Role of Src-induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells.
Albumin transcytosis, a determinant of transendothelial permeability, is mediated by the release of caveolae from the plasma membrane. We addressed the role of Src phosphorylation of the GTPase dynamin-2 in the mechanism of caveolae release and albumin transport. Studies were made in microvascular endothelial cells in which the uptake of cholera toxin subunit B, a marker of caveolae, and (125)I-albumin was used to assess caveolae-mediated endocytosis. Albumin binding to the 60-kDa cell surface albumin-binding protein, gp60, induced Src activation (phosphorylation on Tyr(416)) within 1 min and resulted in Src-dependent tyrosine phosphorylation of dynamin-2, which increased its association with caveolin-1, the caveolae scaffold protein. Expression of kinase-defective Src mutant interfered with the association between dynamin-2, which caveolin-1 and prevented the uptake of albumin. Expression of non-Src-phosphorylatable dynamin (Y231F/Y597F) resulted in reduced association with caveolin-1, and in contrast to WT-dynamin-2, the mutant failed to translocate to the caveolin-rich membrane fraction. The Y231F/Y597F dynamin-2 mutant expression also resulted in impaired albumin and cholera toxin subunit B uptake and reduced transendothelial albumin transport. Thus, Src-mediated phosphorylation of dynamin-2 is an essential requirement for scission of caveolae and the resultant transendothelial transport of albumin.
Shajahan
Ayesha N
AN
Department of Pharmacology, University of Illinois, College of Medicine, M/C 868, 835 S. Wolcott Avenue, Chicago, IL 60612, USA.
Timblin
Barbara K
BK
Sandoval
Raudel
R
Tiruppathi
Chinnaswamy
C
Malik
Asrar B
AB
Minshall
Richard D
RD
D000418:Albumins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000378:metabolism; D034285:Dynamin II / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D008833:Microcirculation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D021381:Protein Transport; D051381:Rats; D012795:Sialoglycoproteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D019061:src-Family Kinases / Q000378:metabolism*
D000418:Albumins; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D005456:Fluorescent Dyes; D012795:Sialoglycoproteins; C065610:sialoglycoprotein gp60; D002772:Cholera Toxin; D019061:src-Family Kinases; D034285:Dynamin II; D034281:Dynamins