Abstract
The molecular mechanisms regulating the exit of caveolin from the Golgi complex are not fully understood. Cholesterol and sphingolipid availability affects Golgi vesiculation events and involves the activity of cytoplasmic phospholipase A(2) (cPLA(2)). We recently demonstrated that high expression levels of annexin A6 (AnxA6) perturb the intracellular distribution of cellular cholesterol, thereby inhibiting caveolin export from the Golgi complex. In the present study we show that in Chinese hamster ovary cells overexpressing AnxA6, sequestration of cholesterol in late endosomes, leading to reduced amounts of cholesterol in the Golgi, inhibits cPLA(2) activity and its association with the Golgi complex. This correlates with the blockage of caveolin export from the Golgi in cells treated with methyl arachidonyl fluorophosphonate, a Ca(2+)-dependent cPLA(2) inhibitor. AnxA6-mediated down-regulation of cPLA(2) activity was overcome upon the addition of exogenous cholesterol or transfection with small interfering RNA targeting AnxA6. These findings indicate that AnxA6 interferes with caveolin transport through the inhibition of cPLA(2).
Highlights
Annexins are a family of Ca2ϩ and membrane-binding proteins involved in membrane trafficking and various other processes including signaling, proliferation, differentiation, and inflammation [1,2,3]
We recently demonstrated that high expression levels of annexin A6 (AnxA6) perturb the intracellular distribution of cellular cholesterol, thereby inhibiting caveolin export from the Golgi complex
Annexin A6 Inhibits cytoplasmic phospholipase A2 (cPLA2) ent study we show that overexpression of AnxA6 indirectly inhibits cPLA2 activity and its association with the Golgi complex through the reduction of cholesterol availability
Summary
Reagents and Antibodies—Nutrient Mixture Ham’s F-12, DMEM, cycloheximide, water-soluble cholesterol, and saponin were from Sigma. [3H]Arachidonic acid ([3H]AA) was from Amersham Biosciences. cPLA2 inhibitors E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS), MAFP, and U18666A were from BIOMOL. After washing with lysis buffer, Sepharose-bound GST-AnxA6 (and GST as control) was incubated with 600 g of cell extract for 2 h at 4 °C, and proteins bound to the column were collected by centrifugation and analyzed by Western blotting. Supernatants of transfected or not transfected cells were incubated with rabbit polyclonal anti-GFP or polyclonal anti-annexin A6 antibodies for 2 h at 4 °C and for 60 min after the addition of protein A-Sepharose. Western Blot Analysis—CHOwt, CHOanx, and HeLa cell lysates, samples from isolated Golgi membranes, and GSTAnxA6 pulldown assays were separated by 10% SDS-PAGE and transferred to Immobilon-P (Millipore) followed by incubation with primary antibodies and the appropriate peroxidase-conjugated secondary antibodies and ECL detection (Amersham Biosciences). Protein content was measured by the method of Lowry et al [23]
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