A rice kinase-protein interaction map.

PubMed

Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

2009-03-01

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

PubMed

Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

1998-10-15

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Effects of prostaglandin F(2alpha)and carbachol on MAP kinases, cytosolic phospholipase A(2)and arachidonic acid release in cat iris sphincter smooth muscle cells.

PubMed

Husain, S; Abdel-Latif, A A

2001-05-01

The signal transduction pathways initiated by Ca(2+)-mobilizing agonists, such as prostaglandin F(2alpha)(PGF(2alpha)) and carbachol (CCh), leading to activation of cytosolic phospholipase A(2)(cPLA(2)) and arachidonic acid (AA) release in a wide variety of tissues remain obscure. To further define the role of protein kinases in receptor mediated stimulation of cPLA(2)and consequently AA release we have investigated the role of mitogen-activated protein (MAP) kinases and protein kinase C (PKC) in PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation and AA release in cat iris sphincter smooth muscle (CISM) cells. The cells were prelabeled with [(3)H]AA for 24 hr and incubated in the absence or presence of the agonist for 5-10 min as indicated. MAP kinases activities and cPLA(2)phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA(2)antibodies. We found that: (a) PGF(2alpha)and CCh increased p38 MAP kinase activity by 197 and 215%, respectively, and increased p42/p44 MAP kinase activity by 200 and 125%, respectively. (b) SB202190, a p38 MAP kinase specific inhibitor, inhibited PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation by 92 and 85%, respectively, and AA release by 62 and 78%, respectively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced cPLA(2)phosphorylation by 70% and AA release by 71%, but had no effect on that of PGF(2alpha). (d) Inhibition of PKC activity by RO 31-8220 inhibited both PGF(2alpha)- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP kinases and cPLA(2)phosphorylation. We conclude from these results that in CISM cells PGF(2alpha)-induced cPLA(2)phosphorylation and AA release is mediated through p38 MAP kinase, but not through p42/p44 MAP kinases, whereas that of CCh is mediated through both p38 MAP kinase and p42/p44 MAP kinases. These effects of PGF(2alpha)and CCh are regulated by the MAP kinases in a PKC-dependent manner. Studies aimed at elucidating the role of

Mitogen-activated protein kinase inhibitors suppress prostaglandin F(2alpha)-induced myosin-light chain phosphorylation and contraction in iris sphincter smooth muscle.

PubMed

Yousufzai, S Y; Gao, G; Abdel-Latif, A A

2000-10-27

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F(2alpha), latanoprost, a prostaglandin F(2alpha) analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-methoxyflavone (PD98059) (10 microM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F(2alpha)- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F(2alpha) increased MAP kinase phosphorylation in a concentration-dependent manner with EC(50) value of 1.1 x 10(-8) M and increased contraction with EC(50) of 0.92 x 10(-9) M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1, 4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F(2alpha)-induced contraction in a concentration-dependent manner with IC(50) values of 2.4, 3.0 and 4.8 microM, respectively. PD98059 had no effect on prostaglandin F(2alpha)- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP(3)) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F(2alpha)-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2- hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 microM), a Ca(2+)-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 microM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F(2alpha)- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in

Regulation of cotton (Gossypium hirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

PubMed

Li, Fangjun; Li, Maoying; Wang, Ping; Cox, Kevin L; Duan, Liusheng; Dever, Jane K; Shan, Libo; Li, Zhaohu; He, Ping

2017-09-01

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

PubMed Central

Teper, Doron; Sunitha, Sukumaran; Martin, Gregory B; Sessa, Guido

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. PMID:26237448

Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin

Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicatemore » that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE{sub 2} and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. - Highlights: • C3G inhibited UVB-induced oxidative damage and inflammation. • C3G inhibited UVB-induced COX-2, iNOS and PGE{sub 2} production. �

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

PubMed Central

Hellesøy, Monica; Lorens, James B.

2015-01-01

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

PubMed Central

2015-01-01

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors. PMID:25075558

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

PubMed

Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis

PubMed Central

Bach, Eviatar; Chaffer, Marcelo; Lai, Wanika; Keefe, Greg; Begg, Douglas J.

2018-01-01

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species. PMID:29581962

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis.

PubMed

Bach, Horacio; Richard-Greenblatt, Melissa; Bach, Eviatar; Chaffer, Marcelo; Lai, Wanika; Keefe, Greg; Begg, Douglas J

2018-01-01

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

PubMed

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

PubMed

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Inhibition of benzopyrene diol epoxide-induced apoptosis by cadmium (II) is AP-1-independent: role of extracelluler signal related kinase

PubMed Central

Mukherjee, Jagat J.; Gupta, Suresh K.; Kumar, Subodh

2010-01-01

Cadmium, a major metal constituent of tobacco smoke, elicits synergistic enhancement of cell transformation when combined with benzo[a]pyrene (BP) or other PAHs. The mechanism underlying this synergism is not clearly understood. We observed that (+/−)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen of BP, induces apoptosis in promotion sensitive mouse epidermal JB6 Cl41 cells at non-cytotoxic concentrations. BPDE also activates AP-1 several folds in AP-1 reporter JB6 cells. Cadmium at non-cytotoxic concentrations inhibits both AP-1 activation and apoptosis in response to BPDE. Since AP-1 is known to be involved in stress-induced apoptosis we investigated whether inhibition of AP-1 by cadmium has any role in the inhibition of BPDE-induced apoptosis. MAP kinases (particularly ERKs, p38 and JNKs) are known to have important role in DNA damage-induced AP-1 activation. We observed that ERK and JNK, but not p38 MAP kinase, are involved in BPDE-induced AP-1 activation. Effect of cadmium on MAP kinases and the effect of inhibition of above three MAP kinases on BPDE-induced AP-1 activation and apoptosis indicate that AP-1 is probably not involved in BPDE-induced apoptosis. Cadmium up-regulates BPDE-activated ERKs and ERK inhibition by U0126 relieves cadmium-mediated inhibition of BPDE-induced apoptosis. We suggest that cadmium inhibits BPDE-induced apoptosis not involving AP-1 but probably through a different mechanism by up-regulating ERK which is known to promote cell survival. PMID:18093576

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak

Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

DOE PAGES

Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak; ...

2014-07-17

Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less

RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia.

PubMed

Chen, Jiwei; Zhang, Qian; Wang, Qigang; Feng, Ming; Li, Yang; Meng, Yonglu; Zhang, Yi; Liu, Guoqin; Ma, Zhimin; Wu, Hongzhi; Gao, Junping; Ma, Nan

2017-02-23

Flower opening is an important process in the life cycle of flowering plants and is influenced by various endogenous and environmental factors. Our previous work demonstrated that rose (Rosa hybrida) flowers are highly sensitive to dehydration during flower opening and the water recovery process after dehydration induced ethylene production rapidly in flower gynoecia. In addition, this temporal- and spatial-specific ethylene production is attributed to a transient but robust activation of the rose MAP KINASE6-ACC SYNTHASE1 (RhMPK6-RhACS1) cascade in gynoecia. However, the upstream component of RhMPK6-RhACS1 is unknown, although RhMKK9 (MAP KINASE KINASE9), a rose homologue of Arabidopsis MKK9, could activate RhMPK6 in vitro. In this study, we monitored RhMKK2/4/5/9 expression, the potential upstream kinase to RhMPK6, in rose gynoecia during dehydration and rehydration. We found only RhMKK9 was rapidly and strongly induced by rehydration. Silencing of RhMKK9 significantly decreased rehydration-triggered ethylene production. Consistently, the expression of several ethylene-responsive genes was down regulated in the petals of RhMKK9-silenced flowers. Moreover, we detected the DNA methylation level in the promoter and gene body of RhMKK9 by Chop-PCR. The results showed that rehydration specifically elevated the DNA methylation level on the RhMKK9 gene body, whereas it resulted in hypomethylation in its promoter. Our results showed that RhMKK9 possibly acts as the upstream component of the RhMKK9-RhMPK6-RhACS1 cascade and is responsible for water recovery-triggered ethylene production in rose gynoecia, and epigenetic DNA methylation is involved in the regulation of RhMKK9 expression by rehydration.

Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa

PubMed Central

Jeffree, Chris E.; Oborny, Radek; Boonyarungsrit, Patid; Read, Nick D.

2012-01-01

In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia. PMID:22900028

Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin.

PubMed

Divya, Sasidharan Padmaja; Wang, Xin; Pratheeshkumar, Poyil; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Hitron, John Andrew; Wang, Lei; Asha, Padmaja; Shi, Xianglin; Zhang, Zhuo

2015-04-01

Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm(2)) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

PubMed Central

Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

2002-01-01

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geiss, Brian J.; Cano, Gina L.; Tavis, John E.

2004-12-05

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22.more » Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.« less

MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

PubMed

Yang, H; Raizada, M K

1998-09-01

Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons

Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Divya, Sasidharan Padmaja; Wang, Xin; Pratheeshkumar, Poyil

Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm{sup 2}) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasiamore » and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E{sub 2} (PGE{sub 2}), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. - Highlights: • Blackberry extract inhibits UVB-induced glutathione

Cyanidin-3-Glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signalling pathways in SKH-1 hairless mice skin

PubMed Central

Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin; Divya, Sasidharan Padmaja; Joseph, Binoy; Hitron, John Andrew; Wang, Lei; Kim, Donghern; Yin, Yuanqin; Roy, Ram Vinod; Lu, Jian; Zhang, Zhuo; Wang, Yitao; Shi, Xianglin

2015-01-01

Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-Glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE2 and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. PMID:25062774

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

PubMed Central

Nebreda, A R; Hunt, T

1993-01-01

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified. Images PMID:8387916

Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xing, Li; Shieh, Huey S.; Selness, Shaun R.

2009-07-24

PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as themore » gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.« less

Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation

PubMed Central

Khan, Mohammed A S; Kang, Jian; Steiner, Theodore S

2004-01-01

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhoea in a number of clinical settings. EAEC diarrhoea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, causes interleukin (IL)-8 release from several epithelial cell lines. The host response to flagellins from E. coli and several other bacteria is mediated by Toll-like receptor 5 (TLR5), which signals through nuclear factor kappa B (NF-κB) to induce transcription of pro-inflammatory cytokines. p38 mitogen-activating protein (MAP) kinase (MAPK) is a member of a family of stress-related kinases that influences a diverse range of cellular functions including host inflammatory responses to microbial products. We studied the role of p38 MAPK in FliC-EAEC-induced IL-8 secretion from Caco-2 human intestinal epithelial cells and THP-1 human monocytic cells. We found that IL-8 secretion from both cell types is dependent on p38 MAPK, which is phospho-activated in response to FliC-EAEC. The role of TLR5 in p38 MAPK-dependent IL-8 secretion was verified in HEp-2 cells transiently transfected with a TLR5 expression construct. Activation of interleukin-1 receptor-associated kinase (IRAK) was also observed in Caco-2 and TLR5-transfected HEp-2 cells after exposure to FliC-EAEC. Finally, we demonstrated that pharmacological inhibition of p38 MAPK reduced IL-8 transcription and mRNA levels, but did not affect NF-κB activation. Collectively, our results suggest that TLR5 mediates p38 MAPK-dependent IL-8 secretion from epithelial and monocytic cells incubated with FliC-EAEC, and that this effect requires IL-8 promoter activation independent of NF-κB nuclear migration. PMID:15270737

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

PubMed Central

Stanko, Vera; Giuliani, Concetta; Retzer, Katarzyna; Djamei, Armin; Wahl, Vanessa; Wurzinger, Bernhard; Wilson, Cathal; Heberle-Bors, Erwin; Teige, Markus; Kragler, Friedrich

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2–AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency. PMID:25064848

Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin.

PubMed

Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin; Divya, Sasidharan Padmaja; Joseph, Binoy; Hitron, John Andrew; Wang, Lei; Kim, Donghern; Yin, Yuanqin; Roy, Ram Vinod; Lu, Jian; Zhang, Zhuo; Wang, Yitao; Shi, Xianglin

2014-10-01

Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE2 and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2'-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes

2',5'-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation.

PubMed

Lee, Yi-Nan; Yeh, Hung-I; Tian, Tin-Yi; Lu, Wen-Wei; Ko, Yu-Shien; Tsai, Cheng-Ho

2002-09-30

We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.

The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

PubMed

Breitenstein, Alexander; Stämpfli, Simon F; Reiner, Martin F; Shi, Yi; Keller, Stephan; Akhmedov, Alexander; Schaub Clerigué, Ariane; Spescha, Remo D; Beer, Hans-Jürg; Lüscher, Thomas F; Tanner, Felix C; Camici, Giovanni G

2017-01-05

Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2 -/- mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2 -/- mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2 -/- and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2 -/- mice (n=7; p<0.05 for wild-type mice and n=5-6; p=NS for JNK2 -/- mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.

Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae.

PubMed

Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

2016-10-26

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

Pharmacophore modeling of diverse classes of p38 MAP kinase inhibitors.

PubMed

Sarma, Rituparna; Sinha, Sharat; Ravikumar, Muttineni; Kishore Kumar, Madala; Mahmood, S K

2008-12-01

Mitogen-activated protein (MAP) p38 kinase is a serine-threonine protein kinase and its inhibitors are useful in the treatment of inflammatory diseases. Pharmacophore models were developed using HypoGen program of Catalyst with diverse classes of p38 MAP kinase inhibitors. The best pharmacophore hypothesis (Hypo1) with hydrogen-bond acceptor (HBA), hydrophobic (HY), hydrogen-bond donor (HBD), and ring aromatic (RA) as features has correlation coefficient of 0.959, root mean square deviation (RMSD) of 1.069 and configuration cost of 14.536. The model was validated using test set containing 119 compounds and had high correlation coefficient of 0.851. The results demonstrate that results obtained in this study can be considered to be useful and reliable tools in identifying structurally diverse compounds with desired biological activity.

Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

PubMed Central

Zaitsevskaya-Carter, T; Cooper, J A

1997-01-01

A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

β-arrestin drives MAP kinase signaling from clathrin-coated structures after GPCR dissociation

PubMed Central

Eichel, K.; Jullié, D.

2016-01-01

β-arrestins critically regulate G protein-coupled receptor (GPCR) signaling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G protein-independent signal via MAP kinase1–3. Despite enormous recent progress toward understanding biophysical aspects of arrestin function4,5, its cell biology remains relatively poorly understood. Two key tenets underlie the present dogma: (1) β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and (2) MAP kinase activation requires endocytosis of formed GPCR - β-arrestin complexes6–9. We show here, using β1-adrenergic receptors, that β-arrestin-2 (Arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin and β-arrestin -dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signaling from CCSs. We propose a β-arrestin signaling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

How MAP kinase modules function as robust, yet adaptable, circuits.

PubMed

Tian, Tianhai; Harding, Angus

2014-01-01

Genetic and biochemical studies have revealed that the diversity of cell types and developmental patterns evident within the animal kingdom is generated by a handful of conserved, core modules. Core biological modules must be robust, able to maintain functionality despite perturbations, and yet sufficiently adaptable for random mutations to generate phenotypic variation during evolution. Understanding how robust, adaptable modules have influenced the evolution of eukaryotes will inform both evolutionary and synthetic biology. One such system is the MAP kinase module, which consists of a 3-tiered kinase circuit configuration that has been evolutionarily conserved from yeast to man. MAP kinase signal transduction pathways are used across eukaryotic phyla to drive biological functions that are crucial for life. Here we ask the fundamental question, why do MAPK modules follow a conserved 3-tiered topology rather than some other number? Using computational simulations, we identify a fundamental 2-tiered circuit topology that can be readily reconfigured by feedback loops and scaffolds to generate diverse signal outputs. When this 2-kinase circuit is connected to proximal input kinases, a 3-tiered modular configuration is created that is both robust and adaptable, providing a biological circuit that can regulate multiple phenotypes and maintain functionality in an uncertain world. We propose that the 3-tiered signal transduction module has been conserved through positive selection, because it facilitated the generation of phenotypic variation during eukaryotic evolution.

Escitalopram Ameliorates Tau Hyperphosphorylation and Spatial Memory Deficits Induced by Protein Kinase A Activation in Sprague Dawley Rats.

