Abstract 1944: Phospholipid scramblase 1 and 3 as novel effectors of cripto-1 signaling

Abstract

Abstract In the present study we performed a yeast two-hybrid (Y2H) screen to search for novel Cripto-1 (CR-1) binding proteins that might modulate CR-1-dependent signaling pathways involving Nodal/Gdf3/Smad 2/3 or GRP78/glypican-1/c-src. Screening a mouse embryo or human colon cDNA prey library with a peptide sequence of CR-1, we isolated six candidate genes, including mNotch3, mFibulin-4, mCHD4, hAHNAK, hHUWE1 and mouse phospholipid scramblase 3 (PLSCR3). PLSCRs consist of a family of four related genes that encode for endofacial transmembrane proteins, and have been proposed to play a role in the redistribution of plasma membrane phospholipids. Recent work on the PLSCRs has revealed their involvment in signal transduction and lipid metabolism. The most studied member of this family, PLSCR1, has been shown to partition into lipid rafts implicated in the regulation and endocytic trafficking of receptor-signaling complexes. We confirmed the results of the Y2H screen since we found that full-length GFP-tagged PLSCR1 and PLSCR3 were able to bind to 3xFLAG-CR-1 in transiently transfected 293T cells, as detected by coimmunoprecipitation and Western blot analysis. In addition, we were able to detect co-localization of these proteins in 293T cells that were transiently expressing GFP-PLSCR1/3 and 3xFLAG-CR-1 by immunocytochemistry. We then evaluated the effect of interaction between CR-1 and PLSCR-1/3 on Nodal signaling. We therefore analyzed the activity of the Nodal/Activin-responsive (n2)7-luciferase reporter ((n2)7-Luc), which contains mouse Fast-1 binding sites from the Nodal left side-specific enhancer, in 293T cells transiently expressing CR1-FLAG, PLSCR1-GFP, PLSCR3-GFP, (n2)7-Luc, TK-renilla, mouse Fast-1, mouse Nodal and human ALK4 expression vectors. Overexpression of PLSCR1 induced a very strong dose-dependent inhibition of CR-1 activation of Nodal-responsive luciferase reporter activity in 293T cells. In contrast, overexpression of PLSCR3 did not have a significant inhibitory effect on CR-1activation of Nodal-responsive luciferase reporter activity in 293T cells. The effect of knockdown of PLSCR-1/3 with specific siRNA on CR-1 ability to induce activation of a Nodal-responsive luciferase reporter assay and to modulate CR-1 Smad-dependent and Smad-independent signaling pathways is currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2011-1944