title: Nuclear translocation of Sgt1 depends on its phosphorylation state.
abstract: 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 Ca(2+)-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-Ca(2+)-mediated Sgt1 dephosphorylation promotes its nuclear translocation, most likely due to disruption of the Sgt1-Hsp90 complex.
authors.affiliation: Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland.
authors.forename: Wiktor
authors.initials: W
authors.lastname: Prus
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zabka
authors.forename: Paweł
authors.initials: P
authors.lastname: Bieganowski
authors.forename: Anna
authors.initials: A
authors.lastname: Filipek
mesh_terms: D021581:Active Transport, Cell Nucleus; D047390:Casein Kinase II / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D014162:Transfection
chemical_list: D018797:Cell Cycle Proteins; D018841:HSP90 Heat-Shock Proteins; C490164:SUGT1 protein, human; D047390:Casein Kinase II