PubMed

Ren, Qing-Guo; Wang, Yan-Juan; Gong, Wei-Gang; Xu, Lin; Zhang, Zhi-Jun

2015-01-01

Here, we investigated the effect of escitalopram pretreatment on protein kinase A (PKA)-induced tau hyperphosphorylation and spatial memory deficits in rats using western blot and behavioral tests, respectively. We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. We measured the total and activity-dependent Ser9-phosphorylated levels of glycogen synthase kinase (GSK)-3β in hippocampal extracts. No significant change in the total level of GSK-3β was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3β. We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). Furthermore, we identified other important kinases and phosphatases, such as protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2, and MAP kinase kinase-1/2, that have previously been reported to play a crucial role in tau phosphorylation; however, we did not detect any significant change in the activation of these kinases or phosphatases in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin. These data provide the first evidence that escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3β signaling pathway.

Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

PubMed Central

Whisenant, Thomas C.; Ho, David T.; Benz, Ryan W.; Rogers, Jeffrey S.; Kaake, Robyn M.; Gordon, Elizabeth A.; Huang, Lan; Baldi, Pierre; Bardwell, Lee

2010-01-01

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates. PMID:20865152

Negative regulatory role of PI3-kinase in TNF-induced tumor necrosis.

PubMed

Matschurat, Susanne; Blum, Sabine; Mitnacht-Kraus, Rita; Dijkman, Henry B P M; Kanal, Levent; De Waal, Robert M W; Clauss, Matthias

2003-10-20

Tissue factor is the prime initiator of blood coagulation. Expression of tissue factor in tumor endothelial cells leads to thrombus formation, occlusion of vessels and development of hemorrhagic infarctions in the tumor tissue, often followed by regression of the tumor. Tumor cells produce endogenous vascular endothelial growth factor (VEGF), which sensitizes endothelial cells for systemically administered tumor necrosis factor alpha (TNF alpha) and synergistically enhances the TNF-induced expression of tissue factor. We have analyzed the pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells (HUVECs) after combined stimulation with TNF and VEGF. By using specific low molecular weight inhibitors, we demonstrated that protein kinase C (PKC), p44/42 and p38 mitogen-activated protein (MAP) kinases, and stress-activated protein kinase (JNK) are essentially involved in the induction of tissue factor. In contrast, the application of wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, led to strongly enhanced expression of tissue factor in TNF- and VEGF-treated cells, implicating a negative regulatory role for PI3-kinase. In vivo, the application of wortmannin promoted the formation of TNF-induced hemorrhages and intratumoral necroses in murine meth A tumors. The co-injection of wortmannin lowered the effective dose of applied TNF. Therefore, it is conceivable that the treatment of TNF-sensitive tumors with a combination of TNF and wortmannin will ensure the selective damage of the tumor endothelium and minimize the risk of systemic toxicity of TNF. TNF-treatment in combination with specific inhibition of PI3-kinase is a novel concept in anti-cancer therapy. Copyright 2003 Wiley-Liss, Inc.

Cot kinase plays a critical role in Helicobacter pylori-induced IL-8 expression.

PubMed

Jang, Sungil; Kim, Jinmoon; Cha, Jeong-Heon

2017-04-01

Helicobacter pylori is a major pathogen causing various gastric diseases including gastric cancer. Infection of H. pylori induces pro-inflammatory cytokine IL-8 expression in gastric epithelial cells in the initial inflammatory process. It has been known that H. pylori can modulate Ras-Raf-Mek-Erk signal pathway for IL-8 induction. Recently, it has been shown that another signal molecule, cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, activates Mek and Erk and plays a role in the Erk pathway, similar to MAP3K signal molecule Raf kinase. Therefore, the objective of this study was to determine whether Cot kinase might be involved in IL-8 induction caused by H. pylori infection. AGS gastric epithelial cells were infected by H. pylori strain G27 or its isogenic mutants lacking cagA or type IV secretion system followed by treatment with Cot kinase inhibitor (KI) or siRNA specific for Cot kinase. Activation of Erk was assessed by Western blot analysis and expression of IL-8 was measured by ELISA. Treatment with Cot KI reduced both transient and sustained Erk activation. It also reduced early and late IL-8 secretion in the gastric epithelial cell line. Furthermore, siRNA knockdown of Cot inhibited early and late IL-8 secretion induced by H. pylori infection. Taken together, these results suggest that Cot kinase might play a critical role in H. pylori type IV secretion apparatus-dependent early IL-8 secretion and CagA-dependent late IL-8 secretion as an alternative signaling molecule in the Erk pathway.

How MAP kinase modules function as robust, yet adaptable, circuits

PubMed Central

Tian, Tianhai; Harding, Angus

2014-01-01

Genetic and biochemical studies have revealed that the diversity of cell types and developmental patterns evident within the animal kingdom is generated by a handful of conserved, core modules. Core biological modules must be robust, able to maintain functionality despite perturbations, and yet sufficiently adaptable for random mutations to generate phenotypic variation during evolution. Understanding how robust, adaptable modules have influenced the evolution of eukaryotes will inform both evolutionary and synthetic biology. One such system is the MAP kinase module, which consists of a 3-tiered kinase circuit configuration that has been evolutionarily conserved from yeast to man. MAP kinase signal transduction pathways are used across eukaryotic phyla to drive biological functions that are crucial for life. Here we ask the fundamental question, why do MAPK modules follow a conserved 3-tiered topology rather than some other number? Using computational simulations, we identify a fundamental 2-tiered circuit topology that can be readily reconfigured by feedback loops and scaffolds to generate diverse signal outputs. When this 2-kinase circuit is connected to proximal input kinases, a 3-tiered modular configuration is created that is both robust and adaptable, providing a biological circuit that can regulate multiple phenotypes and maintain functionality in an uncertain world. We propose that the 3-tiered signal transduction module has been conserved through positive selection, because it facilitated the generation of phenotypic variation during eukaryotic evolution. PMID:25483189

The MAP Kinase Cascade Is Activated prior to the Induction of Gliosis in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model of Dopaminergic Neurotoxicity.

PubMed

Callaghan, James P; Martin, Parthena M; Mass, Marc J

1998-05-01

Injury to the central nervous system (CNS) provokes microglial activation and astrocytic hypertrophy at the site of damage. The signaling events that underlie these cellular responses remain unknown. Recent evidence has implicated tyrosine phosphorylation systems, in general, and the mitogen-activated protein kinase (MAP kinase) cascade, in particular, in the mediation of growth-associated events linked to neural degeneration, such as glial activation. 1 Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, 2 appears to be involved in methamphetamine neurotoxicity. 3 To examine the potential role of these protein kinase pathways in drug-induced damage to the CNS, we used the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to damage nerve terminals in the mouse neostriatum and elicit a glial reaction. The onset of reactive gliosis then was verified by Northern blot analysis of glial fibrillary acidic protein (GFAP) mRNA and qualified by enzyme-linked immunosorbent assay (ELISA) of GFAP (protein). A single administration of MPTP (12.5 mg/kg, subcutaneously (s.c.)) to the C57B1/6J mouse resulted in a 10-fold increase in GFAP mRNA by 1 day and a 4-fold increase in GFAP (protein) by 2 days. To determine the potential role of protein tyrosine phosphorylation and MAP kinase activation in these events, blots of striatal homogenates were probed with antibodies directed against phospho-tyr 204 and phospho-thr 202, residues corresponding to the active sites of p42/44 MAP kinase. After mice were sacrificed by focused microwave irradiation to preserve steady-state phosphorylation, proteins from striatal homogenates were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots of these samples showed a number of phosphotyrosine-labeled bands, but there were no apparent differences between control and MPTP groups. In contrast, phospho-MAP kinase was

High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

PubMed

Zhou, Fasong; Menke, Frank L H; Yoshioka, Keiko; Moder, Wolfgang; Shirano, Yumiko; Klessig, Daniel F

2004-09-01

The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.

Dual modulation of ERK1/2 and p38 MAP kinase activities induced by minocycline reverses the neurotoxic effects of the prion protein fragment 90-231.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Chiovitti, Katia; Villa, Valentina; Simi, Alessandro; Raggi, Federica; Paludi, Domenico; Russo, Claudio; Aceto, Antonio; Florio, Tullio

2009-02-01

Several in vitro and in vivo studies addressed the identification of molecular determinants of the neuronal death induced by PrP(Sc) or related peptides. We developed an experimental model to assess PrP(Sc) neurotoxicity using a recombinant polypeptide encompassing amino acids 90-231 of human PrP (hPrP90-231) that corresponds to the protease-resistant core of PrP(Sc) identified in prion-infected brains. By means of mild thermal denaturation, we can convert hPrP90-231 from a PrP(C)-like conformation into a PrP(Sc)-like structure. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing caspase 3 and p38-dependent apoptosis, while in the native alpha-helix-rich conformation, hPrP90-231 did not induce cell toxicity. The aim of this study was to identify drugs able to block hPrP90-231 neurotoxic effects, focusing on minocycline, a tetracycline with known neuroprotective activity. hPrP90-231 caused a caspase 3-dependent apoptosis via the blockade of ERK1/2 activation and the subsequent activation of p38 MAP kinase. We propose that hPrP90-231-induced apoptosis is dependent on the inhibition of ERK1/2 responsiveness to neurotrophic factors, removing a tonic inhibition of p38 activity and resulting in caspase 3 activation. Minocycline prevented hPrP90-231-induced toxicity interfering with this mechanism: the pretreatment with this tetracycline restored ERK1/2 activity and reverted p38 and caspase 3 activities. The effects of minocycline were not mediated by the prevention of hPrP90-231 structural changes or cell internalization (differently from Congo Red). In conclusion, minocycline elicits anti-apoptotic effects against the neurotoxic activity of hPrP90-231 and these effects are mediated by opposite modulation of ERK1/2 and p38 MAP kinase activities.

TRAF6 and Src kinase activity regulates Cot activation by IL-1.

PubMed

Rodríguez, Cristina; Pozo, Maite; Nieto, Elvira; Fernández, Margarita; Alemany, Susana

2006-09-01

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.

A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.

PubMed Central

Fox, T.; Coll, J. T.; Xie, X.; Ford, P. J.; Germann, U. A.; Porter, M. D.; Pazhanisamy, S.; Fleming, M. A.; Galullo, V.; Su, M. S.; Wilson, K. P.

1998-01-01

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation. PMID:9827991

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

PubMed

Deng, Youping; Xu, Hu; Riedel, Heimo

2007-02-15

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative

Normal p21Ras/MAP kinase pathway expression and function in PBMC from patients with polycystic ovary disease.

PubMed

Buchs, A; Chagag, P; Weiss, M; Kish, E; Levinson, R; Aharoni, D; Rapoport, M J

2004-04-01

Polycystic ovary disease (PCOD) is associated with insulin resistance and increased prevalence of type II diabetes mellitus (T2DM). The p21Ras/MAP kinase is a major intracellular signaling pathway mediating insulin signaling in insulin responsive tissues. The expression, regulation and function of the p21Ras/MAP kinase pathway in PCOD patients were examined. Peripheral blood mononuclear cells (PBMC) were isolated from ten patients with PCOD and ten controls. The expression of p21Ras and its regulatory proteins; hSOS1 and p120GAP were studied. The basal and phytohemaglutinin (PHA) or insulin stimulated phosphorylation of MAP kinase was determined. Expression of p21Ras, and its regulatory proteins hSOS1 and p120GAP were similar in PCOD patients and controls. Basal, PHA and insulin stimulated phosphorylation of MAP kinase, were also comparable in the two groups as well as their PBMC proliferative response. These data indicate that the expression and overall function of the p21Ras/MAP kinase pathway remain intact in non-diabetic patients with PCOD.

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

PubMed Central

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, David G.; Smith, Richard D.; Glass, N. Louise

2014-01-01

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of

Role played by Disabled-2 in albumin induced MAP Kinase signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diwakar, Ramaswamy; Pearson, Alexander L.; Colville-Nash, Paul

2008-02-15

Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGF{beta}-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundancemore » in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGF{beta}-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGF{beta}-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.« less

A Crosslinker Based on a Tethered Electrophile for Mapping Kinase-Substrate Networks

PubMed Central

Riel-Mehan, Megan M; Shokat, Kevan M

2014-01-01

SUMMARY Despite the continuing progress made towards mapping kinase signaling networks, there are still many phosphorylation events for which the responsible kinase has not yet been identified. We are interested in addressing this problem through forming covalent crosslinks between a peptide substrate and the corresponding phosphorylating kinase. Previously we reported a dialdehyde-based kinase binding probe capable of such a reaction with a peptide containing a cysteine substituted for the phosphorylatable ser/thr/tyr residue. Here, we examine the yield of a previously reported dialdehyde-based probe, and report that the dialdehyde based probes possesses a significant limitation in terms of crosslinked kinase-substrate product yield. To address this limitation, we develop a crosslinking scheme based on a kinase activity-based probe, and this new cross-linker provides an increase in efficiency and substrate specificity, including in the context of cell lysate. PMID:24746561

An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis

PubMed Central

Brachmann, Andreas; Schirawski, Jan; Müller, Philip; Kahmann, Regine

2003-01-01

In Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bW and bE. We have identified by RNA fingerprinting a b-regulated gene, kpp6, which encodes an unusual MAP kinase. Kpp6 is similar to a number of other fungal MAP kinases involved in mating and pathogenicity, but contains an additional N-terminal domain unrelated to other proteins. Transcription of the kpp6 gene yields two transcripts differing in length, but encoding proteins of identical mass. One transcript is upregulated by the bW/bE heterodimer, while the other is induced after pheromone stimulation. kpp6 deletion mutants are attenuated in pathogenicity. kpp6T355A,Y357F mutants carrying a non-activatable allele of kpp6 are more severely compromised in pathogenesis. These strains can still form appressoria, but are defective in the subsequent penetration of the plant cuticle. Kpp6 is expressed during all stages of the sexual life cycle except mature spores. We speculate that Kpp6 may respond to a plant signal and regulate the genes necessary for efficient penetration of plant tissue. PMID:12727886

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

PubMed Central

Bogani, Debora; Siggers, Pam; Brixey, Rachel; Warr, Nick; Beddow, Sarah; Edwards, Jessica; Williams, Debbie; Wilhelm, Dagmar; Koopman, Peter; Flavell, Richard A.; Chi, Hongbo; Ostrer, Harry; Wells, Sara; Cheeseman, Michael; Greenfield, Andy

2009-01-01

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

PubMed Central

Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

2016-01-01

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

PubMed

Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

2016-01-01

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants.

PubMed

Chen, Po-Yu; Lee, Kuo-Ting; Chi, Wen-Chang; Hirt, Heribert; Chang, Ching-Chun; Huang, Hao-Jen

2008-08-01

Cross tolerance is a phenomenon that occurs when a plant, in resisting one form of stress, develops a tolerance to another form. Pretreatment with nonlethal heat shock has been known to protect cells from metal stress. In this study, we found that the treatment of rice roots with more than 25 muM of Cu(2+) caused cell death. However, heat shock pretreatment attenuated Cu(2+)-induced cell death. The mechanisms of the cross tolerance phenomenon between heat shock and Cu(2+) stress were investigated by pretreated rice roots with the protein synthesis inhibitor cycloheximide (CHX). CHX effectively block heat shock protection, suggesting that protection of Cu(2+)-induced cell death by heat shock was dependent on de novo protein synthesis. In addition, heat pretreatment downregulated ROS production and mitogen-activated protein kinase (MAPK) activities, both of which can be greatly elicited by Cu(2+) stress in rice roots. Moreover, the addition of purified recombinant GST-OsHSP70 fusion proteins inhibited Cu(2+)-enhanced MAPK activities in an in vitro kinase assay. Furthermore, loss of heat shock protection was observed in Arabidopsis mkk2 and mpk6 but not in mpk3 mutants under Cu(2+) stress. Taken together, these results suggest that the interaction of OsHSP70 with MAPKs may contribute to the cellular protection in rice roots from excessive Cu(2+) toxicity.

