Figure 2.

PKA phosphorylates Dam1p. (A) Alignment of the DAM1 region showing the relevant phosphorylation sites across multiple budding yeast species indicates that Dam1p contains evolutionary conserved PKA sites (S31, S257 is known to be an Ipl1p target) in addition to the well-studied Aurora B/Ipl1p sites. Alignments were performed using UniProt/Align. (B) Purified Dam1p-HBH (JCY986) and nonphosphorylatable dam1-S31A-HBH (JCY988) from asynchronously growing cells were analyzed by Western blot analysis. Blots were treated with anti-His tag antibody to measure total Dam1p and dam1p-S31A (left panel) or an antibody that recognizes phospho-serine/threonine within the consensus PKA site (anti-RRXS^PO-antibody) to measure levels of phosphorylated Dam1p and dam1p-S31A (right panel). (C) In vitro kinase reactions were carried out with recombinant Dam1 complexes and purified GST-Tpk2 or no kinase control followed by western analysis with anti-RRXS^PO-antibody to measure levels of phosphorylation of Dam1p. Samples from the Gst-Tpk2 reactions were also run in another lane and Coomassie-stained to determine which subunit was phosphorylated (right panel). HBH, 6X-histidine-biotin-6X- histidine tag fused to c-terminus of Dam1p or dam1p-S31A. * indicates nonspecific bands.