Natalia Ronkina

The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU-rich element-dependent translation.

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 TTPs 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.

MAPKAP kinases MK2 and MK3 in inflammation: Complex regulation of TNF biosynthesis via expression and phosphorylation of tristetraprolin

Downstream of mitogen-activated protein kinases (MAPKs), three structurally related MAPK-activated protein kinases (MAPKAPKs or MKs) - MK2, MK3 and MK5 - signal to diverse cellular targets. Although there is no known common function for all three MKs, MK2 and MK3 are mainly involved in regulation of gene expression at the post-transcriptional level and are implicated in inflammation and cancer. MK2 and MK3 are phosphorylated and activated by p38(MAPKα,β) and, in turn phosphorylate various substrates involved in diverse cellular processes. In addition to forwarding of the p38-signal by MK2/3, protein complex formation between MK2/3 and p38 mutually stabilizes these enzymes and affects p38(MAPK) signaling in general. Among the substrates of MK2/3, there are mRNA-AU-rich-element (ARE)-binding proteins, such as tristetraprolin (TTP) and hnRNP A0, which regulate mRNA stability and translation in a phosphorylation-dependent manner. Phosphorylation by MK2 stabilizes TTP, releases ARE-containing mRNAs, such as TNF-mRNA, from default translational repression and inhibits their nucleolytic degradation. Here we demonstrate that MK2/3 also contribute to the de novo synthesis of TTP. Whether this contribution proceeds via transcription factors directly targeted by MK2/3 or via chromatin remodeling by the reported binding of MK2/3 to the polycomb repressive complex is still open. A model is proposed, which demonstrates how this new function of transcriptional activation of TTP by MK2/3 cooperates with the role of MK2/3 in post-transcriptional gene expression to limit the inflammatory response.

MK2 and MK3 - a pair of isoenzymes?

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.

Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex

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.

Distinct Functions of the Mitogen-activated Protein Kinase-activated Protein (MAPKAP) Kinases MK2 and MK3 MK2 MEDIATES LIPOPOLYSACCHARIDE-INDUCED SIGNAL TRANSDUCERS AND ACTIVATORS OF TRANSCRIPTION 3 (STAT3) ACTIVATION BY PREVENTING NEGATIVE REGULATORY EFFECTS OF MK3

In LPS-treated macrophages, activation of STAT3 is considered to be crucial for terminating the production of inflammatory cytokines. By analyzing the role of MAPK-activated protein kinase (MK) 2 and MK3 for LPS-induced STAT3 activation in macrophages, the present study provides evidence that MK2 is crucial for STAT3 activation in response to LPS because it prevents MK3 from impeding IFNβ gene expression. Accordingly, LPS-induced IFNβ gene expression is down-regulated in MK2-deficient macrophages and can be reconstituted by additional ablation of the MK3 gene in MK2/3−/− macrophages. This is in contrast to LPS-induced IL-10 expression, which essentially requires the presence of MK2. Further analysis of downstream signaling events involved in the transcriptional regulation of IFNβ gene expression suggests that, in the absence of MK2, MK3 impairs interferon regulatory factor 3 protein expression and activation and inhibits nuclear translocation of p65. This inhibition of p65 nuclear translocation coincides with enhanced expression and delayed degradation of IκBβ, whereas expression of IκBα mRNA and protein is impaired in the absence of MK2. The observation that siRNA directed against IκBβ is able to reconstitute IκBα expression in MK2−/− macrophages suggests that enhanced expression and delayed degradation of IκBβ and impaired NFκB-dependent IκBα expression are functionally linked. In summary, evidence is provided that MK2 regulates LPS-induced IFNβ expression and downstream STAT3 activation as it restrains MK3 from mediating negative regulatory effects on NFκB- and interferon regulatory factor 3-dependent LPS signaling.

Cross talk between the Akt and p38α pathways in macrophages downstream of Toll-like receptor signaling.

