Multiple levels of control regulate the yeast cAMP-response element-binding protein repressor Sko1p in response to stress

The Sko1p transcriptional repressor regulates a subset of osmoinducible str ess defense genes in Saccharomyces cerevisiae by binding to cAMP-responsive elements. We have reported previously that in response to stress Sko1p is phosphorylated by the stress-activated Hog1p mitogen-activated protein kina se, which disrupts its interaction with the Ssn6p . Tup1p corepressor. Here we report that other mechanisms are essential for the regulation of the Sk o1p repressor activity upon stress. The nuclear localization of Sko1p depen ds on the stress-inhibited protein kinase A (PKA). Sko1p is localized in th e nucleus of unstressed cells, and it redistributes to the cytosol upon sev ere salt stress (1 M NaCl). Yeast mutants with low PKA activity localize Sk o1p to the cytoplasm in the absence of stress and exhibit deregulated expre ssion of cAMP-responsive element-regulated genes. The central part (315-486 ) of Sko1p, containing the PKA phosphorylation sites and the basic domain-l eucine zipper domain, is essential for its nuclear localization. Salt-induc ed export of Sko1p from the nucleus is independent of Hog1p and of the Bcy1 p regulatory subunit of PKA. Furthermore, phosphorylation by PKA slightly e nhanced DNA binding affinity of Sko1p in vitro, whereas Sko1p dimerization in vivo is not regulated by stress. Sko1p repressor activity is associated to its binding to the Ssn6p-Tup1p complex. Interestingly, the Sko1p NH2 ter minus (1-315), containing the Hog1p phosphorylation sites, associates in vi vo with Tup1p in the absence of Ssn6p, suggesting that Sko1p represses gene transcription by interacting directly with the Tup1p subunit of the Ssn6p- Tup1p complex.