Identification of a novel interaction between the Ca2+-binding protein S100A11 and the Ca2+- and phospholipid-binding protein annexin A6

Abstract

S100A11 is a member of the S100 family of EF-hand Ca2+-binding proteins, which is expressed in smooth muscle and other tissues. Ca2+ binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca2+-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca2+-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH2 terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH2- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca2+] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma.