S100A1 Is a Novel Molecular Chaperone and a Member of the Hsp70/Hsp90 Multichaperone Complex

Journal of Biological Chemistry

Takata

Tokuda

et al. 2008

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S100A2 and S100A6 interact with several target proteins in a Ca 2؉-regulated manner. However, the exact intracellular roles of the S100 proteins are unclear. In this study we identified Hsp70/ Hsp90-organizing protein (Hop) and kinesin light chain (KLC) as novel targets of S100A2 and S100A6. Hop directly associates with Hsp70 and Hsp90 through the tetratricopeptide (TPR) domains and regulates Hop-Hsp70 and Hop-Hsp90 complex formation. We have found that S100A2 and S100A6 bind to the TPR domain of Hop, resulting in inhibition of the Hop-Hsp70 and Hop-Hsp90 interactions in vitro. Although endogenous Hsp70 and Hsp90 interact with Hop in resting Cos-7 cells, but not with S100A6, stimulation of these cells with ionomycin caused a Hop-S100A6 interaction, resulting in the dissociation of Hsp70 and Hsp90 from Hop. Similarly, glutathione S-transferase pulldown and co-immunoprecipitation experiments revealed that S100A6 binds to the TPR domain of KLC, resulting in inhibition of the KLC-c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1) interaction in vitro. The transiently expressed JIP-1 interacts with KLC in resting Cos-7 cells but not with S100A6. Stimulation of these cells with ionomycin also caused a KLC-S100A6 interaction, resulting in dissociation of JIP-1 from KLC. These results strongly suggest that the S100 proteins modulate Hsp70-Hop-Hsp90 multichaperone complex formation and KLC-cargo interaction via Ca 2؉ -dependent S100 protein-TPR protein complex formation in vivo as well as in vitro. Moreover, we have shown that S100A2 and S100A6 interact with another TPR protein Tom70 and regulate the Tom70-ligand interaction in vitro. Thus, our findings suggest a new intracellular Ca 2؉

Section: Methodsmentioning

confidence: 99%

Section: ؉mentioning

confidence: 99%

Section: Interaction Between Hop and The Members Of The S100 Proteinmentioning

confidence: 99%

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Interactions of S100A2 and S100A6 with the Tetratricopeptide Repeat Proteins, Hsp90/Hsp70-organizing Protein and Kinesin Light Chain

Journal of Biological Chemistry

Takata

Tokuda

et al. 2008

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“…Histidine-tagged PP5 (His-PP5) protein was expressed and purified according to the manufacturer's instructions. S100A1 and S100A4 proteins were expressed and purified as previously described (22,23). Purified recombinant S100A4 proteins were diluted (0.5 mg/ml) and maintained at -30˚C in a freezer for 3 months (air-oxidized).…”

Section: Methodsmentioning

confidence: 99%

Oxidized S100A4 inhibits the activation of protein phosphatase 5 through S100A1 in MKN-45 gastric carcinoma cells

Tsuchiya

Yamaguchi

International Journal of Molecular Medicine

et al. 2014

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Abstract. S100 proteins bind to numerous target proteins, as well as other S100 proteins and activate signaling cascades. S100 proteins can be modified by various post-translational modifications, such as phosphorylation, methylation and acetylation. In addition, oxidation is important for modulating their activities. Previous studies have shown that S100A1 interacts with S100A4 in vitro and in vivo. Due to this potential cross-talk among the S100 proteins, the aim of the present study was to examine whether S100A4 modulates the activity of S100A1. S100A4 was readily oxidized and formed disulfide-linked dimers and oligomers. Although non-oxidized S100A4 bound to protein phosphatase 5 (PP5), the Cu-oxidized S100A4 failed to bind PP5. Instead, the Cu-oxidized S100A4 directly interacted with S100A1 and prevented PP5 activation. Hydrogen peroxide induced S100A4 oxidation in MKN-45 gastric adenocarcinoma cells and decreased S100A1-PP5 interaction, resulted in the inhibition of PP5 activation by S100A1. These data indicate that oxidized S100A4 regulates PP5 activity in a unique manner under oxidative stress conditions. Introduction S100A4 protein is a member of the EF-hand calcium ion-binding protein family, of which >25 members have been found in humans (1). They are 25-65% homologous at the amino acid level, while the sequence of the linker (hinge) region and the C-terminal extension are the most variable among the S100 proteins. Each S100 monomer contains two EF-hand Ca 2+ -binding sites. Ca 2+ -binding causes a conformational change and exposure of a hydrophobic surface allowing interaction with target proteins (2). They do not possess enzymatic activity, but rather regulate the activity of target proteins. Several proteins have been identified as S100A4 targets, including liprin β1 (3), methionine aminopeptidase (4), the p53 tumor suppressor protein (5) and the heavy chain of non-muscle myosin II (6). In addition, S100A4 is capable of forming heterodimers with S100A1 (7,8), which is likely to increase its functional potential. Previous studies have shown that S100A4 interacts with S100A1 via heterodimer formation and that S100A1 can antagonize function of S100A4 (9,10). It has been a general observation that two-hybrid screenings utilizing S100 proteins have primarily detected other S100 family members as targets (11)(12)(13).We have previously demonstrated that S100A1, A2, A6 and S100B interact with the tetratricopeptide repeat (TPR) domain of PP5 in a Ca 2+ -dependent manner and significantly activate its phosphatase activity (14). PP5 is a member of the phosphoprotein phosphatase (PPP) family of a serine/threonine phosphatase (15). PP5 contains a C-terminal catalytic domain and N-terminal three TPR motifs that are unique in the PPP family (16,17). The TPR motif consists of 34 amino acid sequences, and between 3 and 16 copies of the motif are arranged in the proteins as tandem arrays (18,19). This motif can act as an interaction scaffold for protein complex formation. PP5 is a negative regulator of the apoptosi...

“…Histidine-tagged PP5 (His-PP5) protein was expressed and purified according to the manufacturer's protocol. S100 proteins (S100A1, S100A2, S100A6, S100B, and S100P) were expressed and purified as described previously (Yamashita et al 1999;Okada et al 2004). Air-oxidized S100 proteins were prepared by storing the recombinant S100 proteins (0.5 mg/mL) at -30°C for 3 months.…”

Section: Plasmids and Recombinant Proteinsmentioning

confidence: 99%

Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity

Yamaguchi

Tsuchiya

et al. 2016

Tohoku J. Exp. Med.

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Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/ threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calciumbinding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H 2 O 2 ) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H 2 O 2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H 2 O 2 . These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis.