The G2 checkpoint-a node-based molecular switch

Abstract

Tight regulation of the eukaryotic cell cycle is paramount to ensure genomic integrity throughout life. Cell cycle checkpoints are present in each phase of the cell cycle and prevent cell cycle progression when genomic integrity is compromised. The G2 checkpoint is an intricate signaling network that regulates the progression of G2 to mitosis (M). We propose here a node-based model of G2 checkpoint regulation, in which the action of the central CDK1-cyclin B1 node is determined by the concerted but opposing activities of the Wee1 and cell division control protein 25C (CDC25C) nodes. Phosphorylation of both Wee1 and CDC25C at specific sites determines their subcellular localization, driving them either toward activity within the nucleus or to the cytoplasm and subsequent ubiquitin-mediated proteasomal degradation. In turn, this subcellular balance of the Wee1 and CDC25C nodes is directed by the action of the PLK1 and CHK1 nodes via what we have termed the 'nuclear and cytoplasmic decision states' of Wee1 and CDC25C. The proposed node-based model provides an intelligible structure of the complex interactions that govern the decision to delay or continue G2/M progression. The model may also aid in predicting the effects of agents that target these G2 checkpoint nodes.

Keywords: CDC25C; CHK1; G2 checkpoint; PLK1; Wee1; cell cycle.

Figure 1

The nodal basis of the G2 checkpoint. The action of the central CDK1‐cyclin B1 node (clear box) is determined by the concerted but opposing activities of the Wee1 and CDC25C primary regulatory nodes (dashed boxes). In their turn, the PLK1 and CHK1 secondary regulatory nodes (gridded boxes) direct the action of the Wee1 and CDC25C nodes by phosphorylation at specific sites that determine their subcellular localization, either driving them toward nuclear accumulation and activity or cytoplasmic localization and subsequent ubiquitin‐mediated proteasomal degradation.

Figure 2

The PLK1 node. Upon successful DNA repair completion, PLK1 is activated by aurora kinase A (AURKA) in cooperation with hBora through phosphorylation of its Threonine‐210 residue. Following activation, PLK1 can promote G2/M progression by driving nuclear accumulation and CDK1 target activity of CDC25C and signaling Wee1 for ubiquitin‐mediated proteasomal degradation.

Figure 3

The CHK1 node. CHK1 is translocated to the nucleus by p90 RSK phosphorylation. In the nucleus, CHK1 is activated following DNA damage, either directly by ataxia teleangiectasia‐mutated kinase (ATM), or through its downstream target ATM and Rad3‐related kinase (ATR). The inhibitory activity of CHK1 on G2/M progression is threefold: through activating and nuclearly stabilizing Wee1, through deactivating CDC25C by direct phosphorylation, and through promoting cytoplasmic translocation of CDC25C via activation of PP2A/B56δ. In turn, PP2A/B56γ3 can deactivate CHK1 by dephosphorylation, after which it is primed for nuclear export by CDK1. In the cytoplasm, CHK1 is either targeted for proteasomal degradation by SCF^FBX6 or reshuttled to the nucleus by dephosphorylation.

Figure 4

The Wee1 node. Wee1 undergoes nuclear cytoplasmic cycling that is important for determining its inhibitory effect on G2/M progression through the CDK1 node. Wee1 is shuttled into the nucleus by heat‐shock protein 90α (Hsp90α), where it reaches its NDS. Phosphorylation of the Wee1 NDS by CHK1 promotes CDK1‐directed activity of Wee1, while phosphorylation by CDK1 promotes translocation back to the cytoplasm. In the cytoplasm, Wee1 reaches its CDS that is either primed for ubiquitin‐mediated proteasomal degradation by phosphorylation by the PLK1‐CK2 complex or prepared for re‐entry into the nucleus by dephosphorylation by CDC14A.

Figure 5

The CDC25C node. The CDC25C node is characterized by nuclear cytoplasmic cycling that regulates its effect on promoting G2/M progression. Following successful completion of S and G2 phase, CDK2 primes CDC25C for nuclear translocation that is subsequently facilitated by PP1 and the PLK1‐CK2 complex. When it reaches the NDS, targeting by activated CDK1 stimulates activity of CDC25C toward the CDK1‐Cyclin B1 complex while CHK1 drives cytoplasmic translocation both directly and through PP2A/B56δ. Back into the cytoplasm, at the CDS, CDC25C can either be primed for re‐entry into the nucleus by CDK2 or targeted for ubiquitin‐mediated proteasomal degradation.

Figure 6

The CDK1 node. Nuclear cytoplasmic cycling regulates CDK1 activity. The CDK1‐Cyclin B1 is cytoplasmically activated by the CDK7‐Cyclin H complex and subsequently primed for nuclear translocation by PLK1. In the nucleus, the NDS of CDK1 can be deactivated by Wee1‐mediated phosphorylation, a process that is counteracted by CDC25C‐mediated dephosphorylation. Following Wee1‐mediated inactivation, the CDK1‐Cyclin B1 complex is primed for cytoplasmic translocation, most likely through dephosphorylation of Cyclin B1, where it reaches its CDS. Here, targeting by MYT1 further inactivates CDK1 while phosphatase activity enables re‐entry into the nucleus.

Figure 7

Functional implications of the G2 checkpoint balance. The G2 checkpoint can be considered as a scale, where both arms are represented by the primary regulatory Wee1 and CDC25C nodes that pivot around the central CDK1 node and tip toward either active or inactive CDK1. The arms of the scale are in turn balanced by the secondary regulatory CHK1 and PLK1 nodes that act as either weights or hydraulic pumps. Following completion of S and G2 phase, high activity of the CHK1 node tips the scale in favor of inactive CDK1. With increasing DNA repair, CHK1 node activity gradually diminishes while PLK1 node activity complementarily increases. This shift first levels the balances and ultimately tips the G2 checkpoint in favor of CDK1 activity and G2/M progression.

Figure 8

Functional outcome of the decision states involved in the G2 checkpoint. The NDS and CDS can be seen as conformations of a protein that are targeted by competing factors that determine the functional effect of a node on G2 checkpoint. The competing factors can be regarded as magnets that pull with varying strength, thereby routing the protein to a specific conformation. Subsequently are depicted the (A) Wee1 CDS, (B) Wee1 NDS, (C) CDC25C CDS, (D) CDC25C NDS, (E) CHK1 CDS, (F) CHK1 NDS, (G) CDK1 CDS, and (H) CDK1 NDS.