Rationale Lipid rafts are plasma membrane microdomains involved in dynamic membrane signaling and trafficking. Our previous studies (Singleton et al, FASEB J, 2005) have demonstrated that the platelet-derived lipid sphingosine 1-phosphate (S1P) plays a critical regulatory role in maintenance and enhancement of pulmonary vascular barrier function. S1P ligates the S1P receptor 1, which we have previously localized to lipid rafts of human pulmonary artery endothelial cells (HPAECs). Here we use 2-D electrophoresis analysis to characterize changes in tyrosine phosphoprotein content in lipid rafts of HPAECs challenged with S1P.
Methods and Results HPAECs were challenged with 1 μM of S1P for 5 minutes, washed with cold PBS twice, and solublized with 1% Triton X-100 at 4°C. The Triton X-100 insoluble material was subjected to discontinuous OptiprepTM gradient centrigugation and the light density (lipid raft) fractions were collected. Isolated lipid rafts were resolubilized in 50 μL of 7 M urea/2 M thiourea and 50 mM DTT. Isoelectric focusing was carried out on a nonlinear electrophoresis gel strip (pH3-10, 7 cm) with the second dimension carried out on 4-20% SDS-PAGE gel. Analytical gels were stained using Imperial blue (Pierce) and Western blotting was performed using antiphosphotyrosine antibody. Protein levels were not significantly altered in lipid rafts isolated from control vs S1P-treated cells (1 μM, 5 minutes). The 2-D gel images revealed over 30 proteins recruited to lipid rafts after S1P treatment (5 minutes) with antiphosphotyrosine blots showing S1P to increase tyrosine phosphorylation of over 20 proteins, including the src kinase family and cortactin.
Conclusions These results suggest that tyrosine kinases participate in S1P-induced signaling in lipid rafts and that 2-D proteomic analysis is a powerful tool for studying regulation of pulmonary endothelial cells barrier function.
Supported by NIH grant HL58064.