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

PubMed

Chung, Eun Young; Shin, Soon Young; Lee, Young Han

2007-07-05

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

TAO kinases mediate activation of p38 in response to DNA damage

PubMed Central

Raman, Malavika; Earnest, Svetlana; Zhang, Kai; Zhao, Yingming; Cobb, Melanie H

2007-01-01

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM. PMID:17396146

Increased choline kinase activity in 1,2-dimethylhydrazine-induced rat colon cancer.

PubMed

Nakagami, K; Uchida, T; Ohwada, S; Koibuchi, Y; Morishita, Y

1999-11-01

Cancer cells acquire particular characteristics that benefit their proliferation. We previously reported that human colon cancers examined had increased choline kinase activity and phosphocholine levels. The elevated phosphocholine levels were in part due to both activation of choline kinase and increased choline kinase alpha protein levels. In this report, we analyzed choline kinase, which catalyzes the phosphorylation of choline to produce phosphocholine, in rat 1,2-dimethylhydrazine (DMH)-induced colon cancer. This study is the first to demonstrate increased choline kinase alpha enzymatic activity, protein levels, and mRNA levels in DMH-induced colon cancer as well as human colon cancer, although phosphocholine was not increased in DMH-induced rat cancer. The increase in the mRNA level was partly due to an increase in the transcription of the choline kinase alpha gene. The increased choline kinase activity may be a specific characteristic acquired by cancer cells that benefits their proliferation.

The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

PubMed

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.

Mechanistic insights into the urea-induced denaturation of kinase domain of human integrin linked kinase.

PubMed

Syed, Sunayana Begum; Khan, Faez Iqbal; Khan, Sabab Hasan; Srivastava, Saurabha; Hasan, Gulam Mustafa; Lobb, Kevin A; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2018-05-01

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK 193-446 ) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ] 222 (mean residue ellipticity at 222nm), difference absorption coefficient at 292nm (Δε 292 ) and intrinsic fluorescence emission intensity at pH7.5 and 25±0.1°C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0-7.0M. A coincidence of normalized denaturation curves of optical properties ([θ] 222 , Δε 292 and λ max , the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies. Copyright © 2018 Elsevier B.V. All rights reserved.

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

PubMed Central

Ma, H; Gamper, M; Parent, C; Firtel, R A

1997-01-01

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. PMID:9250676

Eicosapentaenoic acid prevents TCDD-induced oxidative stress and inflammatory response by modulating MAP kinases and redox-sensitive transcription factors

PubMed Central

Palanisamy, Kalaiselvi; Krishnaswamy, Rajashree; Paramasivan, Poornima; Chih-Yang, Huang; Vishwanadha, Vijaya Padma

2015-01-01

Background and Purpose Oxidative stress and subsequent activation of inflammatory responses is a widely accepted consequence of exposure to environmental toxins. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a well-known environmental toxin, exerts its toxicity through many signalling mechanisms, with liver being the principal organ affected. However, an effective antidote to TCDD-induced toxicity is unknown. The present study evaluated the effect of eicosapentaenoic acid (EPA), an n3 fatty acid, on TCDD-induced toxicity. Experimental Approach In cultures of HepG2 cells, the EPA/AA ratio was determined using gas chromatography, oxidative stress and inflammatory responses through reactive oxygen species (ROS) levels, antioxidant status, [Ca2+]i, nuclear migration of two redox-sensitive transcription factors, NF-κB p65 and Nrf-2, expression of MAP kinase (p-Erk, p-p38), NF-κB p65, COX-2 and Nrf-2. Cellular changes in ΔΨm, acidic vesicular organelle formation, cell cycle analysis and scanning electron microscopy analysis were performed. Key Results EPA offered significant cytoprotection by increasing EPA/AA ratios in cell membranes, inhibiting ROS generation, enhancing antioxidant status and modulating nuclear translocation of redox-sensitive transcription factors (NF-κB p65 and Nrf-2) and expression of NF-κB p65, COX-2 and Nrf-2. Furthermore, TCDD-induced upstream events of MAPK phosphorylation, the increase in [Ca2+]i levels and cell surface changes in microvilli were significantly inhibited by EPA. EPA treatment maintained ΔΨm and prevented formation of acidic vesicular organelles. Conclusion and Implications The present study demonstrates for the first time some underlying molecular mechanisms of cytoprotection exerted by EPA against TCDD-induced oxidative stress and inflammatory responses. PMID:26177858

Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine.

PubMed

Wentsch, Heike K; Walter, Niklas M; Bührmann, Mike; Mayer-Wrangowski, Svenja; Rauh, Daniel; Zaman, Guido J R; Willemsen-Seegers, Nicole; Buijsman, Rogier C; Henning, Melanie; Dauch, Daniel; Zender, Lars; Laufer, Stefan

2017-05-02

Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

PubMed

Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

2016-09-15

Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases.

PubMed

Rendina, A R; Cleland, W W

1984-10-23

Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE PAGES

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; ...

2014-11-20

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal

The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

PubMed Central

Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

2016-01-01

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

PubMed

Teper, Doron; Girija, Anil Madhusoodana; Bosis, Eran; Popov, Georgy; Savidor, Alon; Sessa, Guido

2018-01-01

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

Activation of the AMP-activated protein kinase-p38 MAP kinase pathway mediates apoptosis induced by conjugated linoleic acid in p53-mutant mouse mammary tumor cells.

PubMed

Hsu, Yung-Chung; Meng, Xiaojing; Ou, Lihui; Ip, Margot M

2010-04-01

Conjugated linoleic acid (CLA) inhibits tumorigenesis and tumor growth in most model systems, an effect mediated in part by its pro-apoptotic activity. We previously showed that trans-10,cis-12 CLA induced apoptosis of p53-mutant TM4t mouse mammary tumor cells through both mitochondrial and endoplasmic reticulum stress pathways. In the current study, we investigated the role of AMP-activated protein kinase (AMPK), a key player in fatty acid metabolism, in CLA-induced apoptosis in TM4t cells. We found that t10,c12-CLA increased phosphorylation of AMPK, and that CLA-induced apoptosis was enhanced by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and inhibited by the AMPK inhibitor compound C. The increased AMPK activity was not due to nutrient/energy depletion since ATP levels did not change in CLA-treated cells, and knockdown of the upstream kinase LKB1 did not affect its activity. Furthermore, our data do not demonstrate a role for the AMPK-modulated mTOR pathway in CLA-induced apoptosis. Although CLA decreased mTOR levels, activity was only modestly decreased. Moreover, rapamycin, which completely blocked the activity of mTORC1 and mTORC2, did not induce apoptosis, and attenuated rather than enhanced CLA-induced apoptosis. Instead, the data suggest that CLA-induced apoptosis is mediated by the AMPK-p38 MAPK-Bim pathway: CLA-induced phosphorylation of AMPK and p38 MAPK, and increased expression of Bim, occurred with a similar time course as apoptosis; phosphorylation of p38 MAPK was blocked by compound C; the increased Bim expression was blocked by p38 MAPK siRNA; CLA-induced apoptosis was attenuated by the p38 inhibitor SB-203580 and by siRNAs directed against p38 MAPK or Bim. Copyright 2009 Elsevier Inc. All rights reserved.

The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

PubMed Central

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo

PubMed Central

Jung, Sung Keun; Lee, Ki Won; Byun, Sanguine; Lee, Eun Jung; Kim, Jong-Eun; Bode, Ann M.; Dong, Zigang

2010-01-01

Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1α expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70S6K in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin. PMID:20008033

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

PubMed Central

Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H.; Hickson, Gilles R.

2014-01-01

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks. PMID:25332165

DNA Protecting Activities of Nymphaea nouchali (Burm. f) Flower Extract Attenuate t-BHP-Induced Oxidative Stress Cell Death through Nrf2-Mediated Induction of Heme Oxygenase-1 Expression by Activating MAP-Kinases

PubMed Central

Ju, Mi-Kyoung

2017-01-01

This study was performed to investigate the antioxidant activities of Nymphaea nouchali flower (NNF) extract and the underlying mechanism using RAW 264.7 cells. The presence of gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate, caffeic acid, quercetin, and apigenin in the NNF was confirmed by high-performance liquid chromatography (HPLC). The extract had a very potent capacity to scavenge numerous free radicals. NNF extract was also able to prevent DNA damage and quench cellular reactive oxygen species (ROS) generation induced by tert-Butyl hydroperoxide (t-BHP) with no signs of toxicity. The NNF extract was able to augment the expression of both primary and phase II detoxifying enzyme, resulting in combat the oxidative stress. This is accomplished by phosphorylation of mitogen-activated protein kinase (MAP kinase) (p38 kinase and extracellular signal-regulated kinase (ERK)) followed by enhancing the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2). This attenuates cellular ROS generation and confers protection from cell death. Altogether, the results of current study revealed that Nymphaea nouchali flower could be a source of natural phytochemicals that could lead to the development of new therapeutic agents for preventing oxidative stress associated diseases and attenuating disease progression. PMID:28956831

Therapeutical relevance of MAP-kinase inhibitors in renal diseases: current knowledge and future clinical perspectives.

PubMed

Grande, M Teresa; López-Novoa, José M

2008-01-01

Renal failure, both acute and chronic, represents an important health problem by its social, sanitary and economic aspects. Mitogen-activated protein kinases (MAPK) are a family of mediators involved in the transduction of extracellular stimuli to intracellular responses. The best studied members of this family are extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), Jun NH(2)-terminal kinase (JNK), p38 kinase and extracellular signal regulated kinases 5 (ERK5) also known as big MAP Kinase 1 (BMK1). MAPKs plays a role in regulating renal function and all these pathways have been demonstrated to be activated in many "in vivo" and cellular models or renal failure. As MAP kinases are key regulators in the control of cell proliferation and cell death, many more or less specific inhibitors of these pathways are being developed for the treatment of tumors. The purpose of this review is to examine the data available on the role of MAPKs activation in "in vivo" models of renal failure, as well as in different renal cell types (especially in mesangial cells, podocytes, tubular epithelial cells and fibroblasts) subjected to stress or damage. We have also reviewed the effect of MAPKs inhibition on renal damage, both "in vivo" and "in vitro". Data collected allow to suggest that therapy of chronic and acute renal disease with MAPKs inhibitors is a promising therapeutic area, although much more basic and clinical studies are necessary before this kind of therapy can be used in the everyday clinic.

Signaling Network Map of Endothelial TEK Tyrosine Kinase

PubMed Central

Sandhya, Varot K.; Singh, Priyata; Parthasarathy, Deepak; Kumar, Awinav; Gattu, Rudrappa; Mathur, Premendu Prakash; Mac Gabhann, F.; Pandey, Akhilesh

2014-01-01

TEK tyrosine kinase is primarily expressed on endothelial cells and is most commonly referred to as TIE2. TIE2 is a receptor tyrosine kinase modulated by its ligands, angiopoietins, to regulate the development and remodeling of vascular system. It is also one of the critical pathways associated with tumor angiogenesis and familial venous malformations. Apart from the vascular system, TIE2 signaling is also associated with postnatal hematopoiesis. Despite the involvement of TIE2-angiopoietin system in several diseases, the downstream molecular events of TIE2-angiopoietin signaling are not reported in any pathway repository. Therefore, carrying out a detailed review of published literature, we have documented molecular signaling events mediated by TIE2 in response to angiopoietins and developed a network map of TIE2 signaling. The pathway information is freely available to the scientific community through NetPath, a manually curated resource of signaling pathways. We hope that this pathway resource will provide an in-depth view of TIE2-angiopoietin signaling and will lead to identification of potential therapeutic targets for TIE2-angiopoietin associated disorders. PMID:25371820

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

PubMed

Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

Conditional Inhibition of Adult Neurogenesis by Inducible and Targeted Deletion of ERK5 MAP Kinase Is Not Associated with Anxiety/Depression-Like Behaviors1,2

PubMed Central

Zou, Junhui; Wang, Wenbin; Pan, Yung-Wei; Abel, Glen M.

2015-01-01

Abstract Although there is evidence that adult neurogenesis contributes to the therapeutic efficacy of chronic antidepressant treatment for anxiety and depression disorders, the role of adult neurogenesis in the onset of depression-related symptoms is still open to question. To address this issue, we utilized a transgenic mouse strain in which adult neurogenesis was specifically and conditionally impaired by Nestin-CreER-driven, inducible knockout (icKO) of erk5 MAP kinase in Nestin-expressing neural progenitors of the adult mouse brain upon tamoxifen administration. Here, we report that inhibition of adult neurogenesis by this mechanism is not associated with an increase of the baseline anxiety or depression in non-stressed animals, nor does it increase the animal’s susceptibility to depression after chronic unpredictable stress treatment. Our findings indicate that impaired adult neurogenesis does not lead to anxiety or depression. PMID:26464972

Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

PubMed

Badrinarayan, Preethi; Sastry, G Narahari

2012-04-01

In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.

p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Guoping; Liu, Dongxu; Liu, Jing

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likelymore » that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.« less

Mitogen-Activated Protein Kinase 14 Promotes AKI

PubMed Central

Husi, Holger; Gonzalez-Lafuente, Laura; Valiño-Rivas, Lara; Fresno, Manuel; Sanz, Ana Belen; Mullen, William; Albalat, Amaya; Mezzano, Sergio; Vlahou, Tonia; Mischak, Harald

2017-01-01

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry–based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity–deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target. PMID:27620989

Staurosporine induces rapid homotypic intercellular adhesion of U937 cells via multiple kinase activation

PubMed Central

Cho, Jae Youl; Katz, David R; Chain, Benjamin M

2003-01-01

Staurosporine is a broad-specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation. In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine-induced aggregation can be distinguished from that induced by the phorbol 12-myristate 13-acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation. Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation. PMID:12970105

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed

Husain, S; Abdel-Latif, A A

1999-08-15

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed Central

Husain, S; Abdel-Latif, A A

1999-01-01

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Johannessen, Cory M; Boehm, Jesse S; Kim, So Young; Thomas, Sapana R; Wardwell, Leslie; Johnson, Laura A; Emery, Caroline M; Stransky, Nicolas; Cogdill, Alexandria P; Barretina, Jordi; Caponigro, Giordano; Hieronymus, Haley; Murray, Ryan R; Salehi-Ashtiani, Kourosh; Hill, David E; Vidal, Marc; Zhao, Jean J; Yang, Xiaoping; Alkan, Ozan; Kim, Sungjoon; Harris, Jennifer L; Wilson, Christopher J; Myer, Vic E; Finan, Peter M; Root, David E; Roberts, Thomas M; Golub, Todd; Flaherty, Keith T; Dummer, Reinhard; Weber, Barbara L; Sellers, William R; Schlegel, Robert; Wargo, Jennifer A; Hahn, William C; Garraway, Levi A

2010-12-16

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity

PubMed Central

Danson, Christopher M.; Pocha, Shirin M.; Bloomberg, Graham B.; Cory, Giles O.

2009-01-01

Summary The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration. PMID:18032787

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity.

PubMed

Danson, Christopher M; Pocha, Shirin M; Bloomberg, Graham B; Cory, Giles O

2007-12-01

The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth.

PubMed

Goschzik, Tobias; Cremer, Harold; Gnanapragassam, Vinayaga S; Horstkorte, Rüdiger; Bork, Kaya; Diestel, Simone

2017-07-01

The cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling.

PubMed

Channavajhala, Padma L; Wu, Leeying; Cuozzo, John W; Hall, J Perry; Liu, Wei; Lin, Lih-Ling; Zhang, Yuhua

2003-11-21

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.

Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Toshihide, E-mail: toshi-su@pharm.teikyo-u.ac.j; Miyazaki, Koichi; Kita, Kayoko

2009-12-15

Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvementmore » of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.« less

Role of Phosphatidylinositol 3-Kinase in Friend Spleen Focus-Forming Virus-Induced Erythroid Disease▿

PubMed Central

Umehara, Daigo; Watanabe, Shinya; Ochi, Haruyo; Anai, Yukari; Ahmed, Nursarat; Kannagi, Mari; Hanson, Charlotte; Ruscetti, Sandra; Nishigaki, Kazuo

2010-01-01

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85α regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85α status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85α status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85α and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85α may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia. PMID:20504929

Substituted N-aryl-6-pyrimidinones: A new class of potent, selective, and orally active p38 MAP kinase inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devadas, Balekudru; Selness, Shaun R.; Xing, Li

2012-02-28

A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-{alpha} in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

PubMed

Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

2016-04-01

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension

Proteolytic Degradation of SCOP in the Hippocampus Contributes to Activation of MAP Kinase and Memory

PubMed Central

Shimizu, Kimiko; Phan, Trongha; Mansuy, Isabelle; Storm, Daniel R.

2007-01-01

Summary Because activation of Erk1/2 MAP kinase (MAPK) is critical for hippocampus-dependent memory, there is considerable interest in mechanisms for regulation of MAPK during memory formation. Here we report that MAPK and CREB-mediated transcription are negatively regulated by SCOP (SCN Circadian Oscillatory Protein) and that SCOP is proteolyzed by calpain when hippocampal neurons are stimulated by BDNF, KCl depolarization, or NMDA. Moreover, training for novel object memory decreases SCOP in the hippocampus. To determine if hippocampus-dependent memory is influenced by SCOP in vivo, we generated a transgenic mouse strain for the inducible overexpression of SCOP in the forebrain. Overexpression of SCOP completely blocked memory for novel objects. We conclude that degradation of SCOP by calpain contributes to activation of MAPK during memory formation. PMID:17382888

Mitogen-activated protein kinase is required for the behavioral desensitization that occurs after repeated injections of angiotensin II

PubMed Central

Vento, Peter J.; Daniels, Derek

2013-01-01

Angiotensin II (AngII) acts on central angiotensin type 1 (AT1) receptors to increase water and saline intake. Prolonged exposure to AngII in cell culture models results in a desensitization of the AT1 receptor that is thought to involve receptor internalization, and a behavioral correlate of this desensitization has been shown in rats after repeated central injections of AngII. Specifically, rats given repeated injections of AngII drink less water than controls after a subsequent test injection of AngII. Under the same conditions, however, repeated injections of AngII have no effect on AngII-induced saline intake. Given earlier studies indicating that separate intracellular signaling pathways mediate AngII-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in AngII-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an AngII test injection in rats given prior treatment with repeated injections of vehicle, AngII, or Sar1,Ile4,Ile8-AngII (SII), an AngII analog that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated AngII injections on water intake. Furthermore, AngII-induced water intake was reduced similarly by repeated injections of AngII or SII. The results suggest that G protein-independent signaling is sufficient to produce behavioral desensitization of the angiotensin system and that the desensitization requires MAP kinase activation. PMID:22581747

Mitogen-activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II.

PubMed

Vento, Peter J; Daniels, Derek

2012-12-01

Angiotensin II (Ang II) acts on central angiotensin type 1 (AT(1)) receptors to increase water and saline intake. Prolonged exposure to Ang II in cell culture models results in a desensitization of the AT(1) receptor that is thought to involve receptor internalization, and a behavioural correlate of this desensitization has been shown in rats after repeated central injections of Ang II. Specifically, rats given repeated injections of Ang II drink less water than control animals after a subsequent test injection of Ang II. In the same conditions, however, repeated injections of Ang II have no effect on Ang II-induced saline intake. Given earlier studies indicating that separate intracellular signalling pathways mediate Ang II-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in Ang II-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an Ang II test injection in rats given prior treatment with repeated injections of vehicle, Ang II or Sar(1),Ile(4),Ile(8)-Ang II (SII), an Ang II analogue that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated Ang II injections on water intake. Furthermore, Ang II-induced water intake was reduced to a similar extent by repeated injections of Ang II or SII. The results suggest that G protein-independent signalling is sufficient to produce behavioural desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.

2′,5′-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways

PubMed Central

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S.; Franklin, Christopher C.; McCord, Joe M.; Day, Brian J.

2011-01-01

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest due to their anti-inflammatory and anti-angiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide (NO) synthase and to induce the expression of heme oxygenase-1 (HO-1). This study examines the mechanisms by which 2′,5′-dihydroxychalcone (2′,5′-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant response elements (MCF-7/AREc32). 2′,5′-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP5+, suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2′,5′-DHC treatment induced the phosphorylation of c-Jun N-terminal kinase (JNK) pathway that was also inhibited by MnTDE-1,3-IP5+. These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, while 2′,5′-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, co-treatment with MnTDE-1,3-IP5+ did not decrease 2′,5′-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein (MAP) kinase pathways showed a role for JNK and p38MAPK in mediating the 2′,5′-DHC-induced Nrf2 response. These findings suggest that the 2′,5′-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. PMID:21712085

Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

PubMed

Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

2013-01-01

Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, Claire; Universite de Toulouse, UPS, IPBS, Toulouse F-31000; Lafosse, Jean-Michel

Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma

PubMed Central

Carpenter, EL; Haglund, EA; Mace, EM; Deng, D; Martinez, D; Wood, AC; Chow, AK; Weiser, DA; Belcastro, LT; Winter, C; Bresler, SC; Asgharzadeh, S; Seeger, RC; Zhao, H; Guo, R; Christensen, JG; Orange, JS; Pawel, BR; Lemmon, MA; Mossé, YP

2013-01-01

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies–as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK. PMID:22266870

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression1[OPEN

PubMed Central

Weimer, Annika K.; Stoppin-Mellet, Virginie; Kosetsu, Ken; Cedeño, Cesyen; Jaquinod, Michel; Njo, Maria; De Milde, Liesbeth; Tompa, Peter; Inzé, Dirk; Beeckman, Tom; Vantard, Marylin

2017-01-01

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1—a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants—is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases. PMID:27879390

Geraniol attenuates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammation in mouse skin: possible role of p38 MAP Kinase and NF-κB.

PubMed

Khan, Abdul Quaiyoom; Khan, Rehan; Qamar, Wajhul; Lateef, Abdul; Rehman, Muneeb U; Tahir, Mir; Ali, Farrah; Hamiza, Oday O; Hasan, Syed Kazim; Sultana, Sarwat

2013-06-01

Abnormal production of reactive oxygen species (ROS) and proinflammatory cytokines often act as trigger for development of most of the chronic human diseases including cancer via up-regulation of transcription factors and activation of MAP kinases. We investigated the protective effects of geraniol (GOH) against 12-O-tetradecanoyl phorbol-13-acetate (TPA) induced oxidative and inflammatory responses, expression of p38MAPK, NF-κB and COX-2 in mouse skin. Animals were divided into four groups I-IV (n=6). Group II and III received topical application of TPA at the dose of 10 nmol/0.2 ml of acetone/animal/day, for two days. Group III was pre-treated with GOH (250 μg) topically 30 min prior to each TPA administration. While group I and IV were given acetone (0.2 ml) and GOH respectively. Our results show that GOH significantly inhibited TPA induced lipid peroxidation (LPO), inflammatory responses, proinflammatory cytokine release, up regulates reduced glutathione (GSH) content and the activity of different antioxidant enzymes. Interestingly, GOH also inhibited TPA induced altered activity of p38MAPK. Further, TPA induced altered expression of NF-κB (p65) and COX-2 was also attenuated by GOH. Thus, our results suggest that GOH attenuates early tumor promotional changes, and it may serve as one of the various ways to prevent carcinogenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of interleukins 2 and 12 on TBT-induced alterations of MAP kinases p38 and p44/42 in human natural killer cells.

PubMed

Aluoch, Aloice O; Whalen, Margaret M

2006-01-01

NK cells are lymphocytes in the non-adaptive immune system that protect the body against intracellular pathogens and eliminate tumor cells. Tributyltin (TBT) is a toxic chemical that has been detected in human foods as well as in human blood. The role of TBT in immunosuppression has been described, including inhibition of the human NK-cell cytotoxic function. Previous studies indicated that exposure of NK cells to TBT for 1 h induced progressive and irreversible inhibition of cytotoxic function. However, it was found that if NK cells were incubated in TBT-free media with either IL-2 or IL-12, loss of cytotoxic function was prevented/reversed within 24 h. Molecular studies established that loss of cytotoxic function is accompanied by alteration of MAP kinases (MAPKs) p38 and p44/42 phosphorylation. This study examined whether interleukin-mediated recovery of cytotoxicity involved reversal of tributyltin-altered p38 and p44/42 phosphorylation. The results indicated that there was no substantial IL-2 prevention/reversal of the TBT-induced alteration of phosphorylation of either p38 or p44/42 after either a 24 or 48 h recovery period. Additionally, IL-12 caused no substantial prevention/reversal of the TBT-induced alteration of phosphorylation of the MAPKs seen after either 24 or 48 h. These data suggest that IL-2 and/or IL-12-mediated recovery of NK cytotoxic function is not a result of prevention/reversal of TBT-induced phosphorylation of p38 and p44/42 MAPKs at the 24 or 48 h time points. Copyright 2005 John Wiley & Sons, Ltd.

Signaling of the ITK (interleukin 2-inducible T cell kinase)-SYK (spleen tyrosine kinase) fusion kinase is dependent on adapter SLP-76 and on the adapter function of the kinases SYK and ZAP70.

PubMed

Hussain, Alamdar; Mohammad, Dara K; Gustafsson, Manuela O; Uslu, Merve; Hamasy, Abdulrahman; Nore, Beston F; Mohamed, Abdalla J; Smith, C I Edvard

2013-03-08

The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor.

Signaling of the ITK (Interleukin 2-inducible T Cell Kinase)-SYK (Spleen Tyrosine Kinase) Fusion Kinase Is Dependent on Adapter SLP-76 and on the Adapter Function of the Kinases SYK and ZAP70*

PubMed Central

Hussain, Alamdar; Mohammad, Dara K.; Gustafsson, Manuela O.; Uslu, Merve; Hamasy, Abdulrahman; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C. I. Edvard

2013-01-01

The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor. PMID:23293025

Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

PubMed

Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

2008-03-01

The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

PubMed Central

Findlay, Greg M.; Yan, Lijun; Procter, Julia; Mieulet, Virginie; Lamb, Richard F.

2007-01-01

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3. PMID:17253963

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

PubMed Central

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

PubMed

May-Dracka, Tricia L; Arduini, Robert; Bertolotti-Ciarlet, Andrea; Bhisetti, Govinda; Brickelmaier, Margot; Cahir-McFarland, Ellen; Enyedy, Istvan; Fontenot, Jason D; Hesson, Thomas; Little, Kevin; Lyssikatos, Joe; Marcotte, Douglas; McKee, Timothy; Murugan, Paramasivam; Patterson, Thomas; Peng, Hairuo; Rushe, Mia; Silvian, Laura; Spilker, Kerri; Wu, Ping; Xin, Zhili; Burkly, Linda C

2018-06-01

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines. Copyright © 2018 Elsevier Ltd. All rights reserved.

FES kinase participates in KIT-ligand induced chemotaxis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voisset, Edwige, E-mail: Edwige.Voisset@inserm.fr; Institut Paoli-Calmettes, Marseille; Universite de la Mediterranee, Aix-Marseille II

2010-02-26

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicatedmore » in cell migration.« less

Tyrosine Kinase Inhibitor-Induced Hypertension.

PubMed

Agarwal, Megha; Thareja, Nidhi; Benjamin, Melody; Akhondi, Andre; Mitchell, George D

2018-06-21

The purpose of this paper is to identify commonly used tyrosine kinase inhibitors (TKIs) that are associated with hypertension, primarily, vascular endothelial growth factor (VEGF) signaling pathway (VSP) inhibitors. We review the incidence, mechanism, and strategies for management of TKI-induced HTN. We hope to provide clinicians with guidance on how to manage similar clinical scenarios. Many of the newer VSP inhibitors are reviewed here, including cediranib, axitinib, pazopanib, and ponatinib. Trials utilizing prophylactic treatment with angiotensin system inhibitors (ASIs) are discussed as well as recent data showing an improvement in overall survival and progression-free survival in patients on ASIs and TKI-induced hypertension. The incidence of TKI-induced HTN among the VEGF inhibitors ranges from 5 to 80% and is dose dependent. Newer generation small-molecule TKIs has a lower incidence. The mechanism of action involves VSP inhibition, leading to decreased nitric oxide and increased endothelin production, which causes vasoconstriction, capillary rarefaction, and hypertension. ASIs and calcium channel blockers are first-line therapy for treatment and are associated with improved overall survival. Nitrates and beta-blockers are associated with in vitro cancer regression; however, there is a paucity of trials regarding their use as an anti-hypertensive agent in the TKI-induced HTN patient population.

Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

PubMed

Singh, Raksha; Lee, Jae-Eun; Dangol, Sarmina; Choi, Jihyun; Yoo, Ran Hee; Moon, Jae Sun; Shim, Jae-Kyung; Rakwal, Randeep; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

2014-01-01

The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lithium and Tamoxifen Modulate Behavior and Protein Kinase C Activity in the Animal Model of Mania Induced by Ouabain

PubMed Central

Dal-Pont, Gustavo C; Resende, Wilson R; Varela, Roger B; Peterle, Bruna R; Gava, Fernanda F; Mina, Francielle G; Cararo, José H; Carvalho, André F; Quevedo, João

2017-01-01

Abstract Background The intracerebroventricular injection of ouabain, a specific inhibitor of the Na+/K+-adenosine-triphosphatase (Na+/K+-ATPase) enzyme, induces hyperactivity in rats in a putative animal model of mania. Several evidences have suggested that the protein kinase C signaling pathway is involved in bipolar disorder. In addition, it is known that protein kinase C inhibitors, such as lithium and tamoxifen, are effective in treating acute mania. Methods In the present study, we investigated the effects of lithium and tamoxifen on the protein kinase C signaling pathway in the frontal cortex and hippocampus of rats submitted to the animal model of mania induced by ouabain. We showed that ouabain induced hyperlocomotion in the rats. Results Ouabain increased the protein kinase C activity and the protein kinase C and MARCKS phosphorylation in frontal cortex and hippocampus of rats. Lithium and tamoxifen reversed the behavioral and protein kinase C pathway changes induced by ouabain. These findings indicate that the Na+/K+-ATPase inhibition can lead to protein kinase C alteration. Conclusions The present study showed that lithium and tamoxifen modulate changes in the behavior and protein kinase C signalling pathway alterations induced by ouabain, underlining the need for more studies of protein kinase C as a possible target for treatment of bipolar disorder. PMID:29020306

Casein kinase 2 inhibition impairs spontaneous and oxytocin-induced contractions in late pregnant mouse uterus.

PubMed

Suhas, K S; Parida, Subhashree; Gokul, Chandrasekaran; Srivastava, Vivek; Prakash, E; Chauhan, Sakshi; Singh, Thakur Uttam; Panigrahi, Manjit; Telang, Avinash G; Mishra, Santosh K

2018-05-01

What is the central question of this study? Does the inhibition of the protein kinase casein kinase 2 (CK2) alter the uterine contractility? What is the main finding and its importance? Inhibition of CK2 impaired the spontaneous and oxytocin-induced contractility in late pregnant mouse uterus. This finding suggests that CK2 is a novel pathway mediating oxytocin-induced contractility in the uterus and thus opens up the possibility for this class of drugs to be developed as a new class of tocolytics. The protein kinase casein kinase 2 (CK2) is a ubiquitously expressed serine or threonine kinase known to phosphorylate a number of substrates. The aim of this study was to assess the effect of CK2 inhibition on spontaneous and oxytocin-induced uterine contractions in 19 day pregnant mice. The CK2 inhibitor CX-4945 elicited a concentration-dependent relaxation in late pregnant mouse uterus. CX-4945 and another selective CK2 inhibitor, apigenin, also inhibited the oxytocin-induced contractile response in late pregnant uterine tissue. Apigenin also blunted the prostaglandin F 2α response, but CX-4945 did not. Casein kinase 2 was located in the lipid raft fractions of the cell membrane, and disruption of lipid rafts was found to reverse its effect. The results of the present study suggest that CK2, located in lipid rafts of the cell membrane, is an active regulator of spontaneous and oxytocin-induced uterine contractions in the late pregnant mouse. © 2018 The Authors. Experimental Physiology © 2018 The Physiological Society.

Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, C.-T.; Department of Biological Sciences, National Sun Yat-sen University, 804, Taiwan; He Shiping

2007-02-01

Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38more » MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.« less

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

PubMed

Fridell, Y W; Jin, Y; Quilliam, L A; Burchert, A; McCloskey, P; Spizz, G; Varnum, B; Der, C; Liu, E T

1996-01-01

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

PubMed Central

Andrusiak, Matthew G.; Jin, Yishi

2016-01-01

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

PubMed Central

Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

2015-01-01

Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

PubMed

Devkota, Sujan; Joseph, Raji E; Boyken, Scott E; Fulton, D Bruce; Andreotti, Amy H

2017-06-13

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP 3 . By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment.

PubMed

Page, Theresa H; Urbaniak, Anna M; Espirito Santo, Ana I; Danks, Lynett; Smallie, Timothy; Williams, Lynn M; Horwood, Nicole J

2018-05-05

Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

PubMed

Andrusiak, Matthew G; Jin, Yishi

2016-04-08

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Simplified molecular input line entry system-based: QSAR modelling for MAP kinase-interacting protein kinase (MNK1).

PubMed

Begum, S; Achary, P Ganga Raju

2015-01-01

Quantitative structure-activity relationship (QSAR) models were built for the prediction of inhibition (pIC50, i.e. negative logarithm of the 50% effective concentration) of MAP kinase-interacting protein kinase (MNK1) by 43 potent inhibitors. The pIC50 values were modelled with five random splits, with the representations of the molecular structures by simplified molecular input line entry system (SMILES). QSAR model building was performed by the Monte Carlo optimisation using three methods: classic scheme; balance of correlations; and balance correlation with ideal slopes. The robustness of these models were checked by parameters as rm(2), r(*)m(2), [Formula: see text] and randomisation technique. The best QSAR model based on single optimal descriptors was applied to study in vitro structure-activity relationships of 6-(4-(2-(piperidin-1-yl) ethoxy) phenyl)-3-(pyridin-4-yl) pyrazolo [1,5-a] pyrimidine derivatives as a screening tool for the development of novel potent MNK1 inhibitors. The effects of alkyl group, -OH, -NO2, F, Cl, Br, I, etc. on the IC50 values towards the inhibition of MNK1 were also reported.

Role of Gab1 in Heart, Placenta, and Skin Development and Growth Factor- and Cytokine-Induced Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Activation

PubMed Central

Itoh, Motoyuki; Yoshida, Yuichi; Nishida, Keigo; Narimatsu, Masahiro; Hibi, Masahiko; Hirano, Toshio

2000-01-01

Gab1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for SH2 and SH3 domains. Gab1 is tyrosine phosphorylated upon stimulation of various cytokines, growth factors, and antigen receptors in cell lines and interacts with signaling molecules, such as SHP-2 and phosphatidylinositol 3-kinase, although its biological roles have not yet been established. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting. Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation. PMID:10779359

Activation of p42/p44 mitogen-activated protein kinase and contraction by prostaglandin F2alpha, ionomycin, and thapsigargin in cat iris sphincter smooth muscle: inhibition by PD98059, KN-93, and isoproterenol.

PubMed

Ansari, H R; Husain, S; Abdel-Latif, A A

2001-10-01

In the present study we investigated the cross talk between the Ca2+ mobilization pathway and the mitogen-activated protein (MAP) kinase pathway and contraction in the cat iris sphincter smooth muscle. Three Ca2+-mobilizing agonists, namely, prostaglandin F2alpha (PGF2alpha), ionomycin, and thapsigargin, and three specific inhibitors, PD98059, a p42/p44 MAP kinase inhibitor; KN-93, a Ca2+-calmodulin-dependent protein kinase II (CaMKII) blocker; and isoproterenol, a cAMP-elevating agent, were used. Changes in tension in response to the agonists were recorded isometrically and MAP kinase phosphorylation and activation were monitored by Western blotting and by in situ myelin basic protein phosphorylation, respectively. We found that 1) stimulation of the sphincter muscle with PGF2alpha, ionomycin, or thapsigargin resulted in rapid phosphorylation and activation of p42/p44 MAP kinase and contraction; and 2) treatment of the muscles with PD98059, KN-93, or isoproterenol resulted in inhibition of the Ca2+-mobilizing agonist-induced responses. The contractile responses induced by PGF2alpha, ionomycin, and thapsigargin were (mg of tension/mg of wet weight tissue) 15.2, 15.4, and 16.2, respectively; the increases in MAP kinase phosphorylation by these agonists were 228, 203, and 190%, respectively; and the increases in MAP kinase activation by the agonists were 212, 191, and 162%, respectively. The stimulatory effects of the agonists on contraction and on MAP kinase phosphorylation and activation were blocked by preincubation of the muscle with PD98059, KN-93, or isoproterenol. These data demonstrate that in the iris sphincter phosphorylation and activation of p42/p44 MAP kinases by PGF2alpha, ionomycin, or thapsigargin require intracellular Ca2+ either from extracellular sources or from internal stores, that CaMKII plays an important role in the regulation of contraction, that CaMKII acts upstream of MAP kinase to control its activation, and that the MAP kinase signaling

Bilirubin induces a calcium-dependent inhibition of multifunctional Ca2+/calmodulin-dependent kinase II activity in vitro.

PubMed

Churn, S B; DeLorenzo, R J; Shapiro, S M

1995-12-01

Excessive bilirubin levels in newborn infants result in long-term neurologic deficits that remain after bilirubin levels return to normal. Much of the observed neurologic deficits can be attributed to bilirubin-induced, delayed neuronal cell death. Inhibition of calcium/calmodulin-dependent kinase II (CaM kinase II) activity that precedes cell death is observed in conditions such as seizure activity, stroke, and glutamate excitotoxicity. Because neonatal bilirubin exposure results in neuronal loss in developing brain systems, we tested whether bilirubin exposure would induce an immediate inhibition of CaM activity, in vitro. P-81 filtration assay of basal and calcium-stimulated kinase activity was performed under standard kinase assay conditions. Bilirubin and/or albumin was added to the reaction vessels to determine the effect of these agents on kinase activity. Bilirubin exposure resulted in a concentration-dependent inhibition of CaM kinase II activity (IC50 = 16.78 microM). At concentrations above 50 microM, bilirubin exposure resulted in a 71 +/- 8% (mean +/- SD) inhibition of kinase activity (p < 0.001, t test, n = 10). Bilirubin exposure did not result in kinase inhibition if excessive bilirubin was removed by albumin binding before stimulation of kinase activity (106.9 +/- 9.6% control activity, n = 5). However, removal of bilirubin by binding with albumin after calcium addition did not restore kinase activity. (36.1 +/- 3.8% control activity, n = 5). Thus, once inhibition was observed, the activity could not be restored by addition of albumin. The data suggest that bilirubin exposure resulted in a calcium-dependent inhibition of CaM kinase II activity that, once induced, was not reversible by removing bilirubin by the addition of albumin. Because inhibition of CaM kinase II activity has been correlated with delayed neuronal cell death in many neuropathologic conditions, bilirubin-induced inhibition of this enzyme may be a cellular mechanism by which

Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase.

PubMed

Qin, Qining; Downey, James M; Cohen, Michael V

2003-02-01

Adenosine and acetylcholine (ACh) trigger preconditioning by different signaling pathways. The involvement of phosphatidylinositol 3-kinase (PI3-kinase), a protein tyrosine kinase, and Src family tyrosine kinase in preconditioning was evaluated in isolated rabbit hearts. Either wortmannin (PI3-kinase blocker), genistein (tyrosine kinase blocker), lavendustin A (tyrosine kinase blocker), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2; Src family tyrosine kinase blocker) was given for 15 min to bracket a 5-min infusion of either adenosine or ACh (trigger phase). The hearts then underwent 30 min of regional ischemia. Infarct size for ACh alone was 9.3 +/- 3.5% of the risk zone versus 34.3 +/- 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30-min ischemic period (mediator phase), ACh-induced protection was not affected (7.4 +/- 2.1% and 9.7 +/- 1.7% infarction, respectively). Adenosine-triggered protection was not blocked by any of the inhibitors. Therefore, PI3-kinase and at least one protein tyrosine kinase, probably Src kinase, are involved in the trigger phase of ACh-induced, but not adenosine-induced, preconditioning. Neither PI3-kinase nor Src kinase is a mediator of the protection of ACh.

Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery.

PubMed

Knock, Greg A; Shaifta, Yasin; Snetkov, Vladimir A; Vowles, Benjamin; Drndarski, Svetlana; Ward, Jeremy P T; Aaronson, Philip I

2008-02-01

We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F(2 alpha) (PGF(2 alpha)) in alpha-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF(2 alpha) were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF(2 alpha) enhanced phosphorylation of three srcFK proteins at tyr-416. In alpha-toxin-permeabilized IPAs, PGF(2 alpha) enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF(2 alpha) enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF(2 alpha)-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF(2 alpha) triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. srcFK are activated by PGF(2 alpha) in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.

Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery

PubMed Central

Knock, Greg A.; Shaifta, Yasin; Snetkov, Vladimir A.; Vowles, Benjamin; Drndarski, Svetlana; Ward, Jeremy P.T.; Aaronson, Philip I.

2008-01-01

Abstract Aims We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. Methods and results Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F2α (PGF2α) in α-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF2α were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF2α enhanced phosphorylation of three srcFK proteins at tyr-416. In α-toxin-permeabilized IPAs, PGF2α enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF2α enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF2α-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF2α triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. Conclusions srcFK are activated by PGF2α in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1. PMID:18032393

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica

2010-10-15

Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conducemore » to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free

Mannheimia haemolytica leukotoxin activates a nonreceptor tyrosine kinase signaling cascade in bovine leukocytes, which induces biological effects.

PubMed

Jeyaseelan, S; Kannan, M S; Briggs, R E; Thumbikat, P; Maheswaran, S K

2001-10-01

The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (DeltaLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The DeltaLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3'-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is

Magnolol suppresses vascular endothelial growth factor-induced angiogenesis by inhibiting Ras-dependent mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling pathways.

PubMed

Kim, Ki Mo; Kim, No Soo; Kim, Jinhee; Park, Jong-Shik; Yi, Jin Mu; Lee, Jun; Bang, Ok-Sun

2013-01-01

Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been reported to possess anticancer activity. Recent studies have also demonstrated that magnolol inhibits cell growth and induces the apoptosis of cancer cells. However, the effects of magnolol on vascular endothelial growth factor (VEGF)-induced angiogenesis in endothelial cells have not been studied. In the present study, we have used human umbilical vein endothelial cells (HUVECs) to investigate the antiangiogenic effect and molecular mechanism of magnolol. Magnolol inhibited the VEGF-induced proliferation, chemotactic motility and tube formation of HUVECs in vitro as well as the vessel sprouting of the aorta ex vivo. Furthermore, magnolol inhibited VEGF-induced Ras activation and subsequently suppressed extracellular signal-regulated kinase (ERK), phosphatidylinositol-3-kinase (PI3K)/Akt and p38, but not Src and focal adhesion kinase (FAK). Interestingly, the knockdown of Ras by short interfering RNA produced inhibitory effects that were similar to the effects of magnolol on VEGF-induced angiogenic signaling events, such as ERK and Akt/eNOS activation, and resulted in the inhibition of proliferation, migration, and vessel sprouting in HUVECs. In combination, these results demonstrate that magnolol is an inhibitor of angiogenesis and suggest that this compound could be a potential candidate in the treatment of angiogenesis-related diseases.

Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.

PubMed

Koli, Swati; Prakash, Atul; Choudhury, Soumen; Mandil, Rajesh; Garg, Satish K

2018-05-21

Adverse effects of mercury on female reproduction are reported; however, its effect on myogenic activity of uterus and mechanism thereof is obscure. Present study was undertaken to unravel the mechanistic pathways of mercuric chloride (HgCl 2 )-induced myometrial contraction in rats. Isometric tension in myometrial strips of rats following in vitro exposure to HgCl 2 was recorded using data acquisition system-based physiograph. HgCl 2 produced concentration-dependent (10 nM-100 μM) uterotonic effect which was significantly (p < 0.05) reduced in Ca 2+ -free solution and inhibited in the presence of nifedipine (1 μM), a L-type Ca 2+ channel blocker, thus suggesting the importance of extracellular Ca 2+ and its entry through L-type calcium channels in HgCl 2 -induced myometrial contractions in rats. Cumulative concentration-response curve of HgCl 2 was significantly (p < 0.05) shifted towards right in the presence of Y-27632 (10 μM), a Rho-kinase inhibitor, suggesting the involvement of Ca 2+ -sensitization pathway in mediating HgCl 2 -induced myometrial contraction. HgCl 2 -induced myometrial contraction was also significantly (p < 0.05) inhibited in the presence of methoctramine or para-fluoro-hexahydro-siladifenidol, a selective M 2 and M 3 receptor antagonists, respectively, which evidently suggest that mercury also interacts with M 2 and M 3 muscarinic receptors to produce myometrial contractions. U-73122 and GF-109203X, the respective inhibitors of PLC and PKC-dependent pathways, downstream to the receptor activation, also significantly (p < 0.05) attenuated the uterotonic effect of HgCl 2 on rat uterus. Taken together, present study evidently reveals that HgCl 2 interacts with muscarinic receptors and activates calcium signaling cascades involving calcium channels, Rho-kinase, protein kinase-C, and phospholipase-C pathways to exert uterotonic effect in rats. Graphical Abstract Graphical abstract depicting the mechanism of mercury-induced

[MAP kinases--molecular transistors in animals and plants].

PubMed

Petersen, Morten; Brodersen, Peter; Mundy, John

2002-06-10

The survival of multicellular organisms depends on the ability of their cells to communicate with each other and to respond to environmental changes. A goal of modern biology is to uncover the processes by which these cellular signals are transduced. Recent studies have shown that MAP-kinases (MAPKs) are important constituents of such signal transduction pathways. MAPKs function as modules in phosphorelay cascades to activate or repress the activity of downstream target proteins. For example, recent research with knockout mice has shown that mammalian MAPKs are involved in the control of neuronal apoptosis and the activation of immune responses. These mammalian MAPKs exert their control by both promoting and inhibiting specific processes. Surprisingly, plants also use MAPKs to control their immune responses, and plant MAPKs also seem to play dual roles as positive and negative regulators. Such mechanistic similarities provide the basis for fruitful conceptual exchange between molecular research on animals and plants.

Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure in Platelets

PubMed Central

Dasgupta, Swapan K.; Le, Anhquyen; Haudek, Sandra B.; Entman, Mark L.; Rumbaut, Rolando E.; Thiagarajan, Perumal

2013-01-01

Background The transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform. Methods and Results ROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an in vivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). Conclusions These studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes. PMID:24358370

Involvement of the Tyr kinase/JNK pathway in carbachol-induced bronchial smooth muscle contraction in the rat.

PubMed

Sakai, Hiroyasu; Watanabe, Yu; Honda, Mai; Tsuiki, Rika; Ueda, Yusuke; Nagai, Yuki; Narita, Minoru; Misawa, Miwa; Chiba, Yoshihiko

2013-05-01

Tyrosine (Tyr) kinases and mitogen-activated protein kinases have been thought to participate in the contractile response in various smooth muscles. The aim of the current study was to investigate the involvement of the Tyr kinase pathway in the contraction of bronchial smooth muscle. Ring preparations of bronchi isolated from rats were suspended in an organ bath. Isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine the phosphorylation of c-Jun N-terminal kinasess (JNKs) in bronchial smooth muscle. To examine the role of mitogen-activated protein kinase(s) in bronchial smooth muscle contraction, the effects of MPAK inhibitors were investigated in this study. The contraction induced by carbachol (CCh) was significantly inhibited by pretreatment with selective Tyr kinase inhibitors (genistein and ST638, n = 6, respectively), and a JNK inhibitor (SP600125, n = 6). The contractions induced by high K depolarization (n = 4), orthovanadate (a potent Tyr phosphatase inhibitor) and sodium fluoride (a G protein activator; NaF) were also significantly inhibited by selective Tyr kinase inhibitors and a JNK inhibitor (n = 4, respectively). However, the contraction induced by calyculin-A was not affected by SP600125. On the other hand, JNKs were phosphorylated by CCh (2.2 ± 0,4 [mean±SEM] fold increase). The JNK phosphorylation induced by CCh was significantly inhibited by SP600125 (n = 4). These findings suggest that the Tyr kinase/JNK pathway may play a role in bronchial smooth muscle contraction. Strategies to inhibit JNK activation may represent a novel therapeutic approach for diseases involving airway obstruction, such as asthma and chronic obstructive pulmonary disease.

