Annexin 2 binding to phosphatidylinositol 4,5-bisphosphate on endocytic vesicles is regulated by the stress response pathway.
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.
Hayes
Matthew J
MJ
Division of Cell Biology, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, United Kingdom.
Merrifield
Christien J
CJ
Shao
Dongmin
D
Ayala-Sanmartin
Jesus
J
Schorey
Crislyn D'Souza
CD
Levine
Tim P
TP
Proust
Jezabel
J
Curran
Julie
J
Bailly
Maryse
M
Moss
Stephen E
SE
D000199:Actins / Q000378:metabolism; D000818:Animals; D017306:Annexin A2 / Q000378:metabolism*; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000502:physiology; D015471:Leukemia, Basophilic, Acute; D009997:Osmotic Pressure; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010873:Pinocytosis / Q000502:physiology; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D022161:Transport Vesicles / Q000378:metabolism*
D000199:Actins; D017306:Annexin A2; D019269:Phosphatidylinositol 4,5-Diphosphate