Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: role in barrier enhancement.
Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-beta-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1microM, 5min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting >3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85alpha phosphatidylinositol 3-kinase (p85alphaPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.
Zhao
Jing
J
Department of Medicine, Pritzker School of Medicine, University of Chicago, Chicago, IL 60637, United States.
Singleton
Patrick A
PA
Brown
Mary E
ME
Dudek
Steven M
SM
Garcia
Joe G N
JG
D003285:Contractile Proteins / Q000378:metabolism; D051356:Cortactin / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells / Q000378:metabolism*; D064448:Filamins; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011651:Pulmonary Artery / Q000166:cytology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism
C495868:CTTN protein, human; D003285:Contractile Proteins; D051356:Cortactin; D064448:Filamins; D008246:Lysophospholipids; D008565:Membrane Proteins; D008840:Microfilament Proteins; D019000:Phosphotyrosine; C060506:sphingosine 1-phosphate; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D009219:Myosin-Light-Chain Kinase; D013110:Sphingosine