Classical Mitogen-activated Protein Kinase Research Articles

Abstract 186: Functional characterization of ERK3 mutants existing in human cancers

Abstract Extracellular signal-Regulated Kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer, and plays an important role in promoting cancer migration and invasion. While the link between ERK3 and cancers has been recognized, little is known about ERK3 mutations in cancer progression. In this study, we have investigated ERK3 mutations on R64 and L290 that have been found in cancers of lung, large intestine and skin (COSMIC database). Importantly, mutations of R64C, R64H, L290P and L290V all are located in the kinase domain of ERK3. In order to characterize these mutations, we generated ERK3 mutants with each of these point mutations, and then overexpressed them in HeLa cells with or without stable knockdown of endogenous ERK3. Notably, we found that in comparison with wild type ERK3, all of these cancer-related mutations lead to reduction of phosphorylation at S189 within the activation loop (an indicator of ERK3 kinase activity). While activation loop phosphorylation is well known to result in activation of the classical MAPKs, its effects on the activity and functions of ERK3 remain elusive. To study the functional impact of these ERK3 mutations on cancer cell invasiveness, we performed trans-well migration assays using HeLa cells overexpressing each of these mutants or the wild type ERK3. In line with previous finding, overexpression of ERK3 increased cell migration. Interestingly, both L290P and L290V mutations significantly increased ERK3's ability in promoting cell migration, whereas R64C and R64H mutations resulted in a decrease in cell migration. These results suggest that S189 phosphorylation within the activation loop does not correlate with ERK3's function in promoting cancer cell motility. To conclude, we report for the first time the functional characterization of cancer-related ERK3 mutations in modulating cancer cell migratory properties. Citation Format: Hadel Alsaran, Lobna Elkhadragy, Weiwen Long. Functional characterization of ERK3 mutants existing in human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 186.

Response of MAPK pathway to iron oxide nanoparticles in vitro treatment promotes osteogenic differentiation of hBMSCs

Iron oxide nanoparticles (IONPs) are generally used in multiple biomedical applications. The tissue repair effect of IONPs had been demonstrated in the previous studies of our group, but the underlying mechanism is unclarified. It is well known that stem cell-based therapies show promising prospect in tissue engineering and regenerative medicine, however, whether IONPs could modulate stem cell fate to promote tissue repair is still unclear. Herein, we found that IONPs could promote osteogenic differentiation of human bone-derived mesenchymal stem cells (hBMSCs) in vitro. To insightfully understand the molecular mechanisms, we performed systematic analyses by use of gene microarray assay and bioinformatics analysis, which revealed that gene expression was widely regulated and classical mitogen-activated protein kinase (MAPK) signal pathway was activated by IONPs treatment. As a result, downstream genes of this pathway were regulated to promote osteogenic differentiation. In summary, the present study elucidates a molecular basis explaining how IONPs effect on hBMSCs, which could have many meaningful impacts for stem cells application in regenerative medicine.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

Exosomes derived from SW480 colorectal cancer cells promote cell migration in HepG2 hepatocellular cancer cells via the mitogen-activated protein kinase pathway.

Exosomes are membrane-derived extracellular vesicles that have recently been recognized as important mediators of intercellular communication. In the present study, we investigated the effects of exosomes derived from SW480 colorectal cancer cells in recipient HepG2 hepatocellular cancer cells. We demonstrated that SW480-derived exosomes were taken up by the recipient HepG2 cells via dynamin-dependent endocytosis and were localized to the HepG2 lysosomes. In addition, SW480-derived exosomes induced the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 following their uptake into HepG2 cells. Of note, these changes occurred during the early phase after exosome treatment. Furthermore, SW480-derived exosomes promoted the migration of recipient HepG2 cells in a wound-healing assay, which was suppressed by pretreatment with U0126, an upstream inhibitor of ERK1/2. These results indicated that SW480-derived exosomes activated a classical mitogen-activated protein kinase pathway in recipient HepG2 cells via dynamin-dependent endocytosis and subsequently enhanced cell migration by ERK1/2 activation. Our results provide new insights into the regulation of cellular functions by exosomes.

