Calcium-regulated Interaction of Sgt1 with S100A6 (Calcyclin) and Other S100 Proteins

Abstract

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of ␤-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins. S100 proteins are a family of small, highly homologous proteins that contain two calcium-binding EF-hand motifs (reviewed in Ref. 1). Because the binding of calcium ions is the most important biochemical feature of S100 proteins, they are thought to function as sensors of calcium ion concentration in the cell. They appear to have several functions, including regulation of protein phosphorylation, cytoskeleton polymerization, and cell cycle progression. S100A6 (calcyclin) is a typical member of the S100 family. The function of S100A6 remains unclear, but evidence suggests that it is involved in cell cycle regulation (2, 3) and exocytosis (4,5). S100A6 may also be involved in tumorigenesis; the protein is overexpressed in several tumors, e.g. melanoma (6).Attempts to elucidate the function of S100A6 have involved the search for its calcium-regulated target proteins. Several such proteins have been identified: glyceraldehyde-3-phosphate dehydrogenase (7), annexin II (8), annexin VI (9), and annexin XI (10). In 1996, a novel mouse protein was identified on the basis of its ability to interact with S100A6 in a calciumdependent manner (11). It was named Calcyclin-Binding Protein (CacyBP) 1 (12). In vitro characterization of the interaction between S100A6 and CacyBP showed that the S100A6-binding domain in CacyBP is comprised of 52 C-terminal ...

Up-regulation, Modification, and Translocation of S100A6 Induced by Exposure to Ionizing Radiation Revealed by Proteomics Profiling

“…The biological functions of S100A6 remains obscure, but some conclusions can be drawn from studying its interacting proteins. The S100A6 interaction with Sgt1, involved in ubiquitin-mediated protein degradation of the known oncogene ␤-catenin, suggests S100A6 involvement in regulation of cell proliferation (23,24). S100A6 also seems to play a key part in other cellular processes such as tropomyosin-related cytoskeleton rearrangements as indicated by interaction and antisense studies (25,26).…”

Calcyclin, a Ca2+ Ion-binding Protein, Contributes to the Anabolic Effects of Simvastatin on Bone

“…But, according to a recent report, S100A6 and other S100 proteins, through their interaction with ubiquitin ligases, can regulate the ubiquitination of ␤-catenin in vivo and thus participate in the process of tumor development and pro- gression (34). Activation of endogenous ␤-catenin signaling in the canonical pathway of wnt induces the expression of alkaline phosphatase and subsequent osteoblast differentiation and, further, participates in BMP-2-mediated signal transduction (35,36).…”

Human Sgt1 Binds HSP90 through the CHORD-Sgt1 Domain and Not the Tetratricopeptide Repeat Domain

“…One such example is the CS domain of SIP, another protein that is part of an E3 protein ubiquitination complex (6,30). Sgt1 and SIP are homologous not only in their CS domains but also in their C-terminal domains, which bind S100 proteins (8,29). Consequently, a homology model was constructed of the CS domain from SIP based on the structure of Sgt-CS domain.…”

“…TPR domains are known as heat shock protein binding domains. The SGS domain was shown to interact with S100 calcium-binding proteins (8). The CS domain has high sequence homology with p23 and is also known as a p23-like domain (9).…”