Anna Filipek

CacyBP/SIP, a Calcyclin and Siah-1-interacting Protein, Binds EF-hand Proteins of the S100 Family

Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating β-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915–926). In murine brain, CacyBP/SIP is expressed at a high level, but S100A6 is expressed at a very low level. Consequently we carried out experiments to determine if CacyBP/SIP binds to other S100 proteins in this tissue. Using CacyBP/SIP affinity chromatography, we found that S100B from the brain extract binds to CacyBP/SIP in a Ca2+-dependent manner. Using a nitrocellulose overlay assay with 125I-CacyBP/SIP and CacyBP/SIP affinity chromatography, we found that this protein binds purified S100A1, S100A6, S100A12, S100B, and S100P but not S100A4, calbindin D9k, parvalbumin, and calmodulin. The interaction of S100 proteins with CacyBP/SIP occurs via its C-terminal fragment (residues 155–229). Co-immunoprecipitation of CacyBP/SIP with S100B from brain and with S100A6 from Ehrlich ascites tumor cells suggests that these interactions are physiologically relevant and that the ubiquitinylation complex involving CacyBP/SIP might be regulated by S100 proteins.

S100A6 - New facts and features

S100A6 (calcyclin) is a 10.5kDa Ca(2+)-binding protein that belongs to the S100 protein family. S100A6 contains two EF-hand motifs responsible for binding of Ca(2+). It also binds Zn(2+) through not yet identified structures. Binding of Ca(2+) induces a conformational change in the S100A6 molecule which in consequence increases its overall hydrophobicity and allows for interaction with target proteins. S100A6 was found in different mammalian and avian (chicken) tissues. A high level of S100A6 is observed in epithelial cells, fibroblasts and in different kinds of cancer cells. The function of S100A6 is not clear at present, but it has been suggested that it may be involved in cell proliferation, cytoskeletal dynamics and tumorigenesis. Additionally, S100A6 might have some extracellular activities. This review presents new facts and features concerning the S100A6 protein.

Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcyclin.

Abstract: A protein target of mouse calcyclin, p30, which we call calcyclin‐binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4‐kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of ∼26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.

Tau Protein Modifications and Interactions: Their Role in Function and Dysfunction

Tau protein is abundant in the central nervous system and involved in microtubule assembly and stabilization. It is predominantly associated with axonal microtubules and present at lower level in dendrites where it is engaged in signaling functions. Post-translational modifications of tau and its interaction with several proteins play an important regulatory role in the physiology of tau. As a consequence of abnormal modifications and expression, tau is redistributed from neuronal processes to the soma and forms toxic oligomers or aggregated deposits. The accumulation of tau protein is increasingly recognized as the neuropathological hallmark of a number of dementia disorders known as tauopathies. Dysfunction of tau protein may contribute to collapse of cytoskeleton, thereby causing improper anterograde and retrograde movement of motor proteins and their cargos on microtubules. These disturbances in intraneuronal signaling may compromise synaptic transmission as well as trophic support mechanisms in neurons.

Ca2+-dependent Translocation of the Calcyclin-binding Protein in Neurons and Neuroblastoma NB-2a Cells

The calcyclin-binding protein (CacyBP) binds calcyclin (S100A6) at physiological levels of [Ca2+] and is highly expressed in brain neurons. Subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca2+] i . Immunostaining indicates that CacyBP is present in the cytoplasm of unstimulated cultured neurons in which resting [Ca2+] i is known to be ∼50 nm. When [Ca2+] i was increased to above 300 nm by KCl treatment, the immunostaining was mainly apparent as a ring around the nucleus. Such perinuclear localization of CacyBP was observed in untreated neuroblastoma NB-2a cells in which [Ca2+] i is ∼120 nm. An additional increase in [Ca2+] i to above 300 nm by thapsigargin treatment did not change CacyBP localization. However, when [Ca2+] i in NB-2a cells dropped to 70 nm, because of BAPTA/AM treatment, perinuclear localization was diminished. Ca2+-induced translocation of CacyBP was confirmed by immunogold electron microscopy and by fluorescence of NB-2a cells transfected with an EGFP-CacyBP vector. Recombinant CacyBP can be phosphorylated by protein kinase C in vitro. In untreated neuroblastoma NB-2a cells, CacyBP is phosphorylated on a serine residue(s), but exists in the dephosphorylated form in BAPTA/AM-treated cells. Thus, phosphorylation of CacyBP occurs in the same [Ca2+] i range that leads to its perinuclear translocation.

Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: a novel mode for S100 protein target recognition.

S100A6 is a member of the S100 subfamily of EF-hand Ca (2+) binding proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP or SIP), a subunit of an SCF-like E3 ubiquitin ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal S100A6 binding region in SIP was mapped to a 31-residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189-219) complex revealed that SIP(189-219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structure-based SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions.

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2+‐binding. The data suggest that Ca2+‐ and Zn2+‐ might trigger the biological activity of calcyclin.

Calcyclin (S100A6) Binding Protein (CacyBP) Is Highly Expressed in Brain Neurons

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0.34 ng/μg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein.

Calcyclin--Ca(2+)-binding protein homologous to glial S-100 beta is present in neurones.

The aim of this work was to confirm the presence of calcylin in brain and to identify calcyclin positive cells. Calcyclin was identified in brain soluble proteins that were bound to phenyl-sepharose in a calcium dependent fashion. Specific antibodies against calcylin were found to stain subsets of brain neurones such as neurones in Ammons horn of hippocampus, granule cells in cerebellum, and neurones in brain stem. Glial cells which contain large amounts of S-100 beta protein were calcyclin negative. These results indicate that calcyclin is present in neurones, but not in glial cells.

Tissue specific distribution of calcyclin — 10.5 kDa Ca2+ -binding protein

Expression of calcyclin in different cell lines and mouse tissues was determined with polyclonal antibodies raised against calcyclin from Ehrlich ascites tumour (EAT) cells. The protein was detected in mouse skeletal and cardiac muscle, in lung, kidney and spleen, and was especially enriched in mouse smooth muscle as well as in rat fibroblasts. No positive immunological reaction was detected in mouse brain, liver and intestine and some tumourigenic cell lines. The level of calcyclin mRNA found in different cells and tissues corresponded well to the calcyclin level estimated by immunoblotting. The calcyclin‐like protein was purified from mouse stomach and appeared to be very similar to the EAT protein.