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Regulation of CHK1 by the Ubiquitin Proteasome
system
Maëlle Cartel, Christine Didier
To cite this version:
Maëlle Cartel, Christine Didier. Regulation of CHK1 by the Ubiquitin Proteasome system. FEBS Journal, Wiley, 2020. �hal-03030938�
Regulation of CHK1 by the Ubiquitin-Proteasome System. Maëlle Cartel 1,2 and Christine Didier 1,2,* 1 Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL 5294, Université de Toulouse, Toulouse, France. 2 Ligue Nationale Contre le Cancer, équipe labellisée 2016. Corresponding Author: Christine Didier, Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL 5294, Toulouse, France. E-mail: christine.didier@inserm.fr Running Title CHK1 Regulation Keywords CHK1, Ubiquitin Proteasome System. Abbreviations ATR, ataxia telangiectasia and Rad3-related; CHFR, checkpoint with forkhead and ring-finger; CHK1, checkpoint kinase 1; CRL, cullin RING ligase; DDR, DNA damage response; DUB, deubiquitylating enzymes; MDM2, mouse double minute 2 homolog; USP, ubiquitin specific proteases.
Abstract (70 words)
The checkpoint kinase 1 (CHK1) is a master regulator of genome integrity in vertebrate cells. Despite its important cell cycle functions, its regulation is still incompletely understood. Cassidy et al. provide novel insights on the regulation of the CHK1 abundance by the HECT E3 ligase HUWE1 during unperturbed cell cycle as well as in response to replicative stress. These results may help us to apprehend the underlying mechanism of tumorigenesis. Introduction/Main text/Conclusion-Discussion Maintenance of genome integrity is crucial for the development, homeostasis, and survival of all organisms, and essential for restricting tumorigenesis. In response to DNA damage, a complex network of proteins sense and mediate repair of damaged DNA or apoptosis. Serine threonine checkpoint kinase 1 (CHK1) is a central protein involved in the DNA damage response (DDR). Its phosphorylation by the upstream kinase ataxia telangiectasia and Rad3-related (ATR) induces its activation in response to DNA damage, and its subsequent phosphorylation of cell cycle substrates leads to DNA repair, cell cycle arrest and cell death in the case of excessive DNA damage (1). Moreover, DDR unrelated functions of CHK1 during DNA replication and mitosis have also recently been described (2,3). This confirms the dual functions of CHK1 and reinforces the attractive notion that CHK1 is a "molecular switch" from normal cell cycle progression to checkpoint arrest. Significant progress has recently been made in our understanding of CHK1 regulation and its implications in cancer etiology and therapy (4). However, areas of darkness remain regarding the regulation of the expression and activation of CHK1, despite its important cell cycle functions. In depth studies are needed
to establish a more integrated view of CHK1 regulation modes depending on the cellular context, normal cell proliferation or under various types of damage and stress.
The work of Cassidy et al. is part of this research and describes for the first time the participation of HUWE1, a HECT domain-containing ubiquitin E3 ligase, in the regulation of the stability of CHK1 in the unperturbed cell cycle and in response to replication stress. It is well known that CHK1 protein levels are tightly regulated by the ubiquitin-proteasome system and any deregulation can have important consequences in terms of proliferation, tumor development and resistance to therapies. To date, two Cullin RING Ligase (CRL) complexes E3s have been described to control CHK1 abundance including the SKP1-Cullin1(Cul1)-Fbx6 complex, preferentially ubiquitylating CHK1 in the nucleus (5), and the other forming the CDT2-Cullin4A(Cul4A)-DDB1 complex preferentially targeting CHK1 in the cytoplasm (6) (referee to Figure 1). However, questions still remain as to the subcellular localization, and the cell cycle phases during which these various E3 ligase complexes intervene. To this purpose, it would be interesting to investigate whether the stability of CHK1 protein is co-ordinately regulated by these multiple E3 ligase complexes and whether crosstalks exist between HUWE1 and other E3 ligases involved in CHK1 regulation, which have probably not yet been identified. Furthermore, although these E3 ligases are mainly involved in the addition of K48-linked poly-ubiquitination, HUWE1 is a multifaceted HECT domain-containing ubiquitin E3 ligase, which catalyses both mono-ubiquitination and K6-, K48- and K-63-linked poly-ubiquitination of its substrates. It would be interesting to specify the context and the conditions that favour the addition of K48 versus K9 or K63 linkages on CHK1 protein, as the latter linkage types create specific poly-ubiquitin topographies involved in a wider variety of regulations, including signal transduction, protein localization, DNA repair, endocytosis, and protein-protein interaction (7). Furthermore, these poly-ubiquitination linkage topographies may depend on the phosphorylation status of the CHK1 protein. On this specific aspect, Cassidy et al. demonstrate that HUWE1 preferentially targets active CHK1 (S296 auto-phosphorylated form, referee to Figure 1B and 7) as the poly-ubiquitinated lysines identified are mainly located in regions of the CHK1 protein that are known to be inaccessible in its closed-inactive conformation (referee to Figure 5C and Table 1). Interestingly, these poly-ubiquitination modifications on CHK1 by HUWE1 E3 ligase are preferentially K48-linked poly-ubiquitin chains, targeting the kinase for proteasome degradation, K6 and K63 ubiquitin linkages have also be detected, consistent with previous analyses (8,9). This opens the way for exciting and potential new mechanisms of CHK1 regulation.
