The Membrane-spanning Domains of Caveolins-1 and -2 Mediate the Formation of Caveolin Hetero-oligomers

In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core ␤-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density gradient ultracentrifugation. Caveolin-1 may modify the biosynthetic pathway of sugar chains via the regulation of the intra-Golgi subcompartment localization of this key glycosyltransferase. N-Acetylglucosaminyltransferase III (GnT-III)1 is one of the Golgi-resident glycosyltransferases that catalyzes the transfer of GlcNAc from an UDP-GlcNAc to a core ␤-mannosyl residue of asparagine-linked glycans (N-glycans) via a ␤1,4-linkage, resulting in the formation of a unique structure, a bisecting GlcNAc (1). Oligosaccharides containing a bisecting GlcNAc (bisected oligosaccharides) do not serve as substrates for GnT-IV and GnT-V, which catalyze the formation of a ␤1,4-branch at an ␣1,3Man and a ␤1,6-branch at an ␣1,6Man, respectively (2, 3). We previously reported that the action of GnT-V was strictly inhibited by the addition of a bisecting GlcNAc at the catalytic step (4). Therefore, the action of GnT-III prior to these enzymes in the Golgi leads to an inhibition of the biosynthesis of multiantennary oligosaccharides. Indeed, the ectopic expression of GnT-III caused an effective reduction in multiantennary oligosaccharides in various types of cells (5, 6). For example, in the case of swine endothelial cells, the expression of GnT-III led to a dramatic decrease in multiantennary oligosaccharides, accompanied by a marked increase in bisected biantennary ones (6). However, there are exceptions, in which a high GnT-III activity did not lead to a decrease in branching. In the case of human hepatoma cells, the overexpression of GnT-III resulted in a dramatic increase in bisected oligosaccharides but did not cause a decrease of the number of antenna (7). It would be expected that GnT-IV and GnT-V act prior to GnT-III in Huh6 and HepG2 cells, because the activities of GnT-III, GnT-IV, and Gn...

Section: Resultssupporting

confidence: 78%

Caveolin-1 Regulates the Functional Localization of N-Acetylglucosaminyltransferase III within the Golgi Apparatus

Sasai¹,

Ikeda

Ihara

et al. 2003

“…The majority of caveolin-1 is found at the cell surface and associated with caveolae (23,31). Some Golgi-associated caveolin-1 is in transit from its site of synthesis in the endoplasmic reticulum to the cell surface (32).…”

Section: Phosphocaveolin-1 Is Co-localized With Focal Complex Moleculmentioning

confidence: 99%

Loss of Caveolin-1 Polarity Impedes Endothelial Cell Polarization and Directional Movement

Beardsley

Fang

Mertz

et al. 2005

137

140

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin ␤ 1 and FAK staining. When cells were migrating, integrin ␤ 1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.

“…This oligomerization event occurs right after or during the synthesis of caveolins at the level of the ER. Although the exact mechanism of how these proteins form oligomeric complexes remains unknown, the interaction between the membrane spanning domains of caveolin-2 and caveolin-1 is known to be required for hetero-oligomer assembly (31). Once these hetero-oligomers reach the transGolgi, they become resistant to Triton X-100 solubilization due to their incorporation into lipid rafts (14).…”

Section: Figmentioning

confidence: 99%

Src-induced Phosphorylation of Caveolin-2 on Tyrosine 19

Lee

Park

Wang

et al. 2002

Self Cite

Caveolin-2 is the least well studied member of the caveolin gene family. It is believed that caveolin-2 is an "accessory protein" that functions in conjunction with caveolin-1. At the level of the ER, caveolin-2 interacts with caveolin-1 to form a high molecular mass heterooligomeric complex that is targeted to lipid rafts and drives the formation of caveolae. However, caveolin-2 is not required for caveolae formation, implying that it may fulfill some unknown regulatory role. Here, we present the first evidence that caveolin-2 is a phosphoprotein. We show that caveolin-2 undergoes Src-induced phosphorylation on tyrosine 19. To study this phosphorylation event in vivo, we generated a novel phospho-specific antibody probe that only recognizes phosphocaveolin-2 (Tyr(P) 19 ). We then used NIH-3T3 cells stably overexpressing c-Src to examine the localization and biochemical properties of phosphocaveolin-2 (Tyr(P) 19 ). Our results indicate that phosphocaveolin-2 (Tyr(P) 19 ) is localized near focal adhesions, remains associated with lipid rafts/caveolae, but no longer forms a high molecular mass hetero-oligomer with caveolin-1. Instead, phosphocaveolin-2 (Tyr(P) 19 ) behaves as a monomer/dimer in velocity gradients. Thus, we conclude that the tyrosine phosphorylation of caveolin-2 (Tyr(P) 19 ) may function as a signal that is recognized by the cellular machinery to induce the dissociation of caveolin-2 from caveolin-1 oligomers. We also demonstrate that (i) insulin-stimulation of adipocytes and (ii) integrin ligation of endothelial cells can both induce the tyrosine phosphorylation of caveolin-2 (Tyr(P) 19 ). During integrin ligation, phosphocaveolin-2 (Tyr(P) 19 ) co-localizes with activated FAK at focal adhesions. Thus, phosphocaveolin-2 (Tyr(P) 19 ) may function as a docking site for Src homology domain-2 (SH2) domain containing proteins during signal transduction. In support of this notion, we identify several SH2 domain containing proteins, namely c-Src, NCK, and Ras-GAP, that interact with caveolin-2 in a phosphorylation-dependent manner. Furthermore, our co-immunoprecipitation experiments show that caveolin-2 and Ras-GAP are constitutively associated in c-Src expressing NIH-3T3 cells, but not in untransfected NIH-3T3 cells.Receptor tyrosine kinases (RTKs) 1 contain one or more autophosphorylation sites. Upon ligand binding, a specific receptor is activated and undergoes autophosphorylation on specific tyrosine residues. One known function for tyrosine phosphorylation is to confer docking sites for Src homology domain-2 (SH2)-containing proteins. Numerous downstream signaling molecules possessing either intrinsic kinase activity or an adapter function are then recruited to the plasma membrane and utilize their SH2 domains to form hetero-oligomeric signaling complexes with activated RTKs.Numerous RTKs, such as epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R), and insulin receptor (Ins-R), as well as non-receptor tyrosine kinases (NRTKs; Src-family tyrosine kinases) are ...