Activation of the Classical Mitogen-Activated Protein Kinases Is Part of the Shiga Toxin-Induced Ribotoxic Stress Response and May Contribute to Shiga Toxin-Induced Inflammation.

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.

PP-063 TWO CASES WITH PRIMARY IMMUNE DEFICIENCY AND NEOPLASTIC LYMPHOID PROLIFERATION

Sphingosine 1-phosphate (S1P) the product of sphingosine kinase (SK) action plays an important role in various pathological conditions like inflammation and cancer. In this study, we show that in the human breast cancer cell line MCF7, epidermal growth factor (EGF) stimulates SK-1 activity in a biphasic manner with a first peak after 15 min and a second delayed activation occurring after 1 h up to 18 h and thereafter declining again. This delayed activation is accompanied by increased mRNA and protein expression of SK-1, but not SK-2. Mechanistically, the transcriptional upregulation is dependent on the classical mitogen-activated protein kinase, protein kinase C (PKC) and the phosphoinositide 3-kinase, since specific inhibitors of these enzymes all abolish the EGF-induced mRNA upregulation and activity of SK-1. Moreover, dexamethasone also suppressed EGF-induced SK-1 mRNA expression and activity which is reversed by the glucocorticoid receptor antagonist RU486. To see whether EGF-induced upregulation of SK-1 is of relevance for tumor progression, we investigated two hallmarks of carcinogenesis, i.e., cell proliferation and migration. Stimulation of cells with EGF leads to enhanced [3H]thymidine incorporation into DNA and also to stimulated migration in a modified Boyden chamber assay. When cells are depleted of SK-1, but not SK-2, by siRNA transfection or by dexamethasone treatment, EGF-induced proliferation and migration are drastically reduced. In summary, these data show that EGF causes an acute stimulation of SK-1 activity and, moreover, triggers a delayed SK activation which is due to increased gene transcription and de novo synthesis of SK-1, which in turn directs cells towards growth and increased motility. Thus, the sphingosine kinase-1 may represent a novel attractive target for cancer therapy.

Direct phosphorylation and activation of a mitogen-activated protein kinase by a calcium-dependent protein kinase in rice.

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.

The non-classical MAP kinase ERK3 controls T cell activation.

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4+ and CD8+ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

The p38 MAPK inhibitor JLU1124 inhibits the inflammatory response induced by lipopolysaccharide through the MAPK-NF-κB pathway in RAW264.7 macrophages

Our previous results showed that JLU1124 is a potent p38 mitogen-activated protein kinase (MAPK) inhibitor. Compared to the classic p38 MAPK inhibitor SB203580, JLU1124 inhibits p38 phosphorylation at low concentrations without cytotoxicity on cells. p38 MAPK is a known target for inflammation treatment. Thus, we became interested in whether JLU1124 has anti-inflammatory effects. We used LPS stimulated RAW264.7 macrophages as a model of inflammation to evaluate the anti-inflammatory effects of JLU1124. Our results showed that p38 phosphorylation, the production of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, the mRNA and protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were enhanced by lipopolysaccharide (LPS). At concentrations of less than 10 μmol/L, JLU1124 inhibits p38 phosphorylation in a dose-dependent manner and significantly suppresses LPS-induced production of NO, IL-6 and TNF-α, and decreases the expressions of iNOS and COX-2 in RAW264.7 macrophages which indicate that JLU1124 has anti-inflammatory effects. However, JLU1124 has no significant effect on the phosphorylation of extracellular signal-regulated kinase1/2 and c-Jun NH2-terminal kinase which was involved in inflammation. Furthermore, our results showed that JLU1124 inhibits NF-κB inhibitor (IκB)α phosphorylation, nuclear translocation and transcriptional activity of NF-κB induced by LPS which may be through suppression of Akt phosphorylation. In conclusion, our study indicates that JLU1124 efficiently inhibits p38 phosphorylation and has anti-inflammatory effects in LPS-treated RAW264.7 macrophages. The anti-inflammatory mechanism of JLU1124 is mainly through decreasing Akt phosphorylation and inhibiting IκBα phosphorylation, thus suppressing NF-κB activation and nuclear translocation.

