Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6.
S100A11 is a member of the S100 family of EF-hand Ca(2+)-binding proteins, which is expressed in smooth muscle and other tissues. Ca(2+) 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 Ca(2+)-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca(2+)-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 NH(2) terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH(2)- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca(2+)] 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.
Chang
Ning
N
Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1.
Sutherland
Cindy
C
Hesse
Eva
E
Winkfein
Robert
R
Wiehler
William B
WB
Pho
Mark
M
Veillette
Claude
C
Li
Susan
S
Wilson
David P
DP
Kiss
Enikõ
E
Walsh
Michael P
MP
D000595:Amino Acid Sequence; D000818:Animals; D017317:Annexin A6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008081:Liposomes; D008297:Male; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D009418:S100 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017317:Annexin A6; D008081:Liposomes; D010743:Phospholipids; D011994:Recombinant Proteins; D009418:S100 Proteins; D002118:Calcium