The mitogen-activated protein kinase (MAPK)-activated protein kinases MK2 and MK3 cooperate in stimulation of tumor necrosis factor biosynthesis and stabilization of p38 MAPK

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.     

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.     

Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.     

Sec6 enhances cell migration and suppresses apoptosis by elevating the phosphorylation of p38 MAPK,MK2, and HSP27

The signaling axis of p38 mitogen-activated protein kinase (p38 MAPK) and MAPK-activated protein kinase 2 (MK2) is the dominant pathway that leads to heat shock protein 27 (HSP27) phosphorylation. After activation of MK2 by p38 MAPK, HSP27 is phosphorylated and depolymerized by MK2, thereby increasing the cell migration and directly interfering with the apoptotic signaling cascades. Sec6 is one of the components of the exocyst complex that is an evolutionarily conserved 8-protein complex. Even though several studies have demonstrated that Sec6 is involved in various cellular physiological functions, the relationship between Sec6 and HSP27 or p38 MAPK during cell migration and apoptosis remains unclear. In the present study, we observed that Sec6 increased the phosphorylation of p38 MAPK through the activation of MAPK kinase 3/6 (MKK3/6). Moreover, Sec6 knockdown suppressed the phosphorylation of HSP27 at Ser⁷⁸ and Ser⁸² sites via suppression of activated MK2. Furthermore, the reduction of phosphorylated HSP27 or p38 MAPK by Sec6 knockdown suppressed cell migration and promoted apoptosis after treatment with tumor necrosis factor-α and cycloheximide. The present study suggested that Sec6 is involved in the enhancement of cell migration and suppression of apoptosis through the activation of HSP27 or p38 MAPK phosphorylation.     

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 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.     

Elimination of protein kinase MK5/PRAK activity by targeted homologous recombination

MK5 (mitogen-activated protein kinase [MAPK]-activated protein kinase 5), also designated PRAK (p38-regulated and -activated kinase), was deleted from mice by homologous recombination. Although no MK5 full-length protein and kinase activity was detected in the MK5 knockout mice, the animals were viable and fertile and did not display abnormalities in tissue morphology or behavior. In addition, these mice did not show increased resistance to endotoxic shock or decreased lipopolysaccharide-induced cytokine production. Hence, MK5 deletion resulted in a phenotype very different from the complex inflammation-impaired phenotype of mice deficient in MK2, although MK2 and MK5 exhibit evolutional, structural, and apparent extensive functional similarities. To explain this discrepancy, we used wild-type cells and embryonic fibroblasts from both MK2 and MK5 knockout mice as controls to reexamine the mechanism of activation, the interaction with endogenous p38 MAPK, and the substrate specificity of both enzymes. In contrast to MK2, which shows interaction with and chaperoning properties for p38 MAPK and which is activated by extracellular stresses such as arsenite or sorbitol treatment, endogenous MK5 did not show these properties. Furthermore, endogenous MK5 is not able to phosphorylate Hsp27 in vitro and in vivo. We conclude that the differences between the phenotypes of MK5- and MK2-deficient mice result from clearly different functional properties of both enzymes.     

MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels.

We demonstrate that lipopolysaccharide-induced tumor necrosis factor (TNF) biosynthesis becomes independent of MAPKAP kinase 2 (MK2) when the AU-rich element (ARE) of the TNF gene is deleted. In spleen cells and macrophages where TNF biosynthesis is restored as a result of this deletion, interleukin (IL)-6 biosynthesis is still dependent on MK2. In MK2-deficient macrophages the half-life of IL-6 mRNA is reduced more than 10-fold, whereas the half-life of TNF mRNA is only weakly decreased. It is shown that the stability of a reporter mRNA carrying the AU-rich 3'-untranslated region (3'-UTR) of IL-6 is increased by MK2. The data provide in vivo evidence that the AU-rich 3'-UTRs of TNF and IL-6 are downstream to MK2 signaling and make MK2 an essential component of mechanisms that regulate biosynthesis of IL-6 at the levels of mRNA stability, and of TNF mainly through TNF-ARE-dependent translational control.     

