The MK2 cascade regulates mGluR-dependent synaptic plasticity and reversal learning

YesThe ability to either erase or update the memories of a previously learned spatial task is an essential process that is required to modify behaviour in a changing environment. Current evidence suggests that the neural representation of such cognitive flexibility involves the balancing of synaptic potentiation (acquisition of memories) with synaptic depression (modulation and updating previously acquired memories). Here we demonstrate that the p38 MAPK/MAPK-activated protein kinase 2 (MK2) cascade is required to maintain the precise tuning of long-term potentiation and long-term depression at CA1 synapses of the hippocampus which is correlated with efficient reversal learning. Using the MK2 knockout (KO) mouse, we show that mGluR-LTD, but not NMDAR-LTD, is markedly impaired in mice aged between 4 and 5 weeks (juvenile) to 7 months (mature adult). Although the amplitude of LTP was the same as in wildtype mice, priming of LTP by the activation of group I metabotropic receptors was impaired in MK2 KO mice. Consistent with unaltered LTP amplitude and compromised mGluR-LTD, MK2 KO mice had intact spatial learning when performing the Barnes maze task, but showed specific deficits in selecting the most efficient combination of search strategies to perform the task reversal. Findings from this study suggest that the mGluR-p38-MK2 cascade is important for cognitive flexibility by regulating LTD amplitude and the priming of LTP.Professor Richard Greene at the University of Bradford - startup fund to setup electrophysiological facility and Wellcome Trust 200646/Z/16/Z to S.A.L.C

Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade

Hypo-osmotic swelling of human Intestine 407 cells leads to a significant increase of intracellular MAPKAP-kinase 2 activity and Hsp27 phosphorylation. Pre-treatment of the cells with the p38 MAP kinase inhibitor SB-203580 blocks this activation, indicating that the hypotonicity-induced activation of MAPKAP kinase 2 is, similarly to that described for hyperosmotic treatment, the result of an activated p38 MAP kinase cascade. The activation of MAPKAP kinase 2 proceeds with kinetics similar to that of one of the first physiological responses of hypo-osmotic treatment, the opening of compensatory Cl- channels. However, inhibition of the p38 MAP kinase cascade does not block the osmo-sensitive anion efflux and, vice versa, activation of p38 MAP kinase by cytokines and anisomycin does not increase the efflux. These results indicate that the p38 MAP kinase cascade is not directly involved in Cl- channel activation but instead may play a role in subsequent cellular repair processes

P38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level

The cytokine tumor necrosis factor (TNF) alpha was found to stimulate the p38 mitogen activated protein (MAP) kinase signalling cascade in human umbilical vein endothelial cells. TNFalpha increased the activity of the p38 substrate MAP kinase-activated-protein (MAPKAP) kinase 2 and the subsequent phosphorylation of the small heat shock protein Hsp27 about two to three fold. This stimulation was blocked almost completely by the specific p38 MAP kinase inhibitor SB203580. This inhibitor also suppressed the TNFalpha-induced surface expression of the endothelial adhesion molecule vascular cell adhesion molecule (VCAM)-1. In contrast, inhibition of p38 MAP kinase had no effect on the stimulated surface expression of the intercellular cell adhesion molecule (ICAM)-1. VCAM-1 mRNA accumulation induced by TNFalpha was not affected by SB203580, suggesting that the p38 MAP kinase signalling cascade regulates the endothelial expression of VCAM-1 at the post-transcriptional level

Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation

Post-translational modifications control the physiological activity of the signal transducer and activator of transcription STAT1. While phosphorylation at tyrosine Y701 is a prerequisite for STAT1 dimerization, its SUMOylation represses the transcriptional activity. Recently, we have demonstrated that SUMOylation at lysine K703 inhibits the phosphorylation of nearby localized Y701 of STAT1. Here, we analysed the influence of phosphorylation of Y701 on SUMOylation of K703 in vivo. For that reason, an Ubc9/substrate dimerization-dependent SUMOylation (USDDS) system was developed, which consists of fusions of the SUMOylation substrate and of the SUMO-conjugating enzyme Ubc9 to the chemically activatable heterodimerization domains FKBP and FRB, respectively. When FKBP fusion proteins of STAT1, p53, CRSP9, FOS, CSNK2B, HES1, TCF21 and MYF6 are coexpressed with Ubc9-FRB, treatment of HEK293 cells with the rapamycin-related dimerizer compound AP21967 induces SUMOylation of these proteins in vivo. For STAT1-FKBP and p53-FKBP we show that this SUMOylation takes place at their specific SUMOylation sites in vivo. Using USDDS, we then demonstrate that STAT1 phosphorylation at Y701 induced by interferon-β treatment inhibits SUMOylation of K703 in vivo. Thus, pY701 and SUMO-K703 of STAT1 represent mutually exclusive modifications, which prevent signal integration at this molecule and probably ensure the existence of differentially modified subpopulations of STAT1 necessary for its regulated nuclear cytoplasmic activation/inactivation cycle

The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in the p38/TNF-α Pathway of Systemic and Cutaneous Inflammation

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-α, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-α levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-α and IL-1β levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-α neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-α production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-α, has a rather elusive role in T-cell-dependent cutaneous inflammation

The MK2 cascade mediates transient alteration in mGluR-LTD and spatial learning in a murine model of Alzheimer's disease

YesA key aim of Alzheimer disease research is to develop efficient therapies to prevent and/or delay the irreversible progression of cognitive impairments. Early deficits in long-term potentiation (LTP) are associated with the accumulation of amyloid beta in rodent models of the disease; however, less is known about how mGluR-mediated long-term depression (mGluR-LTD) is affected. In this study, we have found that mGluR-LTD is enhanced in the APPswe /PS1dE9 mouse at 7 but returns to wild-type levels at 13 months of age. This transient over-activation of mGluR signalling is coupled with impaired LTP and shifts the dynamic range of synapses towards depression. These alterations in synaptic plasticity are associated with an inability to utilize cues in a spatial learning task. The transient dysregulation of plasticity can be prevented by genetic deletion of the MAP kinase-activated protein kinase 2 (MK2), a substrate of p38 MAPK, demonstrating that manipulating the mGluR-p38 MAPK-MK2 cascade at 7 months can prevent the shift in synapse dynamic range. Our work reveals the MK2 cascade as a potential pharmacological target to correct the over-activation of mGluR signalling.Wellcome Trust, Grant/Award Number: 200646/Z/16/

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling

Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function

The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38MAPK, extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38MAPK, ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phosphomimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38MAPK, ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5

The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC50 = 37.5 μM; Ki = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values