Phosphorylation of Caveolin‐1 Tyrosine 14 Leads to Caveolar Coat Destablilzation, Membrane Invagination and Endocytosis

Abstract

Caveolin 1 (Cav-1) is a cholesterol-binding membrane-associated protein required for caveolae formation and transcytosis of macromolecules through the endothelial barrier. Studies from our lab showed that activation of Src via albumin-binding protein gp60, Gβγ or NO leads to phosphorylation of Cav-1 tyrosine 14 and an increase in Cav-1-mediated endocytosis. Here we show that Y14-Cav-1 phosphorylation leads to destabilization of high molecular weight (HMW) Cav-1 oligomers. Using cell fractionation we see a decrease in HMW Cav-1 (> 250 kD) and a corresponding increase in phosphorylated Cav-1 monomers (~ 25 kD) in cells stimulated with albumin or NO. A similar decrease was observed in cells with phospho-mimicking (Y14D), and abolished in cells with phospho-defective (Y14F) mutants of Cav-1. Also Y14D-Cav-1-GFP positive vesicles tracked by TIRF and spinning-disc confocal microscopy were greater in number, velocity and volume when compared to Y14F. When wt Cav-1-CFP- and -YFP were co-transfected in HEK cells, FRET was detected indicative of their close proximity within caveolar coat. Upon stimulation of caveolae-mediated endocytosis with albumin or NO, FRET signal associated with wt Cav-1 rapidly and transiently decreased (~1-3 min) whereas Y14F-Cav-1-FRET was unaffected. We hypothesize that Src-dependent phosphorylation of Cav-1 tyrosine 14 promotes the destabilization of Cav-1 oligomers due to repulsion of neighboring, negatively charged N-terminal p-Y14 residues and this promotes membrane invagination and endocytosis. Support NIH grants T32 HL07239, P01 HL60678 and R01 HL71626