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  • Chk and Chk are functionally redundant protein kinases that

    2021-04-19

    Chk1 and Chk2 are functionally redundant protein kinases that respond to checkpoint signals emanating from the phosphatidylinositol 3-kinase family members ATM (ataxia-telangiectasia mutated) and ATR (Ataxia-telangiectasia and Rad-3 related). A concerted research effort has revealed many mechanistic details of how Chk1 and Chk2 keep a lid on the Pandora's box of DNA damage. Chk1 is activated by bulky DNA lesions and in response to replication fork collapse during S phase of the cell cycle. In contrast, Chk2 responds primarily to DNA double-strand breaks in DNA. The Yaffe group and others have implicated a third kinase pathway, p38/MK2, downstream of ATM and ATR that elicits checkpoint arrest (Bulavin et al., 2001, Manke et al., 2005, Reinhardt et al., 2007). Adding to the intrigue, Chk1, Chk2, and MK2 share identical phosphorylation site preferences and target the same substrates in vitro (O'Neill et al., 2002). So what is the point of having these seemingly redundant checkpoint kinase pathways? This conundrum set the stage for the next chapter in the story. In this issue, Yaffe, Reinhardt, and colleagues shed new mechanistic light on this element of checkpoint control. This elegant, comprehensive, and carefully controlled study demonstrates that Chk1 and MK2 perform different spatial and temporal roles in the establishment of the G2/M checkpoint in p53-deficient cells. This work began with an examination of how each kinase drives rac inhibitor re-entry following checkpoint release. Upon depletion of Chk1 with shRNA in doxorubicin-treated cells, cells failed to establish a complete G2 checkpoint. Conversely, depletion of MK2 led to disruption of long-term G2 checkpoint maintenance. Based on these observations, the authors speculated that Chk1 was necessary for the initiation (and/or early maintenance) of the G2 checkpoint, whereas MK2 is required to sustain this checkpoint at later times. The authors next asked whether these different temporal responses might be due to dynamic changes in subcellular localization of Chk1 or MK2. This turned out to be the case. Following doxorubicin treatment, GFP-tagged MK2 translocated from the nucleus to the cytoplasm while the nuclear localization of GFP-Chk1 was unchanged. These results were substantiated by other techniques including immunofluorescence detection of the endogenous enzymes and a series of convincing cell fractionation studies. Moreover, using a clever reciprocal chimera strategy, Reinhardt and colleagues demonstrated that the Chk1 kinase domain can compensate for the loss of MK2 if targeted to the proper cellular compartment or vice versa. The conclusion of these experiments was that Chk1 and MK2 regulate different temporal phases of checkpoint control through phosphorylation of spatially distinct substrates (Figure 1). Previous studies have demonstrated that MK2 can stabilize mRNAs with AU-rich elements (AREs) in the 3′ untranslated region (UTR) (Gaestel, 2006, Janes et al., 2008, Neininger et al., 2002). Reinhardt and colleagues extended these findings by screening for molecules involved in cell-cycle regulation that contain 3′ AREs. This led to the identification of Gadd45α. DNA damage by doxorubicin led to the accumulation of Gadd45α mRNA, and this increase was blunted by shRNA depletion of MK2. Furthermore, loss of Gadd45α resulted in a checkpoint maintenance failure tightly mirroring the effect seen with the loss of MK2. Subsequent searches for RNA-binding proteins (RBPs) that interact with the 3′UTR of Gadd45α led to the identification of hnRNP A0. The authors then linked posttranscriptional RNA regulation back to kinase signaling by demonstrating that MK2 can phosphorylate hnRNP A0, causing it to bind to the 3′UTR of Gadd45α mRNA stabilizing message levels and resulting in increased Gadd45α proteins levels. At the same time, MK2 phosphorylation of the ribonuclease PARN blocks Gadd45α mRNA degradation. Finally, this manuscript reveals a positive feedback loop where Gadd45α functions through p38/MK2 to maintain Cdc25B/C in the cytoplasm, effectively blocking mitotic entry prior to completion of DNA damage repair (Figure 1).