The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). A gsk-3 null mutant in which these four genes are disrupted showed growth defects on galactose medium. We isolated several multicopy suppressors of this growth defect. Two of them encoded Msn2p and phosphoglucomutase (PGM). Msn2p is a transcription factor that binds to the stress-response element (STRE). PGM is an enzyme that interconverts glucose-1 phosphate and glucose-6 phosphate and is regulated by Msn2p at the transcriptional level. Expression of the mRNAs of PGM2 and DDR2, whose promoter regions possess STRE sequences, on induction by heat shock or salt stress was reduced not only in an msn2 msn4 (msn2 homologue) double mutant but also in the gsk-3 null mutant. STRE-dependent transcription was greatly inhibited in the gsk-3 null mutant or mck1 mds1 double mutant, and this phenotype was suppressed by the expression of Mck1p but not of a kinase-inactive form of Mck1p. Although Msn2p accumulated in the nucleus of the gsk-3 null mutant as well as in the wild-type strain under various stress conditions, its STRE-binding activity was reduced in extracts prepared from the gsk-3 null mutant or mck1 mds1 double mutant. These results suggest that yeast GSK-3 promotes formation of a complex between Msn2p and DNA, which is required for the proper response to different forms of stress. Because neither Msn2p–GSK-3 complex formation nor GSK-3–dependent phosphorylation of Msn2p could be detected, the regulation of Msn2p by GSK-3 may be indirect

The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode glycogen synthase kinase 3 (GSK-3) homologs. The gsk-3 null mutant, in which these four genes are disrupted, shows temperature sensitivity, which is suppressed by the expression of mammalian GSK-3␤ and by an osmotic stabilizer. Suppression of temperature sensitivity by an osmotic stabilizer is also observed in the bul1 bul2 double null mutant, and the temperature sensitivity of the bul1 bul2 double null mutant is suppressed by multiple copies of MCK1. We have screened rog mutants (revertants of gsk-3) which suppress the temperature sensitivity of the mck1 mds1 double null mutant and found that two of them, rog1 and rog2, also suppress the temperature sensitivity of the bul1 bul2 double null mutant. Bul1 and Bul2 have been reported to bind to Rsp5, a hect (for homologous to E6-associated-protein carboxyl terminus)-type ubiquitin ligase, but involvement of Bul1 and Bul2 in protein degradation has not been demonstrated. We find that Rog1, but not Rog2, is stabilized in the gsk-3 null and the bul1 bul2 double null mutants. Rog1 binds directly to Rsp5, and their interaction is dependent on GSK-3. Furthermore, Rog1 is stabilized in the npi1 mutant, in which RSP5 expression levels are reduced. These results suggest that yeast GSK-3 regulates the stability of Rog1 in cooperation with Bul1, Bul2, and Rsp5.

We investigated the status of the components and target genes of the Wnt signaling pathway in Japanese anaplastic thyroid cancers (ATCs) in the present study. Nuclear and cytoplasmic positive staining of beta-catenin, which might indicate the existence of alterations in the Wnt signaling pathway, were found in 40.9% and 63.6% of the 22 ATC samples, respectively. The beta-catenin, adenomatous polyposis coli (APC) and Axin 1 gene mutations were observed in 4.5%, 9.0%, and 81.8% of the 22 ATC samples, respectively. Overexpression of cyclin D1 and c-myc, which are the target genes of the Wnt signaling pathway, was observed in 27.3% and 59.1% of the ATC samples, respectively. There was no significant correlation between nuclear or cytoplasmic positive staining of beta-catenin and nuclear positive staining of cyclin D1 or c-myc. Taken together, the results of beta-catenin immunohistochemistry suggest that alterations in the Wnt signaling pathway are associated with carcinogenesis of ATC, but the frequency of beta-catenin gene mutation in our series is lower than that previously reported. Furthermore, cyclin D1 and c-myc frequently accumulated in ATC, independently of dysfunction in the Wnt signaling pathway.

In Saccharomyces cerevisiae, the overexpression of ROD1 confers resistance to o-dinitrobenzene (o-DNB), a representative of target drugs of glutathione S-transferase. The roles of Rod1 in drug resistance have remained to be determined. We isolated the rog3 mutation as a suppressor mutation of the temperature sensitivity of the strain, in that two of the total four glycogen synthase kinase 3 homologs were deleted. Rog3 is homologous to Rod1, and its overexpression also conferred resistance to o-DNB. Furthermore, these two proteins have PYmotifs, and bound to Rsp5, a hect-type ubiquitin ligase. The rsp5-101 mutant showed sensitivity to o-DNB as did the rod1 mutant, a mutant Rod1 containing altered PY motifs was defective in ability to bind to Rsp5 and in conferring o-DNB resistance. These results suggest that interaction of Rod1 and Rsp5 is important for drug resistance. ß