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

PubMed Central

Thaa, Bastian; Amrun, Siti Naqiah; Simarmata, Diane; Rausalu, Kai; Nyman, Tuula A.; Merits, Andres; McInerney, Gerald M.; Ng, Lisa F. P.

2016-01-01

ABSTRACT Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study, we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen

Lack of phosphoinositide 3-kinase-gamma attenuates ventilator-induced lung injury.

PubMed

Lionetti, Vincenzo; Lisi, Alberto; Patrucco, Enrico; De Giuli, Paolo; Milazzo, Maria Giovanna; Ceci, Simone; Wymann, Matthias; Lena, Annalisa; Gremigni, Vittorio; Fanelli, Vito; Hirsch, Emilio; Ranieri, V Marco

2006-01-01

G protein-coupled receptors may up-regulate the inflammatory response elicited by ventilator-induced lung injury but also regulate cell survival via protein kinase B (Akt) and extracellular signal regulated kinases 1/2 (ERK1/2). The G protein-sensitive phosphoinositide-3-kinase gamma (PI3Kgamma) regulates several cellular functions including inflammation and cell survival. We explored the role of PI3Kgamma on ventilator-induced lung injury. Prospective, randomized, experimental study. University animal research laboratory. Wild-type (PI3Kgamma), knock-out (PI3Kgamma ), and kinase-dead (PI3Kgamma) mice. Three ventilatory strategies (no stretch, low stretch, high stretch) were studied in an isolated, nonperfused model of acute lung injury (lung lavage) in PI3Kgamma, PI3Kgamma, and PI3Kgamma mice. Reduction in lung compliance, hyaline membrane formation, and epithelial detachment with high stretch were more pronounced in PI3Kgamma than in PI3Kgamma and PI3Kgamma (p < .01). Inflammatory cytokines and IkBalpha phosphorylation with high stretch did not differ among PI3Kgamma, PI3Kgamma, and PI3Kgamma. Apoptotic index (terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling) and caspase-3 (immunohistochemistry) with high stretch were larger (p < .01) in PI3Kgamma and PI3Kgamma than in PI3Kgamma. Electron microscopy showed that high stretch caused apoptotic changes in alveolar cells of PI3Kgamma mice whereas PI3Kgamma mice showed necrosis. Phosphorylation of Akt and ERK1/2 with high stretch was more pronounced in PI3Kgamma than in PI3Kgamma and PI3Kgamma (p < .01). Silencing PI3Kgamma seems to attenuate functional and morphological consequences of ventilator-induced lung injury independently of inhibitory effects on cytokines release but through the enhancement of pulmonary apoptosis.

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis

PubMed Central

Franz-Bacon, Karin; DiTirro, Danielle N.; Ly, Tai Wei; Bacon, Kevin B.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease. PMID:27144281

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis.

PubMed

Boehme, Stefen A; Franz-Bacon, Karin; DiTirro, Danielle N; Ly, Tai Wei; Bacon, Kevin B

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.

miR-188 promotes senescence of lineage-negative bone marrow cells by targeting MAP3K3 expression.

PubMed

Zheng, Yue; Liu, Hua; Kong, Ye

2017-08-01

Lineage-negative bone marrow cells (lin-BMCs) have reparative potential for overcoming endothelial dysfunction and reducing cardiovascular risk. Here, we found that miR-188 is upregulated and mitogen-activated protein kinase kinase kinase 3 (MAP3K3) is downregulated in aged lin-BMCs, whereas their expression is reversed in young lin-BMCs. We identified and confirmed MAP3K3 as a direct target of miR-188. MiR-188 overexpression or MAP3K3 silencing in young lin-BMCs increases p16 and p21 expression, enhances cell senescence, and decreases the ability for cell proliferation, migration, and tube formation. Conversely, miR-188 suppression in aged lin-BMCs yields the opposite results. We further found that MAP3K3 is involved in miR-188-induced promotion of lin-BMC senescence. All data reveal that miR-188 induces lin-BMC senescence by targeting MAP3K3 expression, thus, providing new theoretical basis for the prevention and treatment of cardiovascular diseases. © 2017 Federation of European Biochemical Societies.

Membrane androgen receptor characteristics of human ZIP9 (SLC39A) zinc transporter in prostate cancer cells: Androgen-specific activation and involvement of an inhibitory G protein in zinc and MAP kinase signaling.

PubMed

Thomas, Peter; Pang, Yefei; Dong, Jing

2017-05-15

Characteristics of novel human membrane androgen receptor (mAR), ZIP9 (SLC39A9), were investigated in ZIP9-transfected PC-3 cells (PC3-ZIP9). Ligand blot analysis showed plasma membrane [ 3 H]-T binding corresponds to the position of ZIP9 on Western blots which suggests ZIP9 can bind [ 3 H]-T alone, without a protein partner. Progesterone antagonized testosterone actions, blocking increases in zinc, Erk phosphorylation and apoptosis, further evidence that ZIP9 is specifically activated by androgens. Pre-treatment with GTPγS and pertussis toxin decreased plasma membrane [ 3 H]-T binding and blocked testosterone-induced increases in Erk phosphorylation and intracellular zinc, indicating ZIP9 is coupled to an inhibitory G protein (Gi) that mediates both MAP kinase and zinc signaling. Testosterone treatment of nuclei and mitochondria which express ZIP9 decreased their zinc contents, suggesting ZIP9 also regulates free zinc through releasing it from these intracellular organelles. The results show ZIP9 is a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

[Application of Kohonen Self-Organizing Feature Maps in QSAR of human ADMET and kinase data sets].

PubMed

Hegymegi-Barakonyi, Bálint; Orfi, László; Kéri, György; Kövesdi, István

2013-01-01

QSAR predictions have been proven very useful in a large number of studies for drug design, such as kinase inhibitor design as targets for cancer therapy, however the overall predictability often remains unsatisfactory. To improve predictability of ADMET features and kinase inhibitory data, we present a new method using Kohonen's Self-Organizing Feature Map (SOFM) to cluster molecules based on explanatory variables (X) and separate dissimilar ones. We calculated SOFM clusters for a large number of molecules with human ADMET and kinase inhibitory data, and we showed that chemically similar molecules were in the same SOFM cluster, and within such clusters the QSAR models had significantly better predictability. We used also target variables (Y, e.g. ADMET) jointly with X variables to create a novel type of clustering. With our method, cells of loosely coupled XY data could be identified and separated into different model building sets.

New approaches to the treatment of inflammatory disorders small molecule inhibitors of p38 MAP kinase.

PubMed

Peifer, Christian; Wagner, Gerd; Laufer, Stefan

2006-01-01

The therapy of chronic inflammatory diseases like rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) has recently been enriched by the successful launch of the anti-cytokine biologicals Etanercept (tumor necrosis factor (TNF) receptor-p75 Fc fusion protein), Infliximab (chimeric anti-human TNF-alpha monoclonal antibody), Adalimumab (recombinant human anti-human TNF-alpha monoclonal antibody) and Anakinra (recombinant form of human interleukin 1beta (IL-1) receptor antagonist). The success of these novel treatments has impressively demonstrated the clinical benefit that can be gained from therapeutic intervention in cytokine signalling, highlighting the central role of proinflammatory cytokine systems like IL-1alpha and TNF-alpha to be validated targets. However, all of the anti-cytokine biologicals available to date are proteins, and therefore suffering to a varying degree from the general disadvantages associated with protein drugs. Therefore, small molecular, orally active anti-cytokine agents, which target specific pathways of proinflammatory cytokines, would offer an attractive alternative to anti-cytokine biologicals. A number of molecular targets have been identified for the development of such small molecular agents but p38 mitogen-activated protein (MAP) kinase occupies a central role in the regulation of IL-1beta and TNF-alpha signalling network at both the transcriptional and translational level. Since the mid-1990s, an immense number of inhibitors of p38 MAP kinase has been characterised in vitro, and to date several compounds have been advanced into clinical trials. This review will highlight the correlation between effective inhibition of p38 MAP kinase at the molecular target and cellular activity in functional assays of cytokine, particularly TNF-alpha and IL-1beta production. SAR will be discussed regarding activity at the enzyme target, but also with regard to properties required for efficient in vitro and in vivo activity.

Mannheimia haemolytica Leukotoxin Activates a Nonreceptor Tyrosine Kinase Signaling Cascade in Bovine Leukocytes, Which Induces Biological Effects

PubMed Central

Jeyaseelan, S.; Kannan, M. S.; Briggs, R. E.; Thumbikat, P.; Maheswaran, S. K.

2001-01-01

The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (ΔLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The ΔLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3′-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is

Protein Kinase Cδ Suppresses Autophagy to Induce Kidney Cell Apoptosis in Cisplatin Nephrotoxicity

PubMed Central

Pan, Jian; Xiang, Xudong; Liu, Yu; Dong, Guie; Livingston, Man J.; Chen, Jian-Kang; Yin, Xiao-Ming

2017-01-01

Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδ in vitro. Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy–deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy. PMID:27799485

The kinases MEKK2 and MEKK3 regulate transforming growth factor-β-mediated helper T cell differentiation.

PubMed

Chang, Xing; Liu, Fang; Wang, Xiaofang; Lin, Aiping; Zhao, Hongyu; Su, Bing

2011-02-25

Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3) negatively regulated transforming growth factor-β (TGF-β)-mediated Th cell differentiation. Map3k2(-/-)Map3k3(Lck-Cre/-) mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2(-/-)Map3k3(Lck-Cre/-) naive CD4(+) T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-β stimulation in vitro. In addition, Map3k2(-/-)Map3k3(Lck-Cre/-) mice developed more severe experimental autoimmune encephalomyelitis. Map3k2(-/-)Map3k3(Lck-Cre/-) T cells exhibited impaired phosphorylation of SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-β responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-β signaling pathways is important in regulating Th cell differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.

Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes.

PubMed

Li, Luowei; Sampat, Keeran; Hu, Nancy; Zakari, Julia; Yuspa, Stuart H

2006-02-10

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell

Cyanidin-3-glucoside suppresses B[a]PDE-induced cyclooxygenase-2 expression by directly inhibiting Fyn kinase activity.

PubMed

Lim, Tae-Gyu; Kwon, Jung Yeon; Kim, Jiyoung; Song, Nu Ry; Lee, Kyung Mi; Heo, Yong-Seok; Lee, Hyong Joo; Lee, Ki Won

2011-07-15

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) is a well-known carcinogen that is associated with skin cancer. Abnormal expression of cyclooxygenase-2 (COX-2) is an important mediator in inflammation and tumor promotion. We investigated the inhibitory effect of cyanidin-3-glucoside (C3G), an anthocyanin present in fruits, on B[a]PDE-induced COX-2 expression in mouse epidermal JB6 P+ cells. Pretreatment with C3G resulted in the reduction of B[a]PDE-induced expression of COX-2 and COX-2 promoter activity. The activation of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) induced by B[a]PDE was also attenuated by C3G. C3G attenuated the B[a]PDE-induced phosphorylation of MEK, MKK4, Akt, and mitogen-activated protein kinases (MAPKs), but no effect on the phosphorylation of the upstream MAPK regulator Fyn. However, kinase assays demonstrated that C3G suppressed Fyn kinase activity and C3G directly binds Fyn kinase noncompetitively with ATP. By using PP2, a pharmacological inhibitor for SFKs, we showed that Fyn kinase regulates B[a]PDE-induced COX-2 expression by activating MAPKs, AP-1 and NF-κB. These results suggest that C3G suppresses B[a]PDE-induced COX-2 expression mainly by blocking the activation of the Fyn signaling pathway, which may contribute to its chemopreventive potential. Copyright © 2011 Elsevier Inc. All rights reserved.

SRC family kinase (SFK) inhibition reduces rhabdomyosarcoma cell growth in vitro and in vivo and triggers p38 MAP kinase-mediated differentiation

PubMed Central

Casini, Nadia; Forte, Iris Maria; Mastrogiovanni, Gianmarco; Pentimalli, Francesca; Angelucci, Adriano; Festuccia, Claudio; Tomei, Valentina; Ceccherini, Elisa; Di Marzo, Domenico; Schenone, Silvia; Botta, Maurizio; Giordano, Antonio; Indovina, Paola

2015-01-01

Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS. PMID:25762618

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

PubMed

Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

2016-10-01

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

Molecular dynamics simulations and modelling of the residue interaction networks in the BRAF kinase complexes with small molecule inhibitors: probing the allosteric effects of ligand-induced kinase dimerization and paradoxical activation.

PubMed

Verkhivker, G M

2016-10-20

Protein kinases are central to proper functioning of cellular networks and are an integral part of many signal transduction pathways. The family of protein kinases represents by far the largest and most important class of therapeutic targets in oncology. Dimerization-induced activation has emerged as a common mechanism of allosteric regulation in BRAF kinases, which play an important role in growth factor signalling and human diseases. Recent studies have revealed that most of the BRAF inhibitors can induce dimerization and paradoxically stimulate enzyme transactivation by conferring an active conformation in the second monomer of the kinase dimer. The emerging connections between inhibitor binding and BRAF kinase domain dimerization have suggested a molecular basis of the activation mechanism in which BRAF inhibitors may allosterically modulate the stability of the dimerization interface and affect the organization of residue interaction networks in BRAF kinase dimers. In this work, we integrated structural bioinformatics analysis, molecular dynamics and binding free energy simulations with the protein structure network analysis of the BRAF crystal structures to determine dynamic signatures of BRAF conformations in complexes with different types of inhibitors and probe the mechanisms of the inhibitor-induced dimerization and paradoxical activation. The results of this study highlight previously unexplored relationships between types of BRAF inhibitors, inhibitor-induced changes in the residue interaction networks and allosteric modulation of the kinase activity. This study suggests a mechanism by which BRAF inhibitors could promote or interfere with the paradoxical activation of BRAF kinases, which may be useful in informing discovery efforts to minimize the unanticipated adverse biological consequences of these therapeutic agents.

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation.

PubMed

Dickman, M B; Ha, Y S; Yang, Z; Adams, B; Huang, C

2003-05-01

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (Pisum sativum L.).

PubMed

Ortiz-Masia, Dolores; Perez-Amador, Miguel A; Carbonell, Pablo; Aniento, Fernando; Carbonell, Juan; Marcote, Maria J

2008-05-01

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A-D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.

Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

PubMed

Savopoulos, John W; Dowd, Stephen; Armour, Carolyn; Carter, Paul S; Greenwood, Catherine J; Mills, David; Powell, David; Pettman, Gary R; Jenkins, Owen; Walsh, Frank S; Philpott, Karen L

2002-02-01

The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays. Copyright 2002 Elsevier Science (USA).

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

PubMed

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Ferulic acid attenuates focal cerebral ischemia-induced decreases in p70S6 kinase and S6 phosphorylation.

