Submitted on August 16, 2006
Accepted on December 23, 2006
Identification of a novel interaction between the Ca2+-binding protein S100A11 and the Ca2+- and phospholipid-binding protein annexin A6
Ning Chang1, Cindy Sutherland1, Eva Hesse2, Robert Winkfein2, William B Wiehler2, Mark Pho2, Claude Veillette2, Susan Li2, David P. Wilson3, Eniko Kiss2, and Michael P. Walsh4*
1 Biochemistry & Molecular Biology, University of Calgary, Calgary, Canada
2 Calgary, Canada; Biochemistry & Molecular Biology, University of Calgary, Calgary, Canada
3 School of Molecular & Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia
4 Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Canada; Department of Medical Biochemistry, University of Calgary, 3330 Hospital Drive NW, Calgary, T2N 4N1, Canada
* To whom correspondence should be addressed. E-mail: walsh{at}ucalgary.ca.
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 co-precipitation 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 N-terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both N- and C-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.
