Abstract
Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.
Highlights
Annexin 2 is a Ca2؉-binding protein that has an essential role in actin-dependent macropinosome motility
Annexin 2-green fluorescent protein (GFP) and anti-PtdIns-4,5P2 IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P2 synthesis on pinosomes through activation of phosphatidylinositol 5 kinase
Such a role would fit with the observations that annexin 2 is a constituent of the actin tails that propel macropinosomes in rat basophilic leukemia (RBL)1 cells, it is enriched at the point of contact between the actin tail and the vesicle [6]
Summary
Labeling Macropinosomes with Fluorescently Labeled IgE—Alexa568 (Molecular Probes) was conjugated to anti-2,4-dinitrophenol IgE (Sigma) according to the manufacturers’ instructions and stored at Ϫ80 °C. Adherent RBL cells cultured as described previously [10] were chilled to 4 °C in HEPES-buffered saline (HBS). Coverslips were washed three times in PBS, per-. Coverslips were washed again in PBS before incubation with tertiary probes for 1 h at 37 °C in a moist chamber. Coverslips were washed a further three times and mounted in 90% glycerol, 10% PBS, 0.01% n-propylgallate. Cells were fixed and stained using FITC-phalloidin (Sigma) prior to mounting. In experiments to examine the pharmacology of rocketing, potential inhibitors and activators were added to cells 20 min prior to stimulation and maintained at the same concentration throughout stimulation. DNA Constructs—The annexin 2-GFP and GFP-actin fusion constructs have been described previously [6, 10], as have constructs expressing the constitutively active (Q67L) and dominant negative (T27N) mutants of Arf6 [16]
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