Characterization of the metastasis-associated protein, S100A4. Roles of calcium binding and dimerization in cellular localization and interaction with myosin.
Elevated S100A4 protein expression is associated with metastatic tumor progression and appears to be a strong molecular marker for clinical prognosis. S100A4 is a calcium-binding protein that is known to form homodimers and interacts with several proteins in a calcium-dependent manner. Here we show that S100A4 localizes to lamellipodia structures in a migrating breast cancer-derived cell line and colocalizes with a known S100A4-interacting protein, myosin heavy chain IIA, at the leading edge. We demonstrate that S100A4 mutants that are defective in either their ability to dimerize or in calcium binding are unable to interact with myosin heavy chain IIA. An S100A4 mutant that is deficient for calcium binding retains the ability to form homodimers, suggesting that S100A4 can exist as calcium-free or calcium-bound dimers in vivo. However, a calcium-bound S100A4 monomer only interacts with another calcium-bound monomer and not with an S100A4 mutant that does not bind calcium. Interestingly, despite the calcium dependence for interaction with known protein partners, calcium binding is not necessary for localization to lamellipodia. Both wild type and a mutant that is deficient for calcium binding colocalize with known markers of actively forming leading edges of lamellipodia, Arp3 and neuronal Wiskott-Aldrich syndrome protein. These data suggest that S100A4 localizes to the leading edge in a calcium-independent manner, and identification of the proteins that are involved in localizing S100A4 to the lamellipodial structures may provide novel insight into the mechanism by which S100A4 regulates metastasis.
Kim
Edward J
EJ
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Helfman
David M
DM
D051378:Actin-Related Protein 3; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D009362:Neoplasm Metastasis; D011233:Precipitin Tests; D011379:Prognosis; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011554:Pseudopodia / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D051316:Wiskott-Aldrich Syndrome Protein
C495928:ACTR3 protein, human; D051378:Actin-Related Protein 3; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; C077591:S100A4 protein, human; D009218:Myosins; D002118:Calcium