Serine 23 and 36 phosphorylation of caveolin-2 is differentially regulated by targeting to lipid raft/caveolae and in mitotic endothelial cells.
In the present study, using a combination of reconstituted systems and endothelial cells endogenously expressing caveolins, we show that phosphorylation of caveolin-2 at serines 23 and 36 can be differentially regulated by caveolin-1 mediated subcellular targeting to lipid raft/caveolae and in endothelial cells synchronized in mitosis. Detergent insolubility and sucrose flotation gradient experiments revealed that serine 23 phosphorylation of caveolin-2 preferably occurs in detergent-resistant membranes (DRMs), while serine 36 phosphorylation takes place in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of caveolin-1, serine 23-phosphorylated caveolin-2 mostly localizes to plasma membrane, while serine 36-phosphorylated caveolin-2 primarily resides in intracellular compartments. To directly address the role of caveolin-1 in regulating phosphorylation of endogenous caveolin-2, we have used the siRNA approach. The specific knockdown of caveolin-1 in endothelial cells decreases caveolin-2 phosphorylation at serine 23 but not at serine 36. Thus, upregulation of serine 23 phosphorylation of caveolin-2 depends on caveolin-1-driven targeting to plasma membrane lipid rafts and caveolae. Interestingly, although serine 36 phosphorylation does not seem to be regulated in endothelial cells by caveolin-1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36-phosphorylated caveolin-2 in modulating mitosis.
Sowa
Grzegorz
G
Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65212, USA. sowag@health.missouri.edu
Xie
Leike
L
Xu
Li
L
Sessa
William C
WC
D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051243:Caveolin 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008938:Mitosis / Q000502:physiology; D010766:Phosphorylation; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D051242:Caveolin 1; D051243:Caveolin 2; D003902:Detergents; D034741:RNA, Small Interfering; D012694:Serine