Abstract
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.
Highlights
S100 proteins are members of the two EF-hand calcium-binding protein families, which are composed of 20 paralogs in humans [1]
By using the biotinylated S100A6 overlay, we confirmed that recombinant GSTHMG20A-His6 was capable of interacting with S100A6 in a Ca2+ -dependent manner, but not glutathione s-transferase (GST)-His6 protein, on the blotted membrane subsequently subjected to SDS-PAGE
We further analyzed the interaction of highmobility protein group 20A (HMG20A) and various S100 family proteins, including S100A6 under native conditions by using a GST-pulldown assay (Figure 1C)
Summary
S100 proteins are members of the two EF-hand calcium-binding protein families, which are composed of 20 paralogs in humans [1]. To comprehensively analyze target proteins of EF-hand Ca2+ -binding proteins, including calmodulin and S100 proteins, we developed a novel genome-wide screening method based on the protein–protein interactions using the Protein Active Array® (CellFree Sciences, Ehime, Japan), carrying 19,676 recombinant glutathione s-transferase (GST)-fusion proteins [17]. The use of this method resulted in the identification of the striated muscle activator of Rho signaling as a calmodulin interactant, and FOR-20 (FOP-related protein of 20 kDa) as a novel S100A6-interacting protein [18]. We further characterized the biochemical interaction between S100A6 and HMG20A in vitro and in living cells
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