Role of Src-induced Dynamin-2 Phosphorylation in Caveolae-mediated Endocytosis in Endothelial Cells

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 caveolaemediated 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 kinasedefective 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.

Phosphatidylinositol 3-Kinase and Xanthine Oxidase Regulate Nitric Oxide and Reactive Oxygen Species Productions by Apoptotic Lymphocyte Microparticles in Endothelial Cells

“…In the present study, we observed an increase of caveolin-1 expression in MP-treated cells, which was associated with a decrease of eNOS activity. Caveolin-1 is phosphorylated on Tyr 14 by Src family kinases (36 -38), which leads to caveolae fission (39). Here, we reported that MPs increased caveolin-1 expression and decreased its phosphorylation on Tyr 14 by a mechanism insensitive to PI3K and MAPK inhibitors, while the NO production is decreased as reported above.…”

Src kinase regulates the integrity and function of the Golgi apparatus via activation of dynamin 2

“…While tyrosine phosphorylation has been observed in response to various ligands (13)(14)(15)(25)(26)(27)(28), this event is thought to play a role in the regulation of ligand-induced uptake of various receptors. However, the mechanisms involved in regulating Dyn2 function during secretion and, more specifically, whether Src kinase-mediated phosphorylation plays a role in this process have remained largely unexplored.…”

“…Furthermore, there appears to be a selective dynamin dependence for apical vs. basolateral membrane targeted cargo in polarized cell models, where other proteins including CtBP1/BARS have been reported to play a role (10)(11)(12). Moreover, Dyn2 is a substrate of Src and has been shown to be phosphorylated by this kinase during the activation and subsequent endocytic internalization of specific membrane receptors (13)(14)(15). In this study, we provide evidence that Src kinase, through regulation of Dyn2, acts to control Golgi dynamics in both normal and neoplastic cells as well as vesiculation of the TGN during the secretory process.…”

Ethanol‐induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity

“…Because Dyn2 is phosphorylated by an active Src kinase (pSrc) to achieve its biologically active form (pDyn2),16, 34 we quantified the total and phosphorylated forms of Src and Dyn2 in liver homogenates. As shown in Figure 1A, chronic EtOH administration did not affect the total content of either protein (bottom band in each panel).…”

“…Once phosphorylated, Dyn2 catalyzes constriction and scission of endocytic vesicles at the plasma membrane, thereby releasing early endosomes to the cell interior 11, 12, 13. The Src‐kinase is a nonreceptor protein tyrosine kinase that is activated during stress conditions and regulates cytoskeletal‐dependent membrane trafficking by phosphorylation of specific substrates, including Dyn2 14, 15, 16, 17. Inhibition of Dyn2 by small molecule inhibitors impairs lipophagy‐dependent LD breakdown in hepatic cells.…”