Interaction of S100A6 with Target Proteins In Vitro and in Living Cells

Sakane, Kyohei; Yamaguchi, Fuminori; Tsuchiya, Masaharu; Kondo, Rina; Kanayama, Naoki; Hatano, Naoya; Kobayashi, Ryōji; Tokumitsu, Hiroshi

doi:10.1007/978-1-4939-9030-6_23

Cited by 2 publications

(2 citation statements)

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“…S100 proteins (S100A1, A2, A4, A6, A11, A12, and B) were expressed in Escherichia coli BL21 (DE3) using the pET vector and purified by phenyl-sepharose chromatography, as described previously [11]. Recombinant S100A6 was biotinylated with biotinoyl-εaminocaproic acid N-hydroxysuccinimide ester, followed by purification [19]. Recombinant rat calmodulin was expressed in E. coli BL21 (DE3) using the plasmid pET-calmodulin (kindly provided by Dr. Nobuhiro Hayashi, Tokyo Institute of Technology, Yokohama, Japan) [20].…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, 50 µL of glutathione-sepharose beads (50% slurry) were incubated with the sample, followed by incubation for 1 h. After extensive washing, 50 µL of 1× SDS-PAGE sample buffer was added to the beads, followed by heating at 95 • C for 10 min. Samples (10 µL) were analyzed on a Tris-Tricine-SDS-10% polyacrylamide gel using either Coomassie Brilliant Blue staining or immunoblot analysis [19].…”

Section: Gst Precipitation Assaymentioning

confidence: 99%

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Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

et al. 2021

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During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

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Section: Methodsmentioning

confidence: 99%

Section: Gst Precipitation Assaymentioning

confidence: 99%