Phosphorylation of Rph1, a damage-responsive repressor of PHR1 in Saccharomyces cerevisiae, is dependent upon Rad53 kinase

Abstract

Rph1, a Cys2-His2 zinc finger protein, binds to an upstream repressing sequence of the photolyase gene PHR1, and represses its transcription in response to DNA damage in Saccharomyces cerevisiae. In this report, we have demonstrated that the phosphorylation of Rph1 protein was increased in response to DNA damage. The DNA damage-induced phosphorylation of Rph1 was missing in most damage checkpoint mutants including rad9, rad17, mec1 and rad53. These results indicate that Rph1 phosphorylation is under the control of the Mec1-Rad53 damage checkpoint pathway. Rph1 phosphorylation required the kinase activity of Rad53 since it was significantly decreased in rad53 checkpoint mutant. Furthermore, loss of other kinases including Dun1, Tel1 and Chk1, which function downstream of Mec1, did not affect the Rph1 phosphorylation. This contrasts with the derepression of Crt1-regulated genes, which requires both Rad53 and Dun1 protein kinases. These results imply that post-translational modification of Rph1 repressor is regulated by a potentially novel damage checkpoint pathway that is distinct from the RAD53-DUN1-CRT1 cascade implicated in the DNA damage-dependent transcription of ribonucleotide reductase genes.

Figure 1

Rph1 is phosphorylated in response to DNA damage and its phosphorylation occurs on both Ser and Thr residues. (A) Time course analysis of Rph1 phosphorylation after UV-irradiation. Wild-type (WT) cells (W303) were treated with UV (100 J/m²) and total cell extracts were prepared at intervals for 120 min. Eighty micrograms of total cell lysates were applied for immunoblotting. (B) Abolishment of Rph1 phosphorylation by treatment with alkaline phosphatase. The electrophoresis mobility shift of Rph1 protein disappeared with treatment with 60 U of calf intestine alkaline phosphatase (Takara, Japan). –, mock treatment; +, alkaline phosphatase treated. (C) Expression of GST–Rph1 was induced in a selective medium containing 2% galactose. GST–Rph1 overexpressing cells were resuspended in radiolabeled orthophosphate-containing media followed by 1 h incubation. The radiolabeled GST–Rph1 fusion proteins were eluted by immunoprecipitation with α-GST antibody and analyzed by phosphoamino acid assay. (D) Endogeneous Rph1 was radiolabeled with orthophosphate for 1 h as described above. The labeled Rph1 protein was eluted by immunoprecipitation with an anti-Rph1 antibody and analyzed by phosphoamino acid assay. S, Ser; T, Thr; arrow, loading origin.

Figure 2

Loss of checkpoint genes affects phosphorylation of Rph1. Individual cultures from WT (W303), rad17, rad9, rad24, rad9/24, mec3 (A) and mec1, tel1 (B) damage checkpoint mutants cells were grown to mid-log phase, irradiated with UV (100 J/m²) or treated with MMS (0.1%) at 30°C for 1 h and the cell lysates were extracted for immunoblotting. Rph1 phosphorylation appeared to be different patterns in several checkpoint mutants. C, no damage; U, UV-irradiated (100 J/m²); M, MMS-treated (0.1%).

Figure 3

Rph1 phosphorylation is dependent on Rad53 protein kinase. (A) Damage-dependent phosphorylation of Rph1 was checked in the rad53 defective mutant. Wild-type cell and rad53 mutant cell lysates were used for immunoblotting. (B) In vitro kinase assay was performed with GST–Rph1 fusion protein expressed in E.coli. GST–Rph1 proteins bound to glutathione–Sepharose were incubated with total cell lysates treated with UV (100 J/m²) or MMS (0.1%) at 30°C for 1 h and then the degree of phosphorylation was examined by using in vitro kinase assay. (C) Immunoblot analysis showed that Rph1 phosphorylation was diminished in rad53-D339A (a kinase dead mutant) but recovered in Rad53-op (Rad53-overexpressing). C, no damage; U, UV-irradiated (100 J/m²); M, MMS-treated (0.1%).