Regulation of Chk1 by Its C-terminal Domain

Abstract

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ϳ200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration. INTRODUCTIONDNA damage checkpoints halt cell cycle progression to allow time for the repair machinery to correct the lesion. The ultimate targets of these checkpoints are the cyclin-dependent kinases that promote the transition from G1 into S-phase, and from G2 in to mitosis O'Connell and Cimprich, 2005). Two major effectors of the DNA damage checkpoints are the tumor suppressor and transcription factor p53, and the protein serine/threonine kinase Chk1. The importance of checkpoints in genome integrity is highlighted by the frequent inactivation of p53 signaling in tumors, leading to G1/S checkpoint defects and a blockade to apoptosis (Giono and Manfredi, 2006). Conversely, Chk1 and its upstream regulators, which control the G2 DNA damage checkpoint, are rarely (if ever) mutated in tumors, suggesting this pathway may be essential for cancer cell viability. Indeed, inhibition or ablation of Chk1 greatly sensitizes p53 mutant tumor cells to genotoxic therapies (Suganuma et al., 1999;Jackson et al., 2000;Koniaras et al., 2001;Macip et al., 2006). Although Chk1 is essential for embryonic development in the mouse (Liu et al., 2000), this may not be the case for most cells in adult tissues. Therefore, there is intense interest in developing specific Chk1 inhibitors for cancer chemotherapy (Garber, 2005).The G2 DNA damage checkpoint is highly conserved in evolution, and model systems, such as the fission yeast Schizosaccharomyces pombe, have been invaluable in dissecting this signaling pathway. On detection of DNA damage, several protein complexes cooperate to ensure Chk1 activation, leading to a 5-10-fold increase over its basal activity (Capasso et al., 2002;Harvey et al., 2004;Lat...

Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment

“…6B, panel 4). These findings imply that isoform 4 and the more alkaline isoform correspond to the conformational changes induced by phosphorylation of Chk1 at S345 (Kosoy and O'Connell, 2008).…”

“…Given that Chk1 is phosphorylated at S345 by Rad3 kinase whilst bound to damaged chromatin (Capasso et al, 2002;Kosoy and O'Connell, 2008), we analysed the modification status of Chk1 in response to genotoxic and heat stress utilising normal SDS PAGE and isoelectric focusing. We used two different types of genotoxic stress, the chronic modification of Chk1 in cells with hyper-active Cdc2 kinase (Capasso et al, 2002) and the induced modification upon inhibition of topoisomerase 1 [camptothecin (CPT)] (Walworth et al, 1993).…”

Novel Insights into Chk1 Regulation by Phosphorylation

“…However, the pseudo-substrate motif does not appear to be conserved in Chk1 from other species. In addition, the deletion of the Cterminus abrogates Chk1 function in fission yeast (Caparelli and O'Connell, 2013;Kosoy and O'Connell, 2008), suggesting that the regulatory domain may play diverse roles in Chk1 activity. Therefore, much is still unknown about molecular mechanism by which Chk1 is structurally activated by phosphorylation.…”

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

“…However, while deletion of the regulatory domain increases Chk1 activity in vitro (Chen et al 2000), it is essential for Chk1 function in vivo (Kosoy and O'Connell 2008). Further, mutations in the C-terminal domain can either inactivate or super-activate Chk1 function in vivo (Wang and Dunphy 2000;Kosoy and O'Connell 2008;Palermo et al 2008;Pereira et al 2009), suggesting that it contributes more than an inhibitory function to the catalytic domain (Tapia-Alveal et al 2009). …”

“…The library was transformed with pREP41-chk1-E472D (Kosoy and O'Connell 2008), and Chk1-resistant colonies were selected on media lacking thiamine. The pREP41-chk1-E472D was then lost from 500 independent colonies by selection relief, and the plasmid was reintroduced by mating from a wild-type carrier strain.…”