Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target

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Highlights

  • FOR20 is identified as a novel S100A6 target by screening protein microarrays.

  • S100A6 interacts with FOR20 in a Ca2+-dependent manner in vitro and in living cells.

  • S100A6 interacts with FOR20-related centrosomal proteins, FOP and OFD1.

Abstract

S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1–30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, A11, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1–30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function.

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.

Section snippets

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)

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