Mechanistic insight into the Cdc28-related protein kinase Ime2 through analysis of replication protein a phosphorylation

In budding yeast, the meiosis-specific protein kinase Ime2 is required for normal meiotic progression. Current evidence suggests that Ime2 is functionally related to Cdc28, the major cyclin-dependent kinase in yeast that is essential for both cell cycle and meiosis. We have previously reported that a natural target of Ime2 activity is replication protein A (RPA), the cellular single-stranded DNA-binding protein that performs critical functions during DNA replication, repair and recombination. Ime2-dependent RPA phosphorylation first occurs early in meiosis and targets the middle subunit of the RPA heterotrimeric complex (Rfa2). We now demonstrate that Rfa2 serine 27 (S27) is required for Ime2-dependent Rfa2 phosphorylation in vivo. S27 is also required for Rfa2 phosphorylation in vitro catalyzed by immunoprecipitated Ime2. In addition, Ime2 mediates in vitro phosphorylation of a short peptide containing Rfa2 amino acids 23 through 29, thereby providing evidence that S27 itself is the phosphoacceptor. Phosphorylation site mapping supports this conclusion, as mass spectrometry analysis has revealed that at least three residues within Rfa2 amino acids 2 through 35 become phosphorylated specifically during meiosis. Although S27 is embedded in a motif that is recognized by several protein kinases, this sequence is not a typical target of cyclin-dependent kinases. Therefore, the mechanism underlying Ime2 substrate recognition could differ from that of Cdc28.