MAPK-activated protein kinase-2 participates in p38 MAPK-dependent and ERK-dependent functions in human neutrophils

Many neutrophil responses, including chemotaxis, exocytosis, respiratory burst activity and chemokine synthesis, are mediated by p38 MAPK. MAPK-activated protein kinase-2 (MK2) is activated by p38 MAPK in human neutrophils. The present study tested the hypothesis that MK2 mediates multiple p38 MAPK-dependent responses in human neutrophils by comparing the effect of the p38 MAPK inhibitor, SB203580, with an MK2 inhibitory peptide. Both SB203580 and MK2 inhibitory peptide attenuated respiratory burst activity, exocytosis, and chemotaxis. Lipopolysaccharide (LPS)-induced IL-8 production was inhibited by SB203580, but not by the MK2 inhibitory peptide. Inhibition of chemotaxis and respiratory burst activity by SB203580 was less than that of MK2 inhibitory peptide. Inhibition of extracellular signal-regulated kinase (ERK) activity by PD98059 attenuated superoxide release and chemotaxis, and simultaneous treatment with SB203580 and PD98059 demonstrated additive inhibition. ERK phosphorylated MK2 in vitro and activated MK2 in f-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils. These data suggest that MK2 mediates both ERK- and p38 MAPK-dependent neutrophil responses.     

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells.     

p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.     

Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to D-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2(-/-) mice was associated with normal levels of IL-1 beta, IL-6, and IFN-gamma, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1 beta, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2(-/-) mice displayed diminished IFN-gamma synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.     

TRAF6 is a critical signal transducer in IL-33 signaling pathway

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.     

p38 MAP Kinase and MAPKAP Kinases MK2/3 Cooperatively Phosphorylate Epithelial Keratins

The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-Ser⁷³ is catalyzed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38 inhibitors on K8-Ser⁷³ phosphorylation also demonstrated reduced phosphorylations of keratins K18-Ser⁵² and K20-Ser¹³ but not of K8-Ser⁴³¹ or K18-Ser³³. Interestingly, K18-Ser⁵² and K20-Ser¹³ are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-Ser¹³ and K18-Ser⁵² are inhibited by small molecule inhibitors of both p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-Ser¹³ phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-Ser¹³ phosphorylation between the ileum of wild-type and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.     

MAPKAP kinase 2-deficiency prevents neurons from cell death by reducing neuroinflammation--relevance in a mouse model of Parkinson's disease

The inflammatory response in the brain is closely associated with the pathogenesis of degenerative neurological disorders. A role for the p38 stress-activated protein kinase/MAPK-activated protein kinase 2 (MK2) axis in inflammation and apoptosis is well documented. Here, we provide evidence that neurodegeneration can be prevented by eliminating MK2. In primary mesencephalic neuron-glia co-cultures dopaminergic neurons from MK2-deficient (MK2−/−) mice were significantly more resistant to lipopolysaccharide-induced neurotoxicity compared with cells from wild-type mice. This neuroprotection in MK2-deficient cultures was associated with a reduced inflammatory response, especially with reduced production of the inflammatory mediators tumor necrosis factor alpha, keratinocyte-derived chemokine, interleukin-6, and nitric oxide (NO). Interestingly, in primary neuron-enriched cell cultures p38 MAPK, but not MK2, also participates in NO-mediated neuronal cell death. In the MPTP mouse model for Parkinson's disease, MK2-deficient mice show a reduced neuroinflammation and less degeneration of dopaminergic neurons in the substantia nigra after MPTP lesion compared with wild-type mice. In conclusion, our results reveal that MK2 does not directly participate in neuronal cell death, but indirectly contributes to neurodegeneration by the production of neurotoxic substances, such as NO or tumor necrosis factor alpha, from activated glia cells.     

Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.