Regulation of Caveolin‐2 Phosphorylation at Serines 23 and 36

Abstract

Previously, we have shown that caveolin-2 (Cav-2) is phosphorylated under basal conditions at N-terminal serine residues 23 and 36 and that phosphorylation of Cav-2 plays a positive role in caveolae assembly in a reconstituted system, LNCaP cells expressing recombinant caveolins. In the present study, we show that serine phosphorylation of Cav-2 also occurs in endothelial cells (ECs) and is regulated by Cav-1 and subcellular location. More specifically, using adenoviral expression and phospho-specific antibodies to Cav-2, we demonstrate that co-expression of Cav-1 increases by ca. 3-fold phosphorylation of Cav-2 at serine residue 23, and decreases by ca. 2-fold phosphorylation of serine 36 in cells expressing Cav-1 and -2, relative to cells expressing Cav-2 alone. Consistent with the latter, the ratio of serine 23 phosphorylated to total Cav-2 protein in human ECs is 7-fold higher than in cells expressing Cav-2 alone, while the respective ratio of serine 36 phosphorylation is 1.5-fold lower. Subcellular fractionation techniques, separating lipid rafts/caveolae, have determined that serine 23 phosphorylation of Cav-2 preferably occurs in lipid rafts/caveolae. Conversely, serine 36 phosphorylation takes place in non-lipid raft/caveolar compartments. Our subcellular fractionation data are consistent with fluorescence microscopic studies on cells co-expressing Cav-1 and -2, which have shown that vast majority of serine 23-phosphorylated Cav-2 is located to plasma membrane, while serine 36 phosphorylated Cav-2 to perinuclear region. In summary, serine phosphorylation of Cav-2 is a regulated process, largely dependent on Cav-1-driven translocation of Cav-2 from detergent soluble intracellular compartments to detergent insoluble plasma membrane associated lipid rafts and caveolae.