Antagonism of Chk1 Signaling in the G2 DNA Damage Checkpoint by Dominant Alleles of Cdr1

Abstract

Activation of the Chk1 protein kinase by DNA damage enforces a checkpoint that maintains Cdc2 in its inactive, tyrosine-15 (Y15) phosphorylated state. Chk1 downregulates the Cdc25 phosphatases and concomitantly upregulates the Wee1 kinases that control the phosphorylation of Cdc2. Overproduction of Chk1 causes G 2 arrest/delay independently of DNA damage and upstream checkpoint genes. We utilized this to screen fission yeast for mutations that alter sensitivity to Chk1 signaling. We describe three dominant-negative alleles of cdr1, which render cells supersensitive to Chk1 levels, and suppress the checkpoint defects of chk1D cells. Cdr1 encodes a protein kinase previously identified as a negative regulator of Wee1 activity in response to limited nutrition, but Cdr1 has not previously been linked to checkpoint signaling. Overproduction of Cdr1 promotes checkpoint defects and exacerbates the defective response to DNA damage of cells lacking Chk1. We conclude that regulation of Wee1 by Cdr1 and possibly by related kinases is an important antagonist of Chk1 signaling and represents a novel negative regulation of cell cycle arrest promoted by this checkpoint.

Transformation/Transcription Domain-Associated Protein (TRRAP)-Mediated Regulation of Wee1

“…This semi-wee phenotype is less severe than a complete wee phenotype, where cells divide at $8 mm, but nevertheless is indicative of a shortened G2 period of the cell cycle in tra1D cells. Cells with wee or semi-wee phenotypes are checkpoint proficient but resistant to chk1 1 overexpression (Walworth et al 1993;O'Connell et al 1997;Raleigh and O'Connell 2000;Calonge and O'Connell 2006), and with the normal response to DNA damage, we propose this is the reason for Chk1 resistance in tra1 mutants. Notably, both TSA and several of the HDAC mutants also suppressed the semi-wee phenotype of tra1D cells (Table 1).…”

“…1 from pREP41 causes a modest G2 cell delay (Calonge and O'Connell 2006) and was tolerated by both wild-type and tra1D cells. Colony formation in both strains under these conditions was not affected by addition of TSA ( Figure 2).…”

“…We hypothesized that other signaling pathways that control cell cycle progression may be regulated in concert with Chk1 in response to DNA damage. Previously, we had obtained evidence that this was the case for the negative regulators of Wee1, the Cdr1 and Cdr2 kinases (Calonge and O'Connell 2006). To search for additional components that modulate the G2/M transition relevant to Chk1 signaling, we undertook an insertional mutagenesis screen, using the ura4 1 gene as the insertional mutagen, to identify genes required for cell cycle arrest caused by chk1 1 overexpression (see materials and methods).…”

“…Further, several DNA repair mutants cycle normally with active Chk1 (our unpublished observations), suggesting that additional signaling may be regulated by DNA damage that impacts on cell cycle progression. Indeed, we have shown that the changed division response (Cdr) kinases, Cdr1 and Cdr2, act as Chk1 antagonists through their negative regulation of Wee1 (Calonge and O'Connell 2006), and thus it is possible that other pathways controlling cell cycle progression await identification. Cdr1, and possibly Cdr2, directly inhibit Wee1 by phosphorylation (Coleman et al 1993;Kanoh and Russell 1998).…”

Turning off the G2 DNA damage checkpoint

“…We identified dominant-negative mutations in cdr1 that antagonize Chk1: these alleles suppress the DNA damage sensitivity of cells lacking Chk1, and gain-of-function for Cdr1 by itself causes checkpoint defects. Conversely, deletion of cdr1 does not influence Chk1 function, explaining why dominant negative mutants were required for the phenotype [44]; that is, multiple Cdr-like kinases may be regulating Wee1 in response to various stimuli, and as negative regulators of Wee1, the cumulative effect of this is an antagonism of Chk1 as a positive regulator of Wee1. Although not seen in fission yeast, in Xenopus and budding yeast this pathway also impacts on Wee1 stability [45,46].…”