Transformation/transcription domain-associated protein (TRRAP)-mediated regulation of Wee1

Abstract

The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Upregulation of Chk1 by mutation or overexpression bypasses the requirement for upstream regulators or DNA damage to promote a G2 cell cycle arrest. We screened in fission yeast for mutations that rendered cells resistant to overexpressed chk1(+). We identified a mutation in tra1, which encodes one of two homologs of transformation/transcription domain-associated protein (TRRAP), an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed chk1(+), suggesting this phenotype is due to a HAT activity, although expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing chk1(+) and accumulate inactive Wee1 protein. The changed division response (Cdr) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and we show that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation.

Figure 1.—

Tra1 is required for cell cycle arrest by Chk1 overexpression. (A) The indicated strains containing pREP1-Chk1 were grown on media in the presence (promoter repressed) or absence (promoter derepressed) of thiamine for 4 days at 30°. (B) Tra1 is a 3699-amino-acid protein containing the FAT and FATC domains characteristic of the ATM and ATR kinases, but lacks ATP coordinating residues in the PI3K domain (labeled PI3K*) required for kinase activity. ura4 truncates Tra1 at residue 3559 and is expressed in the opposite direction. (C) Western blot analysis shows that expression levels of Chk1 (rabbit anti-Chk1) are unaffected in tra1Δ cells. Actin was used as a loading control. (D) Chk1 activity is also unaffected in tra1Δ cells. Data are mean ± SE; n = 3.

Figure 2.—

The histone deacetylase inhibitor trichostatin A suppresses the Chk1 resistance of tra1Δ. Plates were incubated for 4 days at 30°.

Figure 3.—

Chk1 resistance in tra1Δ is independent of Mik1. (A) Western blot of extracts from cells expressing Myc-tagged Mik1 or an untagged control (no tag) from untreated cells (−) or cells treated with 11 mm HU for 4 hr at 30°. Antitubulin antibodies were used as a loading control. (B) Cells harboring vector (pREP1) or pREP1-Chk1 were grown in the presence or absence of thiamine/MMS for 4 days at 30°. Note that mik1Δ cells are severely growth inhibited by Chk1 overexpression, and this is further exacerbated by MMS.

Figure 4.—

Tra1 is not required for checkpoint arrest, but is required for regulation of the G2/M transition. (A) Tra1 is not required for resistance to MMS. YES plates containing the indicated concentrations of MMS or no drug (control) were inoculated with spots of 10-fold serial dilutions of the indicated strains and were grown at 30° for 4 days. (B) Tra1 is not required for activating phosphorylation on Chk1 in the presence of MMS, showing that signaling through endogenous Chk1 is intact in tra1Δ cells. (C) FACS profiles of DNA content in cycling cells (shaded) or in MMS-treated cells (open). The cell cycle delay (cell elongation) in MMS broadens the profiles of these samples. The lengths of 50 exponentially growing (D) wild-type and (E) tra1Δ cells were determined by microscopy, and representative images are shown. Data are mean ± SD.

Figure 5.—

Wee1 levels accumulate in tra1Δ cells. (A) Western blotting shows that Wee1 protein accumulates in tra1Δ cells, but the levels of Cdc25, Cdc13, tyrosine-15 phosphorylated (Y15^P), and total Cdc2 are not affected. Note that tra1Δ cells are semi-wee (Table 3) and that Y15^P does not accumulate, indicating that the excess Wee1 in tra1Δ cells is not fully active. (B) Cdc13 was immunoprecipitated with a mouse monoclonal anti-Cdc13 antibody and that coprecipitating Cdc2 was detected by Western blotting with a rabbit polyclonal anti-Cdc2 antibody. The anti-rabbit secondary antibody weakly cross-reacts with the mouse IgG heavy chain. Immunoprecipitated Cdc13 comigrates with the IgG heavy chain, which precludes its detection by IP or Western blots, both mouse antibodies. (C) Cdc2 kinase activity in Cdc13 IPs is increased by approximately twofold in tra1Δ cells. Data are mean ± SE; n = 3.

Figure 6.—

Wee1 accumulation in tra1Δ cells is dependent on Cdr1 and Cdr2. (A and B) Western blot for HA-tagged Wee1 in the indicated strains grown to mid-logarithmic phase at 30°. Antitubulin and anti-actin were used as loading controls. Note that increased Wee1 levels are suppressed by cdr1Δ and cdr2Δ.

Figure 7.—

Accumulation of active Cdr1 in tra1 mutants. (A) Western blot analysis of Cdr1 (Flag-tagged) and Cdr2 (HA-tagged) levels. Tubulin and actin are used as loading controls. (B) The ratio of the upper (phosphorylated) to lower (unphosphorylated) Cdr1 was determined by densitometry. Data are mean ± SD; n = 3. (C) tra1Δ cells have an enhanced nitrogen starvation response. FACS profiles of cells grown in 100%, 10%, and 0% nitrogen for 16 hr at 30°.