Differential expression of mitogen activating protein kinases in periodontitis

Following toll-like receptor (TLR) engagement, lipopolysaccharide (LPS) can stimulate the expression of pro-inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase-induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen-activated protein kinases (MAPK) described in mammals, p38, c-Jun N-terminal activated kinases (JNK1-3) and extracellular activated kinases (ERK1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre-clinical animal models. In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho-specific antibodies against p38, JNK and ERK kinases. Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive (p-value < 0.05) for total p38 (correlations ranging 0.49-0.68), phospho-p38 (range 0.44-0.56), and total ERK (range 0.52-0.59) levels, and correlations with JNK levels also supported association (range 0.42-0.59). Phospho-JNK and phospho-ERK showed no significant positive correlation with clinical parameters of disease. These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.

Crystal structure of the p38α MAP kinase in complex with a docking peptide from TAB1

The mitogen-activated protein kinase (MAPK) p38α is a key regulator in many cellular processes, whose activity is tightly regulated by upstream kinases, phosphatases and other regulators. Transforming growth factor-β activated kinase 1 (TAK1) is an upstream kinase in p38α signaling, and its full activation requires a specific activator, the TAK1-binding protein (TAB1). TAB1 was also shown to be an inducer of p38α's autophosphorylation and/or a substrate driving the feedback control of p38α signaling. Here we determined the complex structure of the unphosphorylated p38α and a docking peptide of TAB1, which shows that the TAB1 peptide binds to the classical MAPK docking groove and induces long-range conformational changes on p38α. Our structural and biochemical analyses suggest that TAB1 is a reasonable substrate of p38α, yet the interaction between the docking peptide and p38α may not be sufficient to trigger trans-autophosphorylation of p38α.

Candida albicans and Candida parapsilosis Induce Different T-Cell Responses in Human Peripheral Blood Mononuclear Cells

Candida parapsilosis is the third most frequent cause of candidemia. Despite its clinical importance, little is known about the human immunological response to C. parapsilosis. In this study, we compared the cytokine responses evoked by Candida albicans and C. parapsilosis. C. parapsilosis-stimulated human peripheral blood mononuclear cells (PBMCs) produced similar quantities of tumor necrosis factor α and interleukin 6 and slightly lower amounts of interleukin 1β, compared with C. albicans-stimulated cells. PBMCs stimulated with C. parapsilosis displayed a skewed T-helper cell response, producing more interleukin 10 and less interferon γ than cells stimulated with C. albicans. Notably, C. parapsilosis induced much less interleukin 17 and interleukin 22 production as compared to C. albicans. Inhibition of the 3 classical mitogen-activated protein kinases (p38 kinase, ERK, and JNK) revealed kinase-dependent differences in reductions in cytokine production by the 2 Candida species. Decreased cytokine production after inhibition of dectin 1 revealed that this receptor plays a major role in the recognition of both C. albicans and C. parapsilosis. These data improve understanding of the immune response triggered by C. parapsilosis, a first step for the future design of immunotherapeutic strategies for these infections.

