Interaction of S100A6 with Target Proteins In Vitro and in Living Cells
Abstract
S100A6 is a member of the EF-hand Ca2+-binding protein family, which plays important roles in a wide variety of Ca2+ signaling in the cells, as well as in pathophysiological conditions. Herein, we describe analytical protocols for evaluating the interaction of S100A6 with multiple target proteins in vitro, including biotinylated S100A6 overlay, glutathione-S-transferase (GST)-precipitation, surface plasmon resonance, and a GST-precipitation assay in living cells. These methods will elucidate the detailed molecular mechanisms of S100A6/target interactions and further improve our understanding of the physiological significance of S100A6-mediated Ca2+ signaling. Moreover, they may be used to evaluate other physical S100/target interactions.
Key words
S100A6 Ca2+-binding EF-hand Protein–protein interaction FOR20 Protein phosphatase 5 GST-precipitation assay S100A6-overlay Transfection SPR analysisNotes
Acknowledgment
This work was supported in part by a grant (to H.T.) from the Ryobi Teien Memory Foundation, Japan.
References
- 1.Marenholz I, Heizmann CW, Fritz G (2004) S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochem Biophys Res Commun 322:1111–1122CrossRefGoogle Scholar
- 2.Calabretta B, Battini R, Kaczmarek L, de Riel JK, Baserga R (1986) Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein. J Biol Chem 261:12628–12632PubMedGoogle Scholar
- 3.Tokumitsu H, Mizutani A, Minami H, Kobayashi R, Hidaka H (1992) A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family. J Biol Chem 267:8919–8924PubMedGoogle Scholar
- 4.Filipek A, Jastrzebska B, Nowotny M, Kuznicki J (2002) CacyBP/SIP, a calcyclin and Siah-1-interacting protein, binds EF-hand proteins of the S100 family. J Biol Chem 277:28848–28852CrossRefGoogle Scholar
- 5.Leclerc E, Fritz G, Weibel M, Heizmann CW, Galichet A (2007) S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains. J Biol Chem 282:31317–31331CrossRefGoogle Scholar
- 6.Yamaguchi F, Umeda Y, Shimamoto S, Tsuchiya M, Tokumitsu H, Tokuda M, Kobayashi R (2012) S100 proteins modulate protein phosphatase 5 function: a link between Ca2+ signal transduction and protein dephosphorylation. J Biol Chem 287:13787–13798CrossRefGoogle Scholar
- 7.Sakane K, Nishiguchi M, Denda M, Yamagchi F, Magari M, Kanayama N, Morishita R, Tokumitsu H (2017) Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target. Biochem Biophys Res Commun 491:980–985CrossRefGoogle Scholar
- 8.Billingsley ML, Pennypacker KR, Hoover CG, Brigati DJ, Kincaid RL (1985) A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin. Proc Natl Acad Sci U S A 82:7585–7589CrossRefGoogle Scholar
- 9.Tokumitsu H, Kobayashi R, Hidaka H (1991) A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding-proteins. Arch Biochem Biophys 288:202–207CrossRefGoogle Scholar
- 10.Santamaria-Kisiel L, Rintala-Dempsey AC, Shaw GS (2006) Calcium-dependent and -independent interactions of the S100 protein family. Biochem J 396:201–214CrossRefGoogle Scholar