Institute of Fundamental Technological Research

The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery.

Chaperone proteins, Luciferase, Phosphatases, Enzyme-linked immunoassays, Recombinant proteins, Ligation assay, Plasmid construction, Luciferase assay

Recently we have shown that the Sgt1 (suppressor of G2 allele of Skp1) protein translocates to the nucleus due to heat shock and that the Ca2+-bound form of S100A6 is required for Sgt1 translocation (Prus and Filipek, 2010). In this work we studied the influence of Sgt1 phosphorylation on nuclear translocation. By means of two-dimensional (2D) electrophoresis we showed that in the protein extract of heat-shocked human epidermoid carcinoma (HEp-2) cells a higher level of a basic, most probably non-phosphorylated, form of Sgt1 can be detected. Also, we found a more efficient translocation of Sgt1 induced by heat shock when casein kinase II inhibitor was added to the cells. To confirm the role of Sgt1 phosphorylation/dephosphorylation in its nuclear translocation we transfected cells with non-phosphorylable Sgt1 mutants (S249A, S299A, S249/299A) or a phosphorylation mimic S299D mutant. We found that the levels of S299A and S249/299A mutants were higher than the level of wild type Sgt1 in the nuclear fraction after heat shock. Accordingly, we found that the 139–333 fragment of Sgt1 harboring the mutated residues, but not the 1–138 fragment, translocated to the nucleus upon heat shock. Moreover, we show that S100A6 is required for translocation of the non-phosphorylable Sgt1 mutants and that upon heat shock S100A6 translocates to the nucleus together with Sgt1. In addition, we found that non-phosphorylable Sgt1 mutant interacts with S100A6 more efficiently and at the same time exhibits lower affinity for Hsp90 (heat shock protein 90) than wild type Sgt1. Altogether, our results suggest that S100A6-Ca2+-mediated Sgt1 dephosphorylation promotes its nuclear translocation, most likely due to disruption of the Sgt1-Hsp90 complex.

Sgt1, Protein phosphorylation, Nuclear translocation, S100A6, Heat shock proteins