Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway
Figure 8
Sae2 protein interacts with PBD of Cdc5.
(A) Sae2 protein sequence. The putative Cdc5 phosphorylation sites and PBD binding sites are indicated. (B) Plasmid pEG202-PBD340–705, carrying the polo box domain of Cdc5 (PBD, aa 340 to 705), and pJG4-5-SAE2, carrying the full length SAE2 gene under the GAL1 promoter, were co-transformed with pSH18-34, a β-galactosidase reporter plasmid in the wild type yeast strain EGY48. To assess two-hybrid interaction, these strains were patched on to 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X Gal) plates containing either raffinose (RAF, prey repressed) or galactose (GAL, prey expressed). Accordingly to [50], the strain Y692 (PBD versus Swe1173–400 protein fragment) was used as positive control. (C) Cells of the strain Y202, expressing SAE2-3HA gene, were blocked in G2/M by nocodazole treatment. Whole cell protein extract was prepared and incubated with glutathione-Sepharose beads carrying GST or GST-PBD357–705. Input and pull-down samples were analyzed by western blotting with monoclonal antibody 12CA5 (αHA) or polyclonal antisera raised against GST (αGST). Asterisk denotes bands of GST-PBD degradation or expression of truncated proteins. (D) Schematic model to summarize the results presented in this work. (i) Sae2 transiently binds DSB, regulating ends resection and influencing Mec1-signaling. The checkpoint signal is amplified downstream, regulating several targets, including Cdc5. (ii) After a prolonged checkpoint response, adaptation to damage takes over and Cdc5 is re-activated, likely by an activating kinase (in human cells, it is aurora A [35]); Cdc5 then inhibits checkpoint signaling in a feedback regulatory loop, by likely targeting several factors, including Sae2 whose loading on the irreparable DSB increases, slowing down resection and contributing to counteract the checkpoint signaling (red circles denote phosphorylation). Alternatively, or in addition, Cdc5 function on several targets, including Sae2, is enhanced in the presence of elevated levels of Cdc5, a situation frequently found for Plks in tumor cells.