Moreover, whether other post-transcriptional modifications, partners or adaptor proteins, such as claspin are required for this ubiquitinylation should be investigated in the near future, as suggested by observations from Cassidy et al. (referee to Figure 7).
Ubiquitination modification is a tightly regulated dynamic process, and is antagonized by deubiquitylating enzymes (DUBs). These enzymes can interact with E3 ligases and their substrates and thus regulate their stability and/or their addressee to the proteasome. At least 3 Ubiquitin Specific Proteases (USP), USP1, 3 and 7, have been reported to be able to cleave the ubiquitin chain on CHK1 and promote its stabilization (10, 11, 12, referee to Figure 1). It would be interesting (I) to understand the regulatory mechanisms involved that dictate protein fate; (II) to identify whether a specific deubiquitylating enzyme interacts and works together with HUWE1 E3 ligase, as it has been shown for USP7 and the E3 ubiquitin ligase, Mouse Double Minute 2 homolog, MDM2, and the mitotic E3 ubiquitin ligase, checkpoint with forkhead and Ring-finger, CHFR (13, 14).
Furthermore, the work of Cassidy et al. provides interesting data on the modes of regulation of CHK1 protein stability. Authors have shown that CHK1 itself could control its stability by phosphorylation of the last 60 amino acids of the HECT domain of the HUWE1 E3 ubiquitin ligase (referee to Table 3). It would be important to establish whether CHK1 is able to prime and regulate its own ubiquitinylation in specific contexts, such as unperturbed conditions or in response to a replicative stress. Finally, CHK1 has been found to be overexpressed in a variety of human tumors, and plays an essential role in cellular proliferation and resistance to replicative stress (15). On the other hand, HUWE1 is frequently overexpressed in solid tumors, but can be downregulated in brain tumors (7). Depending on the substrate and the context, HUWE1 is implicated in controlling proliferation and differentiation, apoptosis, DNA repair, and responses to stress. Understanding the cell-specific functions of HUWE1 and its relationship with the CHK1 deserves to be investigated particularly in response to DNA damage conditions. In agreement with this point, Cassidy et al. reported that the interaction between CHK1 and the HECT domain of the HUWE1 E3 ligase is strengthened in response to replicative stress (Figure 7B). Altogether, Cassidy et al., provide interesting new reports and information on the regulation of the CHK1 protein by the HUWE1 E3 ligase and highlight for the first time an auto-regulatory mechanism controlled by CHK1 itself that may help us to understand the underlying mechanism of tumorigenesis. References 1-Guo Z, Kumagai A, Wang SX, Dunphy WG (2000) Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev. 14(21):2745-56.
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Figure Legends
Regulation of CHK1 by the Ubiquitin Proteasome System and cellular fate.
Several transcriptional and post-transcriptional pathways that regulate CHK1 protein have been described, including mechanisms that positively or negatively affect its stability, activity, localization and protein-protein interactions. Deregulation of the ubiquitination and deubiquitination process may underlie the heterogeneity of CHK1 expression observed in tumors. CHK1 is degraded by the Ubiquitin-Proteasome System. Ubiquitin E3 ligases involved in CHK1 ubiquitination and degradation during normal cell cycle progression or in response to DNA damage include two Cullin RING Ligase (CRL) complexes (CDT2-CUL4-DDB1 and SKP1-CUL1-Fbx6) and the HECT E3 ligase HUWE1. Inversely, stabilization of CHK1 by the Ubiquitin Specific Proteases (USP), USP1, USP3 and USP7 has also been described. Acknowledgements The authors would like to thank fellow group members, and Dr Stéphane Manenti for ongoing support and stimulating insights. Authors Contributions MC and CD wrote the manuscript. Conflict of interest The authors declare no conflict of interest.