β2-adrenoceptor agonists can both stimulate and inhibit glucose uptake in mouse soleus muscle through ligand-directed signalling

The β-adrenoceptor agonists BRL37344 and clenbuterol have opposite effects on glucose uptake in mouse soleus muscle, even though the β2-adrenoceptor mediates both effects. Different agonists may direct the soleus muscle β2-adrenoceptor to different signalling mechanisms. Soleus muscles were incubated with 2-deoxy[1-(14)C]-glucose, β-adrenoceptor agonists, other modulators of cyclic AMP, and inhibitors of intracellular signalling. The adenylyl cyclase activator forskolin (1μM), the phosphodiesterase inhibitor rolipram (10μM) and BRL37344 (10, but not 100 or 1,000, nM) increased, whereas clenbuterol (100 nM) decreased, glucose uptake. Forskolin increased, whereas clenbuterol decreased, muscle cyclic AMP content. BRL37344 (10nM) did not increase cyclic AMP. Nevertheless, protein kinase A (PKA) inhibitors prevented the stimulatory effect of BRL37344. Nanomolar but not micromolar concentrations of adrenaline stimulated glucose uptake. After preincubation of muscles with pertussis toxin (100ng/ml), 100nM clenbuterol, 0.1-10μM adrenaline and 100nM BRL37344 stimulated glucose uptake. Clenbuterol increased the proportion of phosphorylated to total β2-adrenoceptor. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and the stress-activated mitogen-activated protein kinase (MAPK), but not of the classical MAPK pathway, prevented stimulation of glucose uptake by BRL37344. Elevation of the cyclic AMP content of soleus muscle stimulates glucose uptake. Clenbuterol, and high concentrations of adrenaline and BRL37344 direct the β2-adrenoceptor partly to Gαi, possibly mediated by β2-adrenoceptor phosphorylation. The stimulatory effect of 10nM BRL37344 requires the activity of PKA, PI3K and p38 MAPK, consistent with BRL37344 directing the β2-adrenoceptor to Gαs. Ligand-directed signalling may explain why β2-adrenoceptor agonists have differing effects on glucose uptake in soleus muscle.

Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

Identification of Map4k4 as a Novel Suppressor of Skeletal Muscle Differentiation

Myoblast differentiation into mature myotubes is a critical step in the development and repair of human skeletal muscle. Here we show that small interfering RNA (siRNA)-based silencing of the Ste20-like mitogen-activated protein 4 kinase 4 (Map4k4) in C2C12 myoblasts markedly enhances expression of myogenic differentiation genes, myoblast fusion, and myotube diameter. In contrast, adenovirus-mediated expression of native Map4k4 in C2C12 cells attenuates each of these processes, indicating that Map4k4 is a negative regulator of myogenic differentiation and hypertrophy. Expression of a Map4k4 kinase-inactive mutant enhances myotube formation, suggesting that the kinase activity of Map4k4 is essential for its inhibition of muscle differentiation. Map4k4 regulation of myogenesis is unlikely to be mediated by classic mitogen-activated protein kinase (MAPK) signaling pathways, because no significant difference in phosphorylation of extracellular signal-regulated kinase (ERK), p38, or c-Jun N-terminal kinase (JNK) is observed in Map4k4-silenced cells. Furthermore, silencing of these other MAPKs does not result in a hypertrophic myotube phenotype like that seen with Map4k4 depletion. Uniquely, Map4k4 silencing upregulates the expression of the myogenic regulatory factor Myf5, whose depletion inhibits myogenesis. Furthermore, Myf5 is required for enhancement of myotube formation in Map4k4-silenced cells, while Myf5 overexpression rescues Map4k4-mediated inhibition of myogenic differentiation. These results demonstrate that Map4k4 is a novel suppressor of skeletal muscle differentiation, acting through a Myf5-dependent mechanism.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Regulation of mitogen activated protein kinases through heterotrimeric G proteins

