Sty1 kinase-dependent phosphosite mapping on Atf1

In the fission yeast Schizosaccharomyces pombe, heterochromatic silencing is quite stable at pericentromeres but unstable at the mating-type (mat) locus under chronic heat stress. We found that the compromised gene silencing at the mat locus at elevated temperature is linked to the phosphorylation status of Atf1 (a member of the ATF/CREB superfamily) by Sty1, one of the mitogen-activated protein kinases (MAPKs). To gain a full picture of Sty1-dependent phosphosite map of Atf1 under heat stress, we purified HA-Atf1 protein from cultures of wild type and sty1Δ strains shifted from 25℃ to either 30℃ or 37℃ for 5 hr followed by immunoprecipitation. Samples were separated by SDS-PAGE, and Coomassie blue-stained gel slices were excised and submitted for mass spectrometry analyses.