3K/AKT pathway-mediated cell proliferation

Date of Award	2014 Jul 4
Original language	English
Supervisor	Tzeng-Horng Leu (Supervisor)