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

The p38/MK2/Hsp25 Pathway Is Required for BMP-2-Induced Cell Migration

Background: Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. Methodology/Principal Findings: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2¿s physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38¿ or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. Conclusions: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration

A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses

Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements

A considerable number of genes that code for AU-rich mRNAs including cytokines, growth factors, transcriptional factors, and certain receptors are involved in both chronic inflammation and cancer. Overexpression of these genes is affected by aberrations or by prolonged activation of several signaling pathways. AU-rich elements (ARE) are important cis-acting short sequences in the 3′UTR that mediate recognition of an array of RNA-binding proteins and affect mRNA stability and translation. This review addresses the cellular and molecular mechanisms that are common between inflammation and cancer and that also govern ARE-mediated post-transcriptional control. The first part examines the role of the ARE-genes in inflammation and cancer and sequence characteristics of AU-rich elements. The second part addresses the common signaling pathways in inflammation and cancer that regulate the ARE-mediated pathways and how their deregulations affect ARE-gene regulation and disease outcome

Processing results from parametric drill hole Kolguev-1-4 drilled on the Peschanoozerskaya Structure on Kolguev Island (Report 5911, Leningrad)

Project 2/819 Proceeding of materials obtained from the parametric drill holes on the Peschanoozerskaya Structure (on Arctic islands). Area of work was the north-eastern part of the Kolguev Island (Barents Sea) in the arch part of the positive Peschanoozerskaya Structure detected during seismic survey. Reasons for drilling: 1. Comprehensive study of the geological-geophysical section of the shelf in the area of the Kolguev Island. Study of stratal waters. 2. Identification of the main regularities and features of the geological structure in the drill section. 3. According to the program of drilling on islands and coasts of Arctic seas for creating a network of geological reference points for reliable referencing of geophysical data. Technical results: The hole has penetrated the section up to 3730 m and stopped in Early Ordovician deposits. Scientific results: 1. A lithologic and stratigraphic sequence of sedimentary rocks (based on different fauna groups) from Early Ordovician to Neogene-Quaternary deposits has been carried out in the studied geological section. Composition of rocks has been studied, rock complexes have been specificated and their geophysical characteristics have been given. 2. Thicknesses of the units are as followed: Ordovician 134 m, Devonian 479 m, Carboniferous 302 m, Permian 1052 m, Triassic 1053 m, Jurassic 247 m, Cretaceous 367 m, Neogene-Quaternary 96 m. 3. According to rock types the section can be divided to three series. The lower series 3117-3730 m chiefly terrigenous series accumulated from the Early Ordovician to the Late Devonian. The middle series 2702-3117 m chiefly carbonaceous series accumulated from the Carboniferous to the Early Permian. The upper series 0-2702 m chiefly terrigenous accumulated from the Early Permian to the Cenozoic. The middle and upper series form a platform mantle; the lower series is transitional between the platform mantle and the folded basement. 4. The series are heterogeneous; each of them can be subdivided. In the base of the lower series (3636-3730 m) Early Ordovician quartzitic sandstones occur. They are overlain by Early Ordovician iron-bearing siltstones occurring in the interval 3596-3636 m. In the lower part of the middle series dolomites with interlayers of anhydrites, limestones and terrigenous mottled rocks occur. In the upper part limestones with interlayers of terrigenous and terrigenous-carbonaceous rocks prevail. The upper series is chiefly composed of terrigenous deposits. Its lower part (the Artinskian and Kungurian stages) is transitional with the significant role of carbonaceous rocks. The upper part comprises silty-sandy and silty-clayey deposits. 5. The hole Kolguev-1-4 has been drilled in the arch part of the positive Peschanoozerskaya structure. That is why thickness of the Triassic deposits in this hole is higher than in the hole Kolguev-3 drilled in the side part of the structure. 6. Angular and stratigraphic disconformity has been identified at the boundary of the Early Ordovician and Late Devonian. The Ordovician deposits have an angle of inclination of 40-50°. The overlain deposits lay almost horizontally. 7. Stratigraphic disconformities also occur at the boundaries Devonian-Carboniferous, Early-Middle Carboniferous, Permian-Triassic, Triassic-Jurassic, and Mesozoic-Cenozoic. 8. Many petrophysical relations are similar to ones indicated earlier in the holes drilled on the Franz Joseph Land and on the continent. 9. The obtained data indicate belonging of the area to the Pechora tectonic depression

Processing results from parametric drill hole Kolguev-3 drilled on the Peschanoozerskaya Structure on Kolguev Island (Report 5911, Leningrad)

Project 2/819 Proceeding of materials obtained from the parametric drill holes on the Peschanoozerskaya Structure (on Arctic islands). Area of work was the north-eastern part of the Kolguev Island (Barents Sea) in the side part of the positive Peschanoozerskaya Structure detected during seismic survey. Reasons for drilling: 1. Comprehensive study of the geological-geophysical section of the shelf in the area of the Kolguev Island. Study of stratal waters. 2. Identification of the main regularities and features of the geological structure in the drill section. 3. According to the program of drilling on islands and coasts of Arctic seas for creating a network of geological reference points for reliable referencing of geophysical data. Technical results: The hole has penetrated the section up to 3507 m and stopped in Late Devonian deposits. Scientific results: 1. A lithologic and stratigraphic sequence of sedimentary rocks (based on different fauna groups) from Late Devonian to Neogene-Quaternary deposits has been carried out in the studied geological section. Composition of rocks has been studied, rock complexes have been specificated and their geophysical characteristics have been given. 2. Thicknesses of the units are as followed: Late Devonian - 7 m, Carboniferous - 320 m, Permian 1088 m, Triassic - 1317 m, Jurassic - 260 m, Cretaceous - 368 m, Neogene-Quaternary - 96 m. 3. According to rock types the section can be divided to two series. The lower series 3133-3507 m corresponds to the middle series in Hole Kolguev-1-4 and chiefly consists of carbonaceous series accumulated from the Carboniferous to the Early Permian. The upper series 0-3133 m corresponds to the upper series in Hole Kolguev-1-4 and chiefly consists of terrigenous accumulated from the Early Permian to the Cenozoic. The two series form a platform mantle. 4. The series are heterogeneous; both of them can be subdivided. In the lower part of the lower series dolomites with interlayers of anhydrites, limestones and terrigenous mottled rocks occur. In the upper part limestones with interlayers of terrigenous and terrigenous-carbonaceous rocks prevail. The upper series is chiefly composed of terrigenous deposits. Its lower part (the Artinskian and Kungurian stages) is transitional with the significant role of carbonaceous rocks. The upper part comprises silty-sandy and silty-clayey deposits. 5. The hole Kolguev-3 has been drilled in the side part of the positive Peschanoozerskaya structure. That is why thickness of the Triassic deposits in this hole is less than in the hole Kolguev-1-4 drilled in the arch part of the structure. 6. Stratigraphic disconformities occur at the boundaries Devonian-Carboniferous, Early-Middle Carboniferous, Permian-Triassic, Triassic-Jurassic, and Mesozoic-Cenozoic. 7. Many petrophysical relations are similar to ones indicated earlier in the holes drilled on the Franz Joseph Land and on the continent. 8. The obtained data indicate belonging of the area to the Pechora tectonic depression

ZDHHC3 tyrosine phosphorylation regulates NCAM palmitoylation

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

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