MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK).Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.Downstream of mitogen-activated protein kinases (MAPKs) different groups of MAPK-activated protein kinases (MAP KAPKs) have been defined (reviewed in reference 28). These enzymes transduce signals to target proteins that are not direct substrates of the MAPKs and, therefore, serve to relay phosphorylation-dependent signaling within MAPK cascades to diverse cellular functions. One of these groups is formed by the three MAPKAPKs, MK2, MK3 (also known as 3pK), and MK5 (also designated PRAK) (reviewed in reference 12). While MK5 is mainly activated by the atypical MAPK ERK3 (29, 30), the remaining two kinases, MK2 and MK3, are directly downstream of the MAPK p38␣/␤ (7,10,24,27,31). Phosphorylation of MK2 and MK3 by p38␣/␤ at two or three major regulatory sites leads to activation and coupled nuclear export of both enzymes, which are localized in the nucleus of resting cells (4,8,26,36,41).A wide variety of substrates has been described for MK2 including proteins interacting with the cytoskeleton, such as small heat shock protein Hsp25 (33); mRNA-binding proteins, such as tristetraprolin (TTP) (6, 32); transcription factors, such as heat shock factor 1 (38); and regulators of the cell cycle and apoptosis, such as Cdc25B/C (23). The phosphorylation site recognition motifs of MK2 and MK3 are similar (20) or even identical (7). Despite the similar recognition motif, not all MK2 substrates have been described as MK3 substrates so far, probably because in most cells MK2 activity dominates and makes analysis of the minor MK3 activity dependent on antibodies which discriminate between both enzymes (7).MK2-deficient mice are more resistant than wild type to endotoxic shock due to impaired production of cytokines such as tumor necrosis factor (T...

TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU–rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE–binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP's own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.

Receptor-interacting protein kinase-1 (RIPK1), a master regulator of cell fate decisions, was identified as a direct substrate of MAPKAP kinase-2 (MK2) by phosphoproteomic screens using LPS-treated macrophages and stress-stimulated embryonic fibroblasts. p38/MK2 interact with RIPK1 in a cytoplasmic complex and MK2 phosphorylates mouse RIPK1 at Ser321/336 in response to pro-inflammatory stimuli, such as TNF and LPS, and infection with the pathogen Yersinia enterocolitica. MK2 phosphorylation inhibits RIPK1 autophosphorylation, curtails RIPK1 integration into cytoplasmic cytotoxic complexes, and suppresses RIPK1-dependent apoptosis and necroptosis. In Yersinia-infected macrophages, RIPK1 phosphorylation by MK2 protects against infection-induced apoptosis, a process targeted by Yersinia outer protein P (YopP). YopP suppresses p38/MK2 activation to increase Yersinia-driven apoptosis. Hence, MK2 phosphorylation of RIPK1 is a crucial checkpoint for cell fate in inflammation and infection that determines the outcome of bacteria-host cell interaction.

Most dendritic cell (DC) responses to Toll-like receptor (TLR) ligands depend on the activation of mitogen-activated protein kinases (MAPKs), but the contributions of the many MAPK-activated kinases (MKs) that act 'downstream' of the MAPKs Erk and p38 are not known. Here we sought to determine which MKs are required for acute TLR-driven, MAPK-dependent DC endocytic responses. Two specific and structurally different inhibitors of the MK Rsk suppressed TLR-induced endocytosis, thus defining in DCs a specific requirement for MKs in TLR responses. In addition, we identify in DCs a previously unknown configuration of the MAPK system whereby Rsk is activated not only by Erk but also by p38 through the intermediates MK2 and MK3. Thus, in DCs, p38 contributes to the activation of all known MK families.

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The MAPK-activated protein kinases MK2 and MK3 form a pair of structurally and functionally closely related enzymes present in mammals and birds. Both protein kinases can bind to p38alpha MAPK and are activated by p38alpha via multiple proline-directed phosphorylations in a stress-dependent manner. Although the expression level and activity of MK2 is always significantly higher than that of MK3, the substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. Functional differences between MK2 and MK3 could result from the more prominent proline-rich SH3-targeting region in MK2, but are not reported so far. Since MK2 and MK3 are the main downstream targets of p38alpha responsible for posttranscriptional stimulation of cytokine biosynthesis, both enzymes are promising targets for the development of small molecule inhibitors which can be used in anti-inflammatory therapy. MK2-knockout mice show decreased LPS-induced cytokine biosynthesis and increased protection against collagen-induced arthritis. Recently generated MK2/3 double knockout mice show further reduction of LPS-induced cytokine production.

Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38α MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38α level and p38-dependent IEG expression. Unexpectedly, restoration of p38α does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induction of SRE-dependent reporter activity is impaired and can only be rescued by catalytically active MK2 in MK2/3-deficient cells. Hence, a new function of MKs in transcriptional activation of IEGs via the p38α-MK2/3-SRF-axis is proposed which probably cooperates with MKs’ role in posttranscriptional gene expression in inflammation and stress response.

The protein kinase TAK1 (transforming growth factor-beta-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38alpha MAPK down-regulates TAK1 and showed that p38alpha MAPK phosphorylates TAB1 at Ser(423) and Thr(431). In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser(372) and Ser(524)) and three on TAB3 (Ser(60), Thr(404) and Ser(506)) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser(372) and Ser(524) of TAB2 are not phosphorylated by pathways dependent on p38alpha/beta MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser(60) and Thr(404) of TAB3 appear to be phosphorylated directly by p38alpha MAPK, whereas Ser(506) is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38alpha MAPK. Studies using TAB1(-/-) MEFs indicate important roles for TAB1 in recruiting p38alpha MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser(60) and Thr(404) and in inhibiting the dephosphorylation of TAB3 at Ser(506). TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFalpha was able to stimulate detectable TAK1 activity in TAB1(-/-) MEFs. Surprisingly, the IL-1 and TNFalpha-stimulated activation of MAPK cascades and IkappaB (inhibitor of nuclear factor kappaB) kinases were similar in TAB1(-/-), MEKK3(-/-) [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFalpha-induced activation of these signalling pathways in TAB1(-/-) and MEKK3(-/-) MEFs.