Here, we analyzed the functional overlap between the two structurally related protein kinases MK2 and MK3 by a gene targeting approach. We provide evidence that both proteins tightly bind to p38, are coexpressed in the cells and tissues analyzed, are activated by the p38 pathway in response to stress (such as LPS or arsenite treatment) with comparable kinetics, and are able to phosphorylate endogenous small heat shock protein Hsp25. It is also demonstrated that in most cells and tissues analyzed (except skeletal muscle), MK3 expression is minor compared to MK2. Furthermore, MK3 enzymatic activity, probably as a result of the relatively low expression of the enzyme, is also minor compared to MK2 in the same cells. This becomes obvious when the activity of both enzymes is monitored by the same antibody against both activated forms of MK2 and MK3 after LPS treatment of peritoneal macrophages and also when the enzymatic activities of WT and MK2-deficient MEFs are compared after arsenite stimulation using an in-gel kinase assay. Hence, it is not surprising that the effects of the deletion of MK3 in the presence of MK2 are minor and difficult to detect.
Therefore, we decided to analyze the role of MK3 in an MK2-deficient background by analyzing how ectopic expression of MK2 and MK3 can rescue MK2 deficiency and by comparing the effects of the MK2 knockout and MK2/MK3 double knockout. We demonstrate that both MK2 and MK3 expression can rescue Hsp25 phosphorylation depending on the catalytic activity of the enzyme, while MK2 and MK3 stabilize p38 to a similar degree as the catalytically inactive mutant of MK2, confirming the idea that binding between both proteins leads to stabilization (17
). Furthermore, stabilization of the same group of LPS-responsive fibroblast transcripts is obtained with ectopic MK2 and MK3, while other transcript stabilities are not changed by both enzymes. It is still open whether the transcript-stabilizing properties of both enzymes in this experimental system result from their catalytic activity or from their stabilization of p38. However, CXCL1 production of these cells can only be significantly increased by catalytically active MK2 and MK3. ARE-containing transcript stability has been previously assessed in a human system by microarray analysis (11
). The study identified several p38 MAPK-stabilized ARE-containing transcripts by means of the p38 inhibitor SB203580. In the experiment shown in Fig. , p38 MAP-stabilized mRNAs can be rescued by MK2 or MK3 (e.g., cxcl1 and Groα) or remain unstable after ActD treatment independent of MK2/MK3 (e.g., JUN B). Although both studies use different experimental designs and cell systems, i.e., pharmacological inhibition of p38 MAPK in a monocytic cell line (THP-1) versus reconstituted MK2-deficient embryonic fibroblasts as shown here, these results suggest that MK2 or MK3 targets a subset of ARE-containing inflammatory mRNAs that are stabilized by the p38 MAPK pathway.
Comparing the MK2 knockout with the MK2/3 double knockout, we could show that MK3 stabilizes the remaining p38 level in the MK2-deficient background and that the role of MK3 in TTP and TNF regulation is qualitatively indistinguishable from the role of MK2. Taking into account that MK2 is an established therapeutic target of inflammatory diseases, such as rheumatoid arthritis (14
), and that small-molecule MK2 inhibitors are under investigation, the finding that MK3 is indistinguishable from MK2 in its inflammatory function would favor MK2/3 dual inhibitors.
The fact that MK2/3-deficient DKO mice are viable and show no obvious defects in embryonic development was unexpected. We anticipated that MK2 and MK3 mutually compensate each other in development, since both enzymes were described to functionally interact with components of the developmentally relevant polycomb complex involved in chromatin remodeling (26
); thus, we expected significant defects in the absence of both enzymes. Possibly a more detailed analysis of, e.g., stem cell development in these animals may be necessary to identify minor but physiologically relevant effects. The further reduction of p38 expression in MK2/3 DKO compared to MK2-deficient tissue is also remarkable. However, the remaining level of p38 is obviously sufficient to compensate the effects of embryonic lethality and placental development described for the p38 knockout mice (1
). This is similar to the finding that hypomorphic alleles of another protein kinase, PDK1, which lead to expression of only 10% of the kinase, can rescue embryonic lethality of that enzyme (18
) and is supported by the observation that arsenite-stimulated ATF1/CREB phosphorylation by the p38 downstream kinases MSK1/2 is not significantly reduced in MK2/3 DKO cells (N. Ronkina, A. Kotlyarov, and M. Gaestel, unpublished data).
From our comparison of MK2 and MK3 here as well as from comparison of their catalytic activities and substrate specificities (7
), their regulation of subcellular localization (8
), and their mode of activation by p38α/β (7
), no significant difference in the functions of both enzymes can be detected so far. This is reminiscent of the situation of p38α and p38β, where p38α displays the major activity and p38β activity is minor and dispensable for signaling of the p38 pathway (3
). In both cases, it is still enigmatic why both related enzymes have been stably maintained during evolution. MK2 and MK3 are both present in birds and mammals, while only one MK2/3 molecule is found in the lower vertebrates (12
). It may well be that there is an exemption from coexpression of both enzymes in one tissue or cell type not tested so far, making only the expressed enzyme essential in these cells. Deletion of the enzyme from these potential cells in mice obviously does not lead to a noticeable phenotype; however, we cannot exclude the possibility that the effect of such specific MK3 function could be detected during a detailed and comprehensive analysis of the MK3-deficient animals. Another possibility could be the activation of one of the two enzymes by an alternative mechanism, activator, and pathway, which is distinct from the p38 pathway and responds to other signals (e.g., of specific Toll-like receptors) and stimuli. However, this pathway and its physiological function are still to be identified.