1. Role of Src-induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells.
- Author
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Shajahan AN, Timblin BK, Sandoval R, Tiruppathi C, Malik AB, and Minshall RD
- Subjects
- Albumins metabolism, Animals, Biological Transport, Blotting, Western, Caveolin 1, Caveolins metabolism, Cells, Cultured, Cholera Toxin metabolism, Dynamins metabolism, Endocytosis, Endothelium, Vascular metabolism, Fluorescent Dyes pharmacology, Microcirculation, Microscopy, Confocal, Microscopy, Fluorescence, Mutation, Phosphorylation, Plasmids metabolism, Precipitin Tests, Protein Transport, Rats, Sialoglycoproteins metabolism, Subcellular Fractions metabolism, Time Factors, Transfection, Dynamin II metabolism, Endothelial Cells metabolism, src-Family Kinases metabolism
- Abstract
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.
- Published
- 2004
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