It has been proposed that MK2 is a third checkpoint kinase, with a function analogous to Chk1 (Manke et al., 2005). The very similar substrate specificity clearly supports this view.
In contrast, we found that genotoxic effects of Chk1 depletion or inhibition are rescued by depletion or inhibition of MK2. These results are in line with a study in HeLa cells that also found an antagonistic relationship between the two kinases (Xiao et al., 2006). This demonstrates that the genotoxic effects of Chk1 impairment depend on MK2 and suggests that a substantial subset of MK2 substrates is not shared by Chk1. How can the antagonistic action of the two kinases despite similar phosphorylation motifs be explained?
One possible mechanism for distinct functions of MK2 and Chk1 was brought up by the discovery that MK2 is exported into the cytoplasm upon certain kinds of genotoxic stress (treatment with cisplatin and doxorubicin) while Chk1 remains nuclear (Reinhardt et al., 2010). It is important to note, however, that neither nucleoside analogs nor UV irradiation were used as sources of DNA damage in that study. Both induce DNA damage signaling very different from that elicited by cisplatin and doxorubicin, and our results show that MK2 is retained in the nucleus following exposure to these agents (Figure V.20).
This finding argues that different subcellular localization cannot account for the different effects we observe for MK2 and Chk1 in the DDR. Rather, the fact that we find a dominant negative effect of ectopically expressed MK2 carrying a mutation of a potential ATM/ATR phosphorylation site and that p38 inhibition does not reduce H2AX phosphorylation following DNA damage suggests a more complex regulation of MK2 localization: Upon general stress, e.g. by sorbitol treatment, MK2 is solely activated by p38, resulting in nuclear export and phosphorylation of cytosolic Hsp27. Upon genotoxic stress caused by UV irradiation or gemcitabine, MK2 is additionally modified by a second pathway (possibly ATM/ATR acting on MK2 T294), which impairs complete nuclear export and thus increases the nuclear activity of MK2, entailing reduced phosphorylation of H2AX.
Another reason for functional divergence between the two kinases might be that their substrate phosphorylation motifs are similar but not identical. While both kinases require a large hydrophobic residue in the -5 position of their substrate and arginine in the -3 position, the other positions, although less important for phosphorylation, display differences: MK2 favors glutamine in the -2 and leucine or asparagine in the -1 site whereas Chk1 prefers tyrosine and glutamate, respectively (Manke et al., 2005). This divergence, however, may account for minor differences in the substrate spectrum but is unlikely to result in completely different functionality.
Finally, if both kinases are localized to the nucleus, share a similar substrate specificity and are activated independently from each other upon DNA damage, it seems most convincing to us that their antagonistic action results from a different spectrum of interaction partners. Apart from their substrate specificity MK2 and Chk1 are structurally unrelated and have different domains that mediate protein-protein interactions. MK2, for instance, can interact with proteins harboring an SH3-domain via its proline-rich N-terminus. However, significant interactions with SH3-domains have not been found for MK2 so far. Interaction with other proteins could also be mediated by p38, which forms a stable complex with MK2. Chk1, on the other hand, contains a PIP-box that facilitates interaction with PCNA. In fact, it has been demonstrated that this PIP-box is required for the DNA damage-induced release of Chk1 from chromatin and also to promote replication fork progression (Speroni et al., 2012). The interaction partners are thus likely responsible to direct MK2 and Chk1 to their specific substrates by either mediating direct interaction between kinase and substrate or by controlling the kinases’ sub-nuclear localization, increasing their local concentration and bringing them in close proximity to potential substrates.
The divergence between MK2 and Chk1 is well exemplified by their separate roles in the regulation of Hsp27 and origin firing.
Hsp27 S82 is exclusively phosphorylated by MKs in response to stress. It is not only localized in the cytoplasm but was also found in sub-nuclear structures (Vos et al., 2009) and could thus be expected to be a substrate of Chk1, as well. However, a dependence of Hsp27 phosphorylation on Chk1 has not been observed so far, and in our hands Chk1 inhibition or depletion does not decrease Hsp27 phosphorylation (Figure V.13). On the contrary, we observe an increase of Hsp27 pS82 upon Chk1 inhibition, which is likely attributed to increased replicative stress. This finding illustrates that, in the case of MK2 and Chk1, identical in vivo substrates cannot be predicted from a shared phosphorylation motif.
The same holds true for their role in origin firing. Chk1 is a master regulator of origin firing whereas MK2 does not seem to play a direct role in this process. This is also supported by our observation that the effect of Wee1 depletion, which also leads to deregulated origin firing (Sorensen and Syljuasen, 2012), does not depend on MK2 (Figure V.14). As detailed before, the increased origin firing observed upon Chk1 depletion or inhibition is caused by activation of previously inactive origin clusters as well as stochastic firing of dormant origins due to replication fork stalling (Blow and Ge, 2009; Ge and Blow, 2010).
Opposed to Wee1, Chk1 has been directly implicated in fork stabilization. Since we could not identify a role for MK2 in the regulation of various factors involved in origin firing (Figure V.19), the rescue of origin firing upon MK2 inhibition is probably indirect and attributed to MK2 activity directed towards fork destabilization by repression of TLS. Thus, origin firing could serve as an example for very distinct functions of MK2 and Chk1, the former destabilizing forks via TLS repression, the latter regulating origin firing and stabilizing forks. The target of MK2 via which it exhibits its control on TLS, however, remains to be identified.
Further support for substantial functional differences between Chk1 and MK2 in spite of similar substrate specificity comes from mice in which loss of Chk1 confers embryonic processes than known so far, and thus new putative substrates emerge.