PubMed

Koh, Phil-Ok

2013-10-25

Ferulic acid exhibits neuroprotective effects against focal cerebral ischemia. PI3/K and Akt signaling pathways play an essential role in protecting against cerebral ischemia. Mammalian target of rapamycin (mTOR), a major downstream target of Akt, regulates p70S6 kinase and S6, both of which are involved in ribosomal biogenesis and protein synthesis. I investigated whether ferulic acid regulates mTOR, p70S6 kinase, and S6 phosphorylation during brain ischemic injury. Rats were treated immediately with vehicle or ferulic acid (100mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brains tissues were removed at 24h after the onset of MCAO and the cerebral cortex regions were collected. Ferulic acid reduced the MCAO-induced infarct volume. I showed previously that ferulic acid prevents the MCAO injury-induced decrease of Akt phosphorylation. In this study, MCAO injury induced decreases in mTOR, p70S6 kinase, and S6 phosphorylation levels, while ferulic acid attenuated the injury-induced decreases. Immunohistochemical staining demonstrated that ferulic acid prevented the MCAO-induced reduction in the number of positive cells for phosphorylated p70S6 kinase and phosphorylated S6. These findings suggest that ferulic acid has a neuroprotective function against focal cerebral ischemia by modulating p70S6 kinase expression and S6 phosphorylation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

PubMed

Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2017-01-01

Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.

Receptor-interacting protein kinases modulate noise-induced sensory hair cell death

PubMed Central

Zheng, H-W; Chen, J; Sha, S-H

2014-01-01

Receptor-interacting protein (RIP) kinases promote the induction of necrotic cell death pathways. Here we investigated signaling pathways in outer hair cells (OHCs) of adult male CBA/J mice exposed to noise that causes permanent threshold shifts, with a particular focus on RIP kinase-regulated necroptosis. One hour after noise exposure, nuclei of OHCs in the basal region of the cochlea displayed both apoptotic and necrotic features. RIP1 and RIP3 protein levels increased and caspase-8 was activated. Treatment with pan-caspase inhibitor ZVAD blocked the activation of caspase-8 and reduced the number of apoptotic nuclei, while increasing levels of RIP1, RIP3, and necrotic OHCs. Conversely, treatment with necrosis inhibitor necrostatin-1 (Nec-1) or RIP3 siRNA (siRIP3) diminished noise-induced increases in RIP1 and RIP3, and decreased necrotic OHC nuclei. This treatment also increased the number of apoptotic nuclei without increasing activation of caspase-8. Consistent with the elevation of levels of RIP1 and RIP3, noise-induced active AMPKα levels increased with ZVAD treatment, but decreased with Nec-1 and siRIP3 treatment. Furthermore, treatment with siRIP3 did not alter the activation of caspase-8, but instead increased activation of caspase-9 and promoted endonuclease G translocation into OHC nuclei. Finally, auditory brainstem response functional measurements and morphological assessment of OHCs showed that ZVAD treatment reduces noise-induced deficits. This protective function is potentiated when combined with siRIP3 treatment. In conclusion, noise-induced OHC apoptosis and necrosis are modulated by caspases and RIP kinases, respectively. Inhibition of either pathway shifts the prevalence of OHC death to the alternative pathway. PMID:24874734

Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells.

PubMed

Zogovic, Nevena; Tovilovic-Kovacevic, Gordana; Misirkic-Marjanovic, Maja; Vucicevic, Ljubica; Janjetovic, Kristina; Harhaji-Trajkovic, Ljubica; Trajkovic, Vladimir

2015-04-01

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and β-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering

Caffeic acid, a phenolic phytochemical in coffee, directly inhibits Fyn kinase activity and UVB-induced COX-2 expression

PubMed Central

Kang, Nam Joo; Lee, Ki Won; Shin, Bong Jik; Jung, Sung Keun; Hwang, Mun Kyung; Bode, Ann M.; Heo, Yong-Seok; Dong, Zigang

2009-01-01

Caffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical present in many foods, including coffee. Recent studies suggested that caffeic acid exerts anticarcinogenic effects, but little is known about the underlying molecular mechanisms and specific target proteins. In this study, we found that Fyn, one of the members of the non-receptor protein tyrosine kinase family, was required for ultraviolet (UV) B-induced cyclooxygenase-2 (COX-2) expression, and caffeic acid suppressed UVB-induced skin carcinogenesis by directly inhibiting Fyn kinase activity. Caffeic acid more effectively suppressed UVB-induced COX-2 expression and subsequent prostaglandin E2 production in JB6 P+ mouse skin epidermal (JB6 P+) cells compared with chlorogenic acid (5-O-caffeoylquinic acid), an ester of caffeic acid with quinic acid. Data also revealed that caffeic acid more effectively induced the downregulation of COX-2 expression at the transcriptional level mediated through the inhibition of activator protein-1 (AP-1) and nuclear factor-κB transcription activity compared with chlorogenic acid. Fyn kinase activity was suppressed more effectively by caffeic acid than by chlorogenic acid, and downstream mitogen-activated protein kinases (MAPKs) were subsequently blocked. Pharmacological Fyn kinase inhibitor (3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine and leflunomide) data also revealed that Fyn is involved in UVB-induced COX-2 expression mediated through the phosphorylation of MAPKs in JB6 P+ cells. Pull-down assays revealed that caffeic acid directly bound with Fyn and non-competitively with adenosine triphosphate. In vivo data from mouse skin also supported the idea that caffeic acid suppressed UVB-induced COX-2 expression by blocking Fyn kinase activity. These results suggested that this compound could act as a potent chemopreventive agent against skin cancer. PMID:19073879

Fenofibrate inhibits aldosterone-induced apoptosis in adult rat ventricular myocytes via stress-activated kinase-dependent mechanisms

PubMed Central

De Silva, Deepa S.; Wilson, Richard M.; Hutchinson, Christoph; Ip, Peter C.; Garcia, Anthony G.; Lancel, Steve; Ito, Masa; Pimentel, David R.; Sam, Flora

2009-01-01

Aldosterone induces extracellular signal-regulated kinase (ERK)-dependent cardiac remodeling. Fenofibrate improves cardiac remodeling in adult rat ventricular myocytes (ARVM) partly via inhibition of aldosterone-induced ERK1/2 phosphorylation and inhibition of matrix metalloproteinases. We sought to determine whether aldosterone caused apoptosis in cultured ARVM and whether fenofibrate ameliorated the apoptosis. Aldosterone (1 μM) induced apoptosis by increasing terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei in ARVM. Spironolactone (100 nM), an aldosterone receptor antagonist, but not RU-486, a glucocorticoid receptor, inhibited aldosterone-mediated apoptosis, indicating that the mineralocorticoid receptor (MR) plays a role. SP-600125 (3 μM)—a selective inhibitor of c-Jun NH2-terminal kinase (JNK)—inhibited aldosterone-induced apoptosis in ARVM. Although aldosterone increased the expression of both stress-activated protein kinases, pretreatment with fenofibrate (10 μM) decreased aldosterone-mediated apoptosis by inhibiting only JNK phosphorylation and the aldosterone-induced increases in Bax, p53, and cleaved caspase-3 and decreases in Bcl-2 protein expression in ARVM. In vivo studies demonstrated that chronic fenofibrate (100 mg·kg body wt−1·day−1) inhibited myocardial Bax and increased Bcl-2 expression in aldosterone-induced cardiac hypertrophy. Similarly, eplerenone, a selective MR inhibitor, used in chronic pressure-overload ascending aortic constriction inhibited myocardial Bax expression but had no effect on Bcl-2 expression. Therefore, involvement of JNK MAPK-dependent mitochondrial death pathway mediates ARVM aldosterone-induced apoptosis and is inhibited by fenofibrate, a peroxisome proliferator-activated receptor (PPAR)α ligand. Fenofibrate mediates beneficial effects in cardiac remodeling by inhibiting programmed cell death and the stress-activated kinases. PMID:19395558

Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

PubMed

Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

2004-07-01

A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

Mangiferin induces apoptosis in multiple myeloma cell lines by suppressing the activation of nuclear factor kappa B-inducing kinase.

PubMed

Takeda, Tomoya; Tsubaki, Masanobu; Kino, Toshiki; Yamagishi, Misa; Iida, Megumi; Itoh, Tatsuki; Imano, Motohiro; Tanabe, Genzoh; Muraoka, Osamu; Satou, Takao; Nishida, Shozo

2016-05-05

Mangiferin is a naturally occurring glucosyl xanthone, which induces apoptosis in various cancer cells. However, the molecular mechanism underlying mangiferin-induced apoptosis has not been clarified thus far. Therefore, we examined the molecular mechanism underlying mangiferin-induced apoptosis in multiple myeloma (MM) cell lines. We found that mangiferin decreased the viability of MM cell lines in a concentration-dependent manner. We also observed an increased number of apoptotic cells, caspase-3 activation, and a decrease in the mitochondrial membrane potential. In addition, mangiferin inhibited the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated inhibitor kappa B (IκB) and increased the expression of IκB protein, whereas no changes were observed in the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase 1/2 (JNK1/2), and mammalian target of rapamycin (mTOR). The molecular mechanism responsible for mangiferin-induced inhibition of nuclear translocation of NF-κB was a decrease in the expression of phosphorylated NF-κB-inducing kinase (NIK). Moreover, mangiferin decreased the expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, and Bcl-xL proteins. Knockdown of NIK expression showed results similar to those observed with mangiferin treatment. Our results suggest that mangiferin induces apoptosis through the inhibition of nuclear translocation of NF-κB by suppressing NIK activation in MM cell lines. Our results provide a new insight into the molecular mechanism of mangiferin-induced apoptosis. Importantly, since the number of reported NIK inhibitors is limited, mangiferin, which targets NIK, may be a potential anticancer agent for the treatment of MM. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Protein kinase Cε regulates nuclear translocation of extracellular signal-regulated kinase, which contributes to bradykinin-induced cyclooxygenase-2 expression.

PubMed

Nakano, Rei; Kitanaka, Taku; Namba, Shinichi; Kitanaka, Nanako; Sugiya, Hiroshi

2018-06-04

The proinflammatory mediator bradykinin stimulated cyclooxygenase-2 (COX-2) expression and subsequently prostaglandin E 2 synthesis in dermal fibroblasts. The involvement of B2 receptors and Gαq in the role of bradykinin was suggested by using pharmacological inhibitors. The PKC activator PMA stimulated COX-2 mRNA expression. Bradykinin failed to induce COX-2 mRNA expression in the presence of PKC inhibitors, whereas the effect of bradykinin was observed in the absence of extracellular Ca 2+ . Bradykinin-induced COX-2 mRNA expression was inhibited in cells transfected with PKCε siRNA. These observations suggest that the novel PKCε is concerned with bradykinin-induced COX-2 expression. Bradykinin-induced PKCε phosphorylation and COX-2 mRNA expression were inhibited by an inhibitor of 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and bradykinin-induced PDK-1 phosphorylation was inhibited by phospholipase D (PLD) inhibitors, suggesting that PLD/PDK-1 pathway contributes to bradykinin-induced PKCε activation. Pharmacological and knockdown studies suggest that the extracellular signal-regulated kinase 1 (ERK1) MAPK signaling is involved in bradykinin-induced COX-2 expression. Bradykinin-induced ERK phosphorylation was attenuated in the cells pretreated with PKC inhibitors or transfected with PKCε siRNA. We observed the interaction between PKCε and ERK by co-immunoprecipitation experiments. These observations suggest that PKCε activation contributes to the regulation of ERK1 activation. Bradykinin stimulated the accumulation of phosphorylated ERK in the nuclear fraction, that was inhibited in the cells treated with PKC inhibitors or transfected with PKCε siRNA. Consequently, we concluded that bradykinin activates PKCε via the PLD/PDK-1 pathway, which subsequently induces activation and translocation of ERK1 into the nucleus, and contributes to COX-2 expression for prostaglandin E 2 synthesis in dermal fibroblasts.

Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

PubMed Central

Ogborne, Richard M.; Rushworth, Stuart A.; O’Connell, Maria A.

2008-01-01

The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2. PMID:18586007

PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

PubMed Central

Shi, Yu; He, Mao-xian

2016-01-01

The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway

PubMed Central

Kanda, Yasunari; Mizuno, Katsushige; Kuroki, Yasutomi; Watanabe, Yasuhiro

2001-01-01

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood.We have previously reported that thrombin activates p38 mitogen-activated protein kinase (p38 MAPK) by a tyrosine kinase-dependent mechanism, and that p38 MAPK has a role in thrombin-induced mitogenic response in rat VSMC.In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin-induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose-dependent manner, whereas it had no effect on the response to platelet-derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA-AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C.We next examined the effect of AG1478 on thrombin-induced DNA synthesis. AG1478 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. In contrast, PDGF-induced DNA synthesis was not affected by AG1478.In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin-induced proliferation of VSMC. PMID:11309236

Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors

PubMed Central

Ravikumar, Balaguru; Parri, Elina; Timonen, Sanna; Airola, Antti; Wennerberg, Krister

2017-01-01

Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001) between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel-based modeling approach

Cefradine blocks solar-ultraviolet induced skin inflammation through direct inhibition of T-LAK cell-originated protein kinase

PubMed Central

Ke, Changshu; Zhang, Guiping; Xiao, Juanjuan; Wu, Dan; Zeng, Xiaoyu; Chen, Jingwen; Guo, Jinguang; Zhou, Jie; Shi, Fei; Zhu, Feng

2016-01-01

Skin inflammation, and skin cancer induced by excessive solar ultraviolet (SUV) is a great threat to human health. SUV induced skin inflammation through activating p38 mitogen-activated protein kinase (p38) and c-Jun N-termeinal kinases (JNKs). T-LAK cell-originated protein kinase (TOPK) plays an important role in this process. Herein, the clinical data showed TOPK, phospho-p38, phospho-JNKs were highly expressed in human solar dermatitis. Ex vivo studies showed that SUV induced the phosphorylation of p38 and JNKs in HaCat and JB6 cells in a dose and time dependent manner. Molecule docking model indicated cefradine, an FDA-approved cephalosporin antibiotic, directly binds with TOPK. The result of in vitro binding assay verified cefradine can directly bind with TOPK. In vitro kinase results showed cefradine can inhibit TOPK activity. Ex vivo studies further showed cefradine inhibited SUV-induced the phosphorylation level of p38, JNKs and H2AX through inhibiting TOPK activity in a dose and time dependent manner, and cefradine inhibited the secretion of IL6 and TNF-α in HaCat and JB6 cells. In vivo studies showed that cefradine down-regulated SUV-induced the phosphorylation of p38, JNKs and H2AX and inhibited the secretion of IL6 and TNF-α in Babl/c mice. These results indicated that cefradine can inhibit SUV-induced skin inflammation by blocking TOPK signaling pathway, and TOPK is an effective target for suppressing inflammation induced by SUV irradiation. PMID:27016423

Species differences in the effects of prostanoids on MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in bovine and cat iris sphincter smooth muscle.

PubMed

Kaddour-Djebbar, I; Ansari, H R; Akhtar, R A; Abdel-Latif, A A

2005-01-01

There is evidence from our own laboratory and that of others that EP-receptor ligands are strong contractile agonists in bovine iris sphincter and that FP-receptor agonists are strong contractile agonists in cat iris sphincter. Here, we have investigated the effects of prostaglandin (PG) receptor agonists of the FP-, EP-, TP- and DP-class on myosin light chain (MLC) phosphorylation, p42/p44 MAP kinase phosphorylation and contraction in the iris sphincter of bovine and cat. Using three signal transduction mechanism assays, namely MLC phosphorylation, MAP kinase phosphorylation and contraction, we demonstrated that in bovine iris sphincter the rank order of potency of the PG agonists in the contractile and MLC phosphorylation assays is as follows: E2>U46619>F2alpha>D2, and in cat F2alpha>D2>E2>U46619. In the MAP kinase assay, in bovine iris sphincter the rank order of potency is E2>F2alpha and in cat F2alpha>E2. These conclusions are supported by the following findings: (1) In the contractile assay, in the bovine sphincter the EC50s for PGF2alpha, PGE2, U46619 and PGD2 were found to be 1.4x10(-7), 5.0x10(-9), 9.0x10(-9) and 1.3x10(-6)M, respectively, and the corresponding values in the cat were 1.9x10(-8), 2.3x10(-7), 1.5x10(-6) and 6.9x10(-8)M, respectively. (2) In the MLC phophorylation assay, in the bovine sphincter PGF2alpha, PGE2, U46619 and PGD2 increased MLC phophorylation by 118%, 165%, 153% and 72%, respectively, and the corresponding values in cat were 175%, 99%, 90% and 95%, respectively. (3) In the MAP kinase assay, in the bovine iris sphincter PGF2alpha and PGE2, increased MAP kinase phosphorylation by 276% and 328%, respectively, and the corresponding values in cat were 308% and 245%, respectively. The data presented demonstrate pronounced species differences in the effects of the prostanoids on the MLC kinase signaling pathway in bovine and cat irides and furthermore confirm the existence of FP-receptors in that of the bovine.

