Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins.
Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.
Gimona
M
M
Cold Spring Harbor Laboratory, NY 11724, USA.
Lando
Z
Z
Dolginov
Y
Y
Vandekerckhove
J
J
Kobayashi
R
R
Sobieszek
A
A
Helfman
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015415:Biomarkers; D001717:Birds; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D009418:S100 Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014335:Tropomyosin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D003432:Cross-Linking Reagents; C030564:S-100 calcium-binding protein alpha subunit; D009418:S100 Proteins; D014335:Tropomyosin; D002118:Calcium