FIGURE 2.

Phosphorylation of ScMaf1, Maf1-id, and Maf1-id S388A. A, phosphorylation of Maf1-id in PKA and SCH9 ATP analog sensitive (−as) strains. Wild type and −as strains were grown to OD ∼0.5 and treated with the ATP analog 1NM-PP1 before extract preparation and separation by phos-tag SDS-PAGE as in Fig. 1E. Note the slowest migrating band in all lanes results from phosphorylation at an unknown site that is insensitive to inhibition of Sch9 and PKA. B, phosphorylation of WT Maf1 FL (full-length), Maf1-id, and S388A mutant proteins in vitro. Expression of GST-Tpk1, GST-Sch9, and GST-Cka1 kinases was induced by addition of 4% galactose to log phase yeast cells grown in YP raffinose medium. Each kinase was purified on a glutathione-Sepharose column. Recombinant WT Maf1FL, Maf1-id, and S388A mutant proteins (500 ng) were incubated with the yeast-purified kinase and [γ-³²P]ATP for 1 h before separation by standard SDS-PAGE. An autoradiogram (upper panel), Western detection of each kinase using anti-GST antibody (middle panel) and Western detection of each recombinant Maf1 protein using anti-HIS antibody (lower panel) are shown for each kinase reaction.