Novel receptor-like protein kinases induced by Erwinia carotovora and short oligogalacturonides in potato.

PubMed

Montesano, M; Kõiv, V; Mäe, A; Palva, E T

2001-11-01

summary Identification of potato genes responsive to cell wall-degrading enzymes of Erwinia carotovora resulted in the isolation of cDNA clones for four related receptor-like protein kinases. One of the putative serine-threonine protein kinases might have arisen through alternative splicing. These potato receptor-like kinases (PRK1-4) were highly equivalent (91-99%), most likely constituting a family of related receptors. All PRKs and four other plant RLKs share in their extracellular domain a conserved bi-modular pattern of cysteine repeats distinct from that in previously characterized plant RLKs, suggesting that they represent a new class of receptors. The corresponding genes were rapidly induced by E. carotovora culture filtrate (CF), both in the leaves and tubers of potato. Furthermore, the genes were transiently induced by short oligogalacturonides. The structural identity of PRKs and their induction pattern suggested that they constitute part of the early response of potato to E. carotovora infection.

Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity.

PubMed

Szczepankiewicz, Bruce G; Kosogof, Christi; Nelson, Lissa T J; Liu, Gang; Liu, Bo; Zhao, Hongyu; Serby, Michael D; Xin, Zhili; Liu, Mei; Gum, Rebecca J; Haasch, Deanna L; Wang, Sanyi; Clampit, Jill E; Johnson, Eric F; Lubben, Thomas H; Stashko, Michael A; Olejniczak, Edward T; Sun, Chaohong; Dorwin, Sarah A; Haskins, Kristi; Abad-Zapatero, Cele; Fry, Elizabeth H; Hutchins, Charles W; Sham, Hing L; Rondinone, Cristina M; Trevillyan, James M

2006-06-15

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.

The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

PubMed

Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

2010-06-01

Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

PubMed

Dwyer, Amy R; Mouchemore, Kellie A; Steer, James H; Sunderland, Andrew J; Sampaio, Natalia G; Greenland, Eloise L; Joyce, David A; Pixley, Fiona J

2016-07-01

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1. © Society for Leukocyte Biology.

Dermatophytes activate skin keratinocytes via mitogen-activated protein kinase signaling and induce immune responses.

PubMed

Achterman, Rebecca R; Moyes, David L; Thavaraj, Selvam; Smith, Adam R; Blair, Kris M; White, Theodore C; Naglik, Julian R

2015-04-01

Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation. Copyright © 2015, Achterman et al.

Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae

PubMed Central

Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

2015-01-01

The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed. PMID:25628332

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

NASA Technical Reports Server (NTRS)

Pavalko, Fredrick M.; Gerard, Rita L.; Ponik, Suzanne M.; Gallagher, Patricia J.; Jin, Yijun; Norvell, Suzanne M.

2003-01-01

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

A Conserved p38 Mitogen-Activated Protein Kinase Pathway Regulates Drosophila Immunity Gene Expression

PubMed Central

Han, Zhiqiang Stanley; Enslen, Hervé; Hu, Xiaodi; Meng, Xiangjun; Wu, I-Huan; Barrett, Tamera; Davis, Roger J.; Ip, Y. Tony

1998-01-01

Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-κB and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. We have investigated the possible involvement of the p38 MAP kinase pathway in the Drosophila immune response. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide. PMID:9584193

RelB-induced expression of Cot, an MAP3K family member, rescues RANKL-induced osteoclastogenesis in alymphoplasia mice by promoting NF-κB2 processing by IKKα.

PubMed

Taniguchi, Rei; Fukushima, Hidefumi; Osawa, Kenji; Maruyama, Toshimasa; Yasuda, Hisataka; Weih, Falk; Doi, Takahiro; Maki, Kenshi; Jimi, Eijiro

2014-03-14

The alternative nuclear factor-κB (NF-κB) pathway, mainly the RelB-p52 heterodimer, plays important roles in bone metabolism through an unknown mechanism. We have previously reported that alymphoplasia (aly/aly) mice, which lack active NF-κB-inducing kinase (NIK), show mild osteopetrosis due to the inhibition of osteoclastogenesis. p100 retains RelB in the cytoplasm and inhibits RANKL-induced osteoclastogenesis in aly/aly cells. Furthermore, the overexpression of RelB in aly/aly cells rescues RANKL-induced osteoclastogenesis by inducing p100 processing. In contrast, the overexpression of p65 in aly/aly cells has no effect. However, the overexpression of RelB fails to rescue RANKL-induced osteoclastogenesis in the presence of p100ΔGRR, which cannot be processed to p52, suggesting that p100 processing is a key step in RelB-rescued, RANKL-induced osteoclastogenesis in aly/aly cells. In this study, Cot (cancer Osaka thyroid), an MAP3K, was up-regulated by RelB overexpression. Analysis of the Cot promoter demonstrated that p65 and RelB bound to the distal NF-κB-binding site and that RelB but not p65 bound to the proximal NF-κB-binding site in the Cot promoter. The knocking down of Cot expression significantly reduced the RANKL-induced osteoclastogenesis induced by RelB overexpression. The phosphorylation of IKKα at threonine 23 and its kinase activity were indispensable for the processing of p100 and osteoclastogenesis by RelB-induced Cot. Finally, constitutively activated Akt enhanced osteoclastogenesis by RelB-induced Cot, and a dominant-negative form of Akt significantly inhibited it. Taken together, these results indicate that the overexpression of RelB restores RANKL-induced osteoclastogenesis by activation of Akt/Cot/IKKα-induced p100 processing.

RelB-induced Expression of Cot, an MAP3K Family Member, Rescues RANKL-induced Osteoclastogenesis in Alymphoplasia Mice by Promoting NF-κB2 Processing by IKKα*

PubMed Central

Taniguchi, Rei; Fukushima, Hidefumi; Osawa, Kenji; Maruyama, Toshimasa; Yasuda, Hisataka; Weih, Falk; Doi, Takahiro; Maki, Kenshi; Jimi, Eijiro

2014-01-01

The alternative nuclear factor-κB (NF-κB) pathway, mainly the RelB-p52 heterodimer, plays important roles in bone metabolism through an unknown mechanism. We have previously reported that alymphoplasia (aly/aly) mice, which lack active NF-κB-inducing kinase (NIK), show mild osteopetrosis due to the inhibition of osteoclastogenesis. p100 retains RelB in the cytoplasm and inhibits RANKL-induced osteoclastogenesis in aly/aly cells. Furthermore, the overexpression of RelB in aly/aly cells rescues RANKL-induced osteoclastogenesis by inducing p100 processing. In contrast, the overexpression of p65 in aly/aly cells has no effect. However, the overexpression of RelB fails to rescue RANKL-induced osteoclastogenesis in the presence of p100ΔGRR, which cannot be processed to p52, suggesting that p100 processing is a key step in RelB-rescued, RANKL-induced osteoclastogenesis in aly/aly cells. In this study, Cot (cancer Osaka thyroid), an MAP3K, was up-regulated by RelB overexpression. Analysis of the Cot promoter demonstrated that p65 and RelB bound to the distal NF-κB-binding site and that RelB but not p65 bound to the proximal NF-κB-binding site in the Cot promoter. The knocking down of Cot expression significantly reduced the RANKL-induced osteoclastogenesis induced by RelB overexpression. The phosphorylation of IKKα at threonine 23 and its kinase activity were indispensable for the processing of p100 and osteoclastogenesis by RelB-induced Cot. Finally, constitutively activated Akt enhanced osteoclastogenesis by RelB-induced Cot, and a dominant-negative form of Akt significantly inhibited it. Taken together, these results indicate that the overexpression of RelB restores RANKL-induced osteoclastogenesis by activation of Akt/Cot/IKKα-induced p100 processing. PMID:24488495

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

PubMed Central

Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

Midazolam suppresses interleukin-1β-induced interleukin-6 release from rat glial cells

PubMed Central

2011-01-01

Background Peripheral-type benzodiazepine receptor (PBR) expression levels are low in normal human brain, but their levels increase in inflammation, brain injury, neurodegenerative states and gliomas. It has been reported that PBR functions as an immunomodulator. The mechanisms of action of midazolam, a benzodiazepine, in the immune system in the CNS remain to be fully elucidated. We previously reported that interleukin (IL)-1β stimulates IL-6 synthesis from rat C6 glioma cells and that IL-1β induces phosphorylation of inhibitory kappa B (IκB), p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription (STAT)3. It has been shown that p38 MAP kinase is involved in IL-1β-induced IL-6 release from these cells. In the present study, we investigated the effect of midazolam on IL-1β-induced IL-6 release from C6 cells, and the mechanisms of this effect. Methods Cultured C6 cells were stimulated by IL-1β. IL-6 release from C6 cells was measured using an enzyme-linked immunosorbent assay, and phosphorylation of IκB, the MAP kinase superfamily, and STAT3 was analyzed by Western blotting. Results Midazolam, but not propofol, inhibited IL-1β-stimulated IL-6 release from C6 cells. The IL-1β-stimulated levels of IL-6 were suppressed by wedelolactone (an inhibitor of IκB kinase), SP600125 (an inhibitor of SAPK/JNK), and JAK inhibitor I (an inhibitor of JAK 1, 2 and 3). However, IL-6 levels were not affected by PD98059 (an inhibitor of MEK1/2). Midazolam markedly suppressed IL-1β-stimulated STAT3 phosphorylation without affecting the phosphorylation of p38 MAP kinase, SAPK/JNK or IκB. Conclusion These results strongly suggest that midazolam inhibits IL-1β-induced IL-6 release in rat C6 glioma cells via suppression of STAT3 activation. Midazolam may affect immune system function in the CNS. PMID:21682888

GhMAP3K65, a Cotton Raf-Like MAP3K Gene, Enhances Susceptibility to Pathogen Infection and Heat Stress by Negatively Modulating Growth and Development in Transgenic Nicotiana benthamiana.

PubMed

Zhai, Na; Jia, Haihong; Liu, Dongdong; Liu, Shuchang; Ma, Manli; Guo, Xingqi; Li, Han

2017-11-21

Mitogen-activated protein kinase kinase kinases (MAP3Ks), the top components of MAPK cascades, modulate many biological processes, such as growth, development and various environmental stresses. Nevertheless, the roles of MAP3Ks remain poorly understood in cotton. In this study, GhMAP3K65 was identified in cotton, and its transcription was inducible by pathogen infection, heat stress, and multiple signalling molecules. Silencing of GhMAP3K65 enhanced resistance to pathogen infection and heat stress in cotton. In contrast, overexpression of GhMAP3K65 enhanced susceptibility to pathogen infection and heat stress in transgenic Nicotiana benthamiana . The expression of defence-associated genes was activated in transgenic N. benthamiana plants after pathogen infection and heat stress, indicating that GhMAP3K65 positively regulates plant defence responses. Nevertheless, transgenic N. benthamiana plants impaired lignin biosynthesis and stomatal immunity in their leaves and repressed vitality of their root systems. In addition, the expression of lignin biosynthesis genes and lignin content were inhibited after pathogen infection and heat stress. Collectively, these results demonstrate that GhMAP3K65 enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development in transgenic N. benthamiana plants.

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.

PubMed

Kisfalvi, Krisztina; Hurd, Cliff; Guha, Sushovan; Rozengurt, Enrique

2010-05-01

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

A Comparative Study of the Aneugenic and Polyploidy-inducing Effects of Fisetin and Two Model Aurora Kinase Inhibitors

PubMed Central

Gollapudi, P.; Hasegawa, L.S.; Eastmond, D.A.

2014-01-01

Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements. PMID:24680981

Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae.

PubMed

Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

2015-04-01

The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C delta degradation.

PubMed

Sumitomo, M; Shen, R; Goldberg, J S; Dai, J; Navarro, D; Nanus, D M

2000-12-01

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase Cdelta (PKCdelta) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Prl cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKCdelta using Rottlerin at concentrations that inhibit PKCdelta but not PKCalpha, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKCdelta is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKCdelta protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKCdelta degradation.

Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Black, Adrienne T.; Joseph, Laurie B.; Casillas, Robert P.

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 {mu}M) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase,more » thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A{sub 4} (LTA{sub 4}) hydrolase and leukotriene C{sub 4} (LTC{sub 4}) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA{sub 4} hydrolase and LTC{sub 4} synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.« less

A mitotically inheritable unit containing a MAP kinase module

PubMed Central

Kicka, Sébastien; Bonnet, Crystel; Sobering, Andrew K.; Ganesan, Latha P.; Silar, Philippe

2006-01-01

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate. PMID:16938837

A mitotically inheritable unit containing a MAP kinase module.

PubMed

Kicka, Sébastien; Bonnet, Crystel; Sobering, Andrew K; Ganesan, Latha P; Silar, Philippe

2006-09-05

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate.

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Haipeng; Xu Beibei; Sheveleva, Elena

2008-10-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression.more » LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca{sup 2+} concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.« less

Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calabrese, G.; Sallese, M.; Stornaiuolo, A.

1994-09-01

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment ofmore » the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.« less

SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway

PubMed Central

Li, Yingqiu; Hu, Junru; Vita, Randi; Sun, Binggang; Tabata, Hiroki; Altman, Amnon

2004-01-01

Protein kinase C-θ (PKCθ) plays an important role in T-cell activation via stimulation of AP-1 and NF-κB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCθ-interacting kinase. SPAK interacted with PKCθ (but not with PKCα) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCθ, but not by PKCα. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCθ-deficient T cells. Transfected SPAK synergized with constitutively active PKCθ to activate AP-1, but not NF-κB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCθ-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCθ- and TCR/CD28-induced AP-1, but not NF-κB, activation. These results define SPAK as a substrate and target of PKCθ in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-κB) activation. PMID:14988727

The P38alpha and P38delta MAP kinases may be gene therapy targets in the future treatment of severe burns.

PubMed

Wang, Shuyun; Huang, Qiaobing; Guo, Xiaohua; Brunk, Ulf T; Han, Jiahuai; Zhao, Keseng; Zhao, Ming

2010-08-01

Microvascular barrier damage, induced by thermal injury, imposes life-threatening problems owing to the pathophysiological consequences of plasma loss and impaired perfusion that finally may lead to multiple organ failure. The aim of the present study was to define the signaling role of selected mitogen-activated protein kinases (MAPKs) in general vessel hyperpermeability caused by burns and to look for a potential gene therapy. Rearrangement of cytoskeletons and cell tight junctions were evaluated by phalloidin labeling of actin and immunocytochemical demonstration of the ZO-1 protein, whereas blood vessel permeability was evaluated by a fluorescence ratio technique. The p38 MAPK inhibitor SB203580 largely blocked burn serum-induced stress-fiber formation and tight-junction damage. Using the adenoviral approach to transfect dominant negative forms of p38 MAPKs, we found that p38alpha and p38delta had similar effects. The in vivo part of the study showed that transfection of these two constructs significantly lowered general venular hyperpermeability and enhanced the survival of burned animals. Because the p38 MAPK pathway seems to play a crucial role in burn-induced vascular hyperpermeability, general transfection with p38 MAP dominant negative constructs might become a new therapeutic method to block burn-induced plasma leakage.

Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190.

PubMed

Gardner, Samantha; Gross, Sean M; David, Larry L; Klimek, John E; Rotwein, Peter

2015-10-01

The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38α/β is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached ∼90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis. Copyright © 2015 the American Physiological Society.

A small-molecule inhibitor of NF-κB-inducing kinase (NIK) protects liver from toxin-induced inflammation, oxidative stress, and injury.

PubMed

Ren, Xiaomeng; Li, Xinzhi; Jia, Linna; Chen, Deheng; Hou, Hai; Rui, