Yap4 PKA- and GSK3-dependent phosphorylation affects its stability but not its nuclear localization.
Yap4 is a nuclear-resident transcription factor induced in Saccharomyces cerevisiae when exposed to several stress conditions, which include mild hyperosmotic and oxidative stress, temperature shift or metal exposure. This protein is also phosphorylated. Here we report that this modification is driven by PKA and GSK3. In order to ascertain whether Yap4 is directly or indirectly phosphorylated by PKA, we searched for stress and PKA-related kinases that could phosphorylate Yap4. We show that phosphorylation is independent of the kinases Rim15, Yak1, Sch9, Slt2, Ste20 and Ptk2. In addition, we showed that Yap4 phosphorylation is also abrogated in the triple GSK3 mutant mck1 rim11 yol128c. Furthermore, our data reveal that Yap4 nuclear localization is independent of its phosphorylation state. This protein has several putative phosphorylation sites, but only the mutation of residues T192 and S196 impairs its phosphorylation under different stress conditions. The ability of the non-phosphorylated forms of Yap4 to partially rescue the hog1 severe sensitivity phenotype is not affected, suggesting that Yap4 activity is maintained in the absence of phosphorylation. However, this modification seems to be required for stability of the protein, as the non-phosphorylated form has a shorter half-life than the phosphorylated one.
Genomics and Stress Laboratory, Instituto de Tecnologia Química e Biológica, Av. da República, Apt. 127, 2781-901 Oeiras, Portugal.
Jorge
J
Pereira
Catarina
C
Pimentel
Catarina
C
Amaral
Regina A
RA
Menezes
Claudina
C
Rodrigues-Pousada
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005800:Genes, Fungal; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C422266:Cin5 protein, S cerevisiae; D017931:DNA Primers; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D017868:Cyclic AMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3