ABSTRACT The stimulation of Toll-like receptors (TLRs) on macrophages by pathogen-associated molecular patterns (PAMPs) results in the activation of intracellular signaling pathways that are required for initiating a host immune response. Both phosphatidylinositol 3-kinase (PI3K)–Akt and p38 mitogen-activated protein kinase (MAPK) signaling pathways are activated rapidly in response to TLR activation and are required to coordinate effective host responses to pathogen invasion. In this study, we analyzed the role of the p38-dependent kinases MK2/3 in the activation of Akt and show that lipopolysaccharide (LPS)-induced phosphorylation of Akt on Thr308 and Ser473 requires p38α and MK2/3. In cells treated with p38 inhibitors or an MK2/3 inhibitor, phosphorylation of Akt on Ser473 and Thr308 is reduced and Akt activity is inhibited. Furthermore, BMDMs deficient in MK2/3 display greatly reduced phosphorylation of Ser473 and Thr308 following TLR stimulation. However, MK2/3 do not directly phosphorylate Akt in macrophages but act upstream of PDK1 and mTORC2 to regulate Akt phosphorylation. Akt is recruited to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the membrane, where it is activated by PDK1 and mTORC2. Analysis of lipid levels in MK2/3-deficient bone marrow-derived macrophages (BMDMs) revealed a role for MK2/3 in regulating Akt activity by affecting availability of PIP3 at the membrane. These data describe a novel role for p38α-MK2/3 in regulating TLR-induced Akt activation in macrophages.

p38(MAPK)/MK2-dependent phosphorylation controls cytotoxic RIPK1 signalling in inflammation and infection

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. p38MAPK/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 p38MAPK/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.

Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production

The p38 MAPK (mitogen-activated protein kinase)/MK2 [MAPKAP (MAPK-activated protein) kinase-2] signalling pathway is a major regulator of stress- and cytokine-induced gene expression at the transcriptional and post-transcriptional level. Using phosphoproteomics we identified the ER (endoplasmic reticulum)-associated ubiquitin-conjugating enzyme Ube2j1 as a potential substrate of MK2. We demonstrate that Ube2j1 is phosphorylated in a cytokine-, cytosolic stress- and LPS (lipopolysaccharide)-induced manner. The cytosolic stress-induced phosphorylation of Ube2j1 proceeds at Ser(184), a site described previously to be phosphorylated in response to ER stress, which is located in a perfect MK2 consensus motif. The cytosolic stress-induced phosphorylation of Ube2j1, but not its ER-stress-induced phosphorylation is sensitive to p38/MK2 inhibitors and abrogated in MK2/MK3-deficient cells. In a pull-down assay we demonstrate the interaction of MK2 with Ube2j1 in HEK (human embryonic kidney)-293T cells. Furthermore, MK2 is able to phosphorylate recombinant Ube2j1, but not the S184A mutant in an in vitro kinase assay. These findings strongly suggest that MK2 directly phosphorylates Ube2j1 at Ser(184) upon p38-activating stress in vivo. However, ectopically expressed Ube2j1-S184A mutant displays ubiquitinating activity towards the model substrate ER-synthesized T-cell receptor-α similar to that of the wild-type protein. Interestingly, Ube2j1 is phosphorylated in response to LPS also in macrophages and contributes to MK2-dependent TNFα biosynthesis by a so far unknown mechanism.

Deletion of MK2 signalling in vivo inhibits small Hsp phosphorylation but not diabetic nephropathy

It is supposed that some stress-induced heat shock proteins (Hsps) are regulated through e.g. stimulation of the p38MAPK/MK(MAPKAP)-2 signalling pathway. It has been postulated from in vitro experiments that phosphorylation of Hsp25(rodents)/Hsp27(human), the major phosphorylation substrate of MK2, is responsible for mesangial contractility and glomerular hyperfiltration in the diabetic kidney. To verify this hypothesis in vivo we studied the renal function of nondiabetic and streptozotocin (STZ)-induced, diabetic MK2(-/-) mice in comparison to wild-type (WT) control mice. Following 8 weeks of hyperglycaemia, light microscopy showed increased glomerulosclerosis and tubulointerstitial renal fibrosis in both diabetic study groups. Protein analysis demonstrated that Hsp25 phosphorylation is stimulated upon high-glucose condition but inhibited in the diabetic MK2(-/-) mice. However, we found the kidney-body weight ratio significantly increased in diabetic WT and MK2(-/-) mice. No difference regarding the increased expression of the extracellular matrix proteins and TGF-beta1 between both diabetic study groups was observed. Importantly, diabetic MK2(-/-) mice showed no protection against renal hyperfiltration in the diabetic state and the development of diabetic albuminuria. Although activation of p38MAPK has been previously shown in diabetes mellitus, our results indicate that blockade of the downstream MK2/Hsp25 signalling pathway does not interfere with the development of early diabetic nephropathy.

ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

ABSTRACT The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis.