Biochemical and Biophysical Research Communications
Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target
Introduction
S100A6 (also known as calcyclin), a member of the S100 family of EF-hand calcium-binding proteins [1], [2], was first discovered as the growth-regulated gene, 2A9. In quiescent fibroblasts, S100A6 mRNA levels are increased by treatment with serum, PDGF, and EGF [3]. The physiological functions of S100A6 were thought to be mediated by interactions with its target proteins [4]. Previous studies using biochemical approaches identified a number of intracellular target proteins for S100A6 including glyceraldehyde-3-phosphate dehydrogenase, annexin II, annexin VI [5], annexin XI [calcyclin-associated protein 50kDa (CAP-50)] [6], caldesmon [7], tropomyosin [8], calponin, CacyBP/SIP (calcyclin-binding protein and siah-1-Interacting Protein) [9], [10], Hsp70/Hsp90-organizing protein, kinesin light chain, translocase of outer mitochondrial membrane 70 [11], protein phosphatase 5 (PP5) [12], FK506-binding protein 38 [13], and C-terminus of Hsc70-interacting protein [14], consistent with its primarily cytoplasmic location. However, the extracellular localization of S100A6 was also shown to be important for modulating the viability of neuroblastoma cell line SH-SY5Y via interactions with the receptor for advanced glycation end products, RAGE [15]. Although many S100A6 target proteins have been identified, only a single structure of the S100A6 complex, which contains a 31-residue CacyBP/SIP fragment (Ser189–Arg219), has been resolved by NMR spectroscopy [16]. Therefore, a consensus primary binding sequence has not been reported unlike for calmodulin (CaM), a major eukaryotic Ca2+-dependent signal transducer. Structural studies have revealed many types of interactions between CaM and its target proteins [17], which can be distinguished from interactions between S100A6 and its targets: The Ca2+-bound form of CaM binds to an amphipathic alpha helix with two anchoring hydrophobic residues that span 10 to 16 amino acid residues of some CaM targets, including CaM-dependent protein kinases [18]. Recently, a tricopeptide repeat (TPR) domain was identified as a common feature of the Ca2+-dependent S100A6-binding regions of PP5 [12], Hsp70/Hsp90-organizing protein, kinesin light chain, and translocase of outer mitochondrial membrane 70 [11]. The growing number of S100A6 target proteins may indicate the increasing number of physiological functions ascribed to S100A6 and the existence of novel Ca2+/S100A6-dependent signaling pathways. Recently, we developed a novel genome-wide screening method for the identification of CaM-binding targets using the Protein Active Array® (Cell Free Sciences Co., Ltd, Ehime, Japan) carrying 19,676 recombinant GST-fusion proteins. The use of this method resulted in the identification of novel human CaM targets including the striated muscle activator of Rho signaling and the Lhx2 transcription factor [19]. In this report, we screened 19,676 human full-length cDNA-derived GST-fusion proteins using a biotinylated S100A6-binding assay and discovered FOR20 (FOP-related protein of 20 kDa) as a novel S100A6-binding protein. We further characterized the biochemical interaction between S100A6 and FOR20 in vitro and in living cells.
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Materials
S100 proteins (S100A1, A2, A4, A6, A11, A12, and B) were expressed in Escherichia coli BL21 (DE3) using the pET vector and purified as described previously [11](Supplemental Data Fig. S2). GST-PP5 was expressed and purified as described previously [12]. Recombinant S100A6 was biotinylated with biotinoyl-ε-aminocaproic acid N-hydroxysuccinimide ester followed by purification using PD-10 chromatography. Human FOR20 cDNA (NM_144600) was obtained by PCR using HeLa cell cDNA as a template with a
Screening of S100A6 targets with human protein array
To identify the S100A6 target proteins, we screened a protein array system containing 19,676 human full-length cDNA-derived recombinant GST-fusion proteins with biotinylated S100A6. Quantitative analysis of biotinylated S100A6 showed that subnanogram levels of the protein were readily detectable using an avidin-HRP-mediated ECL method (Fig. 1A). We then incubated the human protein array first with 0.36 μg/mL biotinylated S100A6 in the presence of 2 mM CaCl2 and then with avidin-HRP, followed by
Discussion
In this report, we identified FOR20 as a novel S100A6-binding protein by screening human protein arrays with a biotinylated S100A6 probe. This interactome approach with high-throughput and high-sensitivity, which we used to identify novel human CaM-binding proteins [19], has helped identify other S100 target proteins. FOR20 has been identified as a centrosomal protein, which may play a role in primary cilium formation although the regulatory mechanism of FOR20 is still unclear [22]. This study
Funding
This research is partially supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Japan Agency for Medical Research and Development (to MD and RM).
Conflict of interest
The authors declare that they have no conflicts of interest.
References (28)
- et al.
S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature)
Biochem. Biophys. Res. Commun.
(2004) - et al.
Binding of S100 proteins to RAGE: an update
Biochimica Biophysica Acta-Molecular Cell Res.
(2009) - et al.
Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein
J. Biol. Chem.
(1986) - et al.
S100A6-new facts and features
Biochem. Biophys. Res. Commun.
(2009) - et al.
Identification of annexin II, annexin VI and glyceraldehyde-3-phosphate dehydrogenase as calcyclin-binding proteins in bovine heart
Int. J. Biochem.
(1993) - et al.
A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family
J. Biol. Chem.
(1992) - et al.
Chicken gizzard calcyclin–distribution and potential target proteins
Biochem. Biophys. Res. Commun.
(1996) - et al.
Ca2+-dependent binding of calcyclin to muscle tropomyosin
Biochem. Biophys. Res. Commun.
(1996) - et al.
CacyBP/SIP, a calcyclin and Siah-1-interacting protein, binds EF-hand proteins of the S100 family
J. Bio Chem.
(2002) - et al.
Interactions of S100A2 and S100A6 with the tetratricopeptide repeat proteins, Hsp90/Hsp70-organizing protein and kinesin light chain
J. Biol. Chem.
(2008)
S100 proteins modulate protein phosphatase 5 function: a link between Ca2+ signal transduction and protein dephosphorylation
J. Biol. Chem.
Ca2+/S100 proteins act as upstream regulators of the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein)
J. Biol. Chem.
S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains
J. Biol. Chem.
Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen
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2021, Cell CalciumCitation Excerpt :To investigate the multifunctional roles of S100A2, we set up genome-wide protein–protein interaction (PPI) screening using human protein arrays containing 19,676 recombinant human proteins to identify potential S100A2 target proteins. By screening the human protein arrays with biotinylated calmodulin and S100A6, the striated muscle activator of Rho signaling [19] and FOR20 were identified as novel calmodulin- and S100A6-binding proteins [20], respectively, which indicates that genome-wide PPI screening is an effective and time-saving method for comprehensive identification of potential interactants for various Ca2+ mediators. In this report, we identified multiple candidates for S100A2 target proteins through comprehensive PPI screening and characterized TPPP/p25 (tubulin polymerization-promoting protein) as a novel S100A2 target that may be a target for multiple S100 proteins.
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