In this study, we sought to resolve the molecular basis of a key step in the checkpoint-dependent activation of Chk1 in response to genomic stress. The activation of Chk1 involves phosphorylation-dependent docking of Chk1 with its cognate mediator protein (Claspin) and recognition of the resulting Claspin–Chk1 complex by ATR–ATRIP. In particular, phosphorylation of Claspin on multiple residues in its CKAD mediates the binding of Chk1. Direct biochemical evidence has indicated the presence of Claspin in this complex enhances the ability of ATR–ATRIP to phosphorylate Chk1 on key residues necessary for the ultimate activation of Chk1 (Kumagai et al., 2004
; Lindsey-Boltz et al., 2009
). Thus, the kinase that phosphorylates Claspin on the CKAD plays a key role in this pathway.
To address the identification of this kinase(s) systematically, we initially performed a kinome-wide RNAi screen in Drosophila
S2 cells. Various RNAi screens in these cells have been used to identify numerous regulators in diverse cellular pathways (Bettencourt-Dias et al., 2004
; Bjorklund et al., 2006
; Boutros and Ahringer, 2008
). For our purposes, the fact that Drosophila
contains approximately one-third as many kinases as humans was a considerable advantage. S2 cells appear to possess checkpoint-signaling pathways generally similar to those in other metazoan cells (de Vries et al., 2005
; Yi et al., 2009
). In particular, the Drosophila
Chk1 homologue Grapes exhibits a conserved checkpoint function in S2 cells. It functions downstream of Mei-41 (the Drosophila
homologue of ATR) and is critical for a proper checkpoint response to genotoxic stress.
Nonetheless, the reagents available for the study of checkpoint responses in Drosophila S2 cells are still relatively limited. To circumvent this problem, we introduced the Xenopus version of Chk1 into the Drosophila cells as a more readily traceable marker to monitor the checkpoint response. We were able to establish a system in which phosphorylation of this reporter could be induced following treatment with a variety of replication inhibitors. Moreover, this response seems to have molecular features similar to those present in vertebrate cells. For example, RNAi-mediated knockdown of Drosophila homologues of ATR and Claspin abolished checkpoint-dependent phosphorylation of the exogenously introduced Chk1 reporter molecule.
The results of our screen indicated that knockdown of several casein kinases led to the reduced phosphorylation of Chk1. Following this lead, we next employed a candidate-based cDNA overexpression approach to further pinpoint a specific kinase that can directly phosphorylate the CKAD of Claspin. From these tests, we eventually found that Drosophila Gish, a homologue of CK1γ, could phosphorylate the CKAD of Claspin quite effectively both in vitro and in the Drosophila tissue culture cells. At this juncture, we chose to extend our studies to human cells. Interestingly, humans possess three different versions of CK1γ, namely, CK1γ1, CK1γ2, and CK1γ3. All three proteins have very similar central kinase domains but are significantly different in their N- and C-terminal extensions. We observed that all three kinases could phosphorylate the CKAD relatively well in vitro. However, our further characterizations established that CK1γ1 appears to be the form primarily responsible for the phosphorylation of CKAD in human cells. Upon expression in human cells, only CK1γ1 could phosphorylate the CKAD of Claspin in vivo. More importantly, siRNA-mediated knockdown of CK1γ1 resulted in greatly reduced phosphorylation of Claspin, whereas knockdowns of CK1γ2 and CK1γ3 did not have a significant effect. As expected from the known function of the phosphorylation of Claspin, cells with diminished levels of CK1γ1 were greatly compromised in their ability to carry out the activation of Chk1 in response to a variety of genotoxic agents, including APH, HU, and UV. Furthermore, these cells had the physiological defects characteristic of cells with impairment of the Chk1-mediated signaling pathway. In particular, these cells showed reduced survival following treatment with genotoxic agents, impaired recovery of stalled replication forks, a defective G2/M checkpoint response, and spontaneous DNA damage in the absence of exogenous stress. Taken together, our results indicated CK1γ1 is an important regulator in Chk1-mediated cellular checkpoint responses.
The casein kinase 1 family of serine/threonine kinases is highly conserved and ubiquitously expressed. The functions of CK1 encompass a wide variety of processes, including cell proliferation, cell division, apoptosis, circadian rhythms, and others (Gross and Anderson, 1998
; Knippschild et al., 2005
; Cheong and Virshup, 2011
). In mammals, this family contains at least seven members (α, β, δ, ε, γ1, γ2, and γ3) with multiple splicing variants. All of the CK1 proteins share significant homology in the central kinase domain (53–98% identical), but differ significantly in the flanking N- and C-terminal sequences, which most likely confer unique properties to the various kinases. In the case of CK1γ1, we identified and isolated multiple splicing variants of this kinase from human U2OS cells. These isoforms contain distinct C-terminal sequences (ranging from 3 to 106 amino acids), display discrete subcellular localizations, and exhibit differences in their abilities to phosphorylate the CKAD. A similar phenomenon has also been observed in various organisms in the case of CK1α, which contains four types of isoforms with distinct localizations and kinase activities (Zhang et al., 1996
; Green and Bennett, 1998
; Burzio et al., 2002
). These findings strongly suggest various isoforms of CK1 may participate in distinct cellular activities due to differences in access to and/or affinity for substrates.
Interestingly, we initially found it difficult to achieve good rescue of CK1γ1-depleted cells with vectors encoding an siRNA-resistant version of the major published form of CK1γ1. However, as described in the last part of Results
, there are multiple forms of CK1γ1 in human cells. We were able to clone and express three additional versions of CK1γ1, which we designated as isoforms B, C, and D to distinguish them from the published form (isoform A). In cellular localization studies, we found that isoform A had the expected prominent localization to cell membranes (Davidson et al., 2005
). However, isoforms B and C resided in both the nucleus and cytoplasm, whereas isoform D was mainly cytoplasmic. In coexpression studies, we found that isoforms A, B, and C (but not D) could phosphorylate the CKAD of Claspin, although isoforms B and C were more effective than isoform A in this in vivo assay. Ultimately, we were able to rescue depletion of CK1γ1 by introducing siRNA-resistant forms of isoforms A, B, and C into the cells. It is straightforward to understand how the nuclear isoforms could regulate Claspin, but it is intriguing that a membrane-bound enzyme is also partially responsible. Conceivably, some fraction of this isoform could be absent from the membrane. It is also possible that Claspin could shuttle between the nucleus and cytoplasm and thus be subject to regulation by enzymes in both locations. In this regard, a recent study has shown that perturbed cell-surface signaling through the Sonic Hedgehog (Shh) pathway inhibits ATR-mediated signaling by disrupting the interaction between Claspin and Chk1 (Leonard et al., 2008
It will be important to understand the mechanisms that control the phosphorylation of Claspin by CK1γ1 (see ). It is known that phosphorylation of the CKAD in Xenopus egg extracts is dependent upon ATR. However, ATR itself is unable to phosphorylate the critical sites in the CKAD directly. One possibility would be that ATR might somehow regulate the activity of CK1γ1. However, we have been able to detect only a subtle increase in the activity of CK1γ1 in response to genomic stress. Moreover, when we mutated the only apparent potential target site for ATR (Ser361) to Ala in CK1γ1, we could not observe a change in its kinase activity toward the CKAD upon coexpression in U2OS cells (unpublished data). Another possibility is that ATR might regulate the accessibility of Claspin to CK1γ1. Further studies will be required to understand the dynamics of this phosphorylation.
In summary, we identified a conserved casein kinase, Gish/CK1γ1, from Drosophila and humans as a specific enzyme that controls phosphorylation of the CKAD of Claspin. Functional studies have revealed that this kinase is critical for mediating activation of Chk1 and ensuring a proper checkpoint response under conditions of genotoxic stress. Further studies of its regulation and function should help us gain more insight into the molecular basis of checkpoint responses.