P226 SRp40 MEDIATES TGF-INDUCED POST-TRANSCRIPTIONAL SPLICING OF FIBRONECTIN IN RENAL EPITHELIAL CELLS Phanish, M, Shirali, S, Dockrell, M SWT Institute for Renal Research, St Helieer Hospital, Carshalton More than two thirds of the human protein-coding genes undergo alternative splicing, facilitating the production of several polypeptides from a single gene. Fibronectin (Fn), the important exctracellular matrix protein, is a good example of post-transcriptional splicing. The solubility and activity of fibronectin is highly dependent on the splicing that occurs. Compelling evidence suggests that the isoform containing Extra Domain A (EDA+) has profibrotic activity and targeting its production may be an anti-fibrotic strategy. Our group has previously produced evidence for a putative role for the arginine serine rich protein SRp40 in mediating EDA+ Fn production from human tubule epithelial cells in response to TGF1 and have a suggested a possible role for PI-3kinase. Cdc2-like kinases (Clks) have been implicated as one of the key kinases that regulate SR protein activity; and evidence suggests that Clk may be either up-stream or down-stream of PI-3kinase depending on the cellular context. Here we investigate the regulation of SRp40 by TGF and assess possible roles for Clk and PI3 kinase in EDA+ Fn production. Primary and transformed human PTEC were used. Intracellular SRp40 localisation was assessed by immunofluorescent microscopy. Cell were treated with TGF1 (2.5ng/ml) for 648 hr, before fixing. To investigate the role of Clk, cells were treated with TGF1 +/- a Clk inhibitor, Tg-0003, 10 M for 48 h and EDA+ Fn production assessed by Western Blotting. Direct association of phoshpho-Akt (pAkt) and SRp40 was investigated by treating cells with TGF1 +/- Ly294002 (5M), the PI-3K inhibitor. Cells were subsequently lysed and the lysates subjected to immuno-precipitation by an SRp40 antibody and the IP subjected to immunoblotting. TGF1 induced a time dependent intracellular relocalisation of SRp40 as determined by immunofluorescent microscopy. An apparent shift to a more nuclear localisation was observed 24 h after TGF1 treatment which then returned to basal conditions by 48h. This intracellular redistribution appeared to be PI3kinase dependent as it was modified by a submaximal dose of the PI3-kinase inhibitor LY294002. To investigate whether Clk might be involved in the process cells were treated with TGF +/- Tg003; however Tg003 had no effect on TGF-induced EDA+ Fn production. We then investigated whether Akt the down stream target of PI3 kinase may have a direct interaction with SRp40. Immunoprecipitation experiments demonstrated that TGF1 caused an association of phospho-Akt and SRp40 which could be inhibited by pre-treatment with the PI3-kinase inhibitor LY294002. Previously our group has demonstrated a requirement of SRp40 expression for TGF1induced EDA+ Fn expression. Here we demonstrate that TGF1 induced an intracellular redistribution of SRp40 in PI3 kinase dependent manner. We also provided evidence for the activation of SRp40 being independent of Clk activity but that TGF1 treatment is associated with a direct interaction of SRp40 pAKT.