Heterotrimeric G proteins are known as G proteins, consist of α, β, and γ subunits. G protein mediated signaling is employed by virtually all cells in the mammalian organism and is centrally involved in diverse physiological functions such as perception of sensory information, modulation of synaptic transmission, hormone release and actions, regulation of cell contraction and migration, or cell growth and differentiation. The amino acid identity of the α subunits has been used as basis for the classification of G proteins into four families Gs, Gi, Gq, and G12. G proteins stimulate distinct downstream effectors including enzymes, ion channels and small GTPase, thus regulating multiple signaling pathways including those involved in the activation of mitogen-activated protein kinase pathways. Mitogen-activated protein kinases are a family that constitute signaling pathways involved in processes that control gene expression, cell division, cell survival, apoptosis, metabolism, differentiation and motility. The mitogen-activated protein kinase family includes ERK1/2, c-Jun N-terminal kinazlar 1, 2 ve 3, p38MAPK a, b, g, and d, and ERK5 as classical mitogen-activated protein kinases, and ERK3, ERK4 NLK, and ERK7 as atypical mitogen-activated protein kinases. Each of mitogen-activated protein kinase pathways consists of three distinct kinases, namely an upstream respectively; mitogen-activated protein kinase kinase kinase , mitogen-activated protein kinase kinase and mitogen-activated protein kinase

Analysis of critical molecules and signaling pathways in osteoarthritis and rheumatoid arthritis

Osteoarthritis (OA) and rheumatoid arthritis (RA) are the most prevalent forms of arthritis in the elderly. This study aimed to explore the molecular mechanisms of these diseases and identify underlying therapeutic targets. Using GSE1919 microarray data sets downloaded from the Gene Expression Omnibus database, we screened differentially expressed genes (DEGs) in OA and RA cells. The underlying molecular mechanisms of these crucial genes were investigated by Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Small molecule expression and SNP analysis were also conducted by searching CMap and dbSNP databases. More than 320 genes changed in the arthritic cells and there were only 196 DEGs between OA and RA. OA and RA activated the classic mitogen-activated protein kinase signaling pathway, insulin signaling pathway, antigen processing and presentation and intestinal immune network for IgA production. Graft-versus-host disease and autoimmune thyroid disease-related pathways were also activated in OA and RA. Parthenolide and alsterpaullone may be treatments for OA and RA and insulin-like growth factor 1, collagen α2(I) chain and special AT-rich sequence-binding protein 2 may be critical SNP molecules in arthritis. Our findings shed new light on the common molecular mechanisms of OA and RA and may provide theoretical support for further clinical therapeutic studies.

Extracellular regulated kinase5 is expressed in fetal mouse submandibular glands and is phosphorylated in response to epidermal growth factor and other ligands of the ErbB family of receptors

Growth factors and their receptors regulate development of many organs through activation of multiple intracellular signaling cascades including a mitogen-activated protein kinase (MAPK). Extracellular regulated kinases (ERK)1/2, classic MAPK family members, are expressed in fetal mouse submandibular glands (SMG), and stimulate branching morphogenesis. ERK5, also called big mitogen-activated protein kinase 1, was recently found as a new member of MAPK super family, and its biological roles are still largely unknown. In this study, we investigated the expression and function of ERK5 in developing fetal mouse SMGs. Western blotting analysis showed that the expression pattern of ERK5 was different from the pattern of ERK1/2 in developing fetal SMGs. Both ERK1/2 and ERK5 were phosphorylated after exposure to ligands of the ErbB family of receptor tyrosine kinases (RTKs). Phosphorylation of ERK1/2 was strongly induced by epidermal growth factor (EGF) in SMG rudiments at embryonic day 14 (E14), E16 and E18. However, ERK5 phosphorylation induced by EGF was clearly observed at E14 and E16, but not at E18. Branching morphogenesis of cultured E13 SMG rudiments was strongly suppressed by administration of U0126, an inhibitor for ERK1/2 activation, whereas the phosphorylation of ERK5 was not inhibited by U0126. BIX02188, a specific inhibitor for ERK5 activation, also inhibited branching morphogenesis in cultured SMG rudiments. These results show that EGF-responsive ERK5 is expressed in developing fetal mouse SMG, and suggest that both ERK1/2 and ERK5 signaling cascades might play an important role in the regulation of branching morphogenesis.

Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove.

Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear "docking" motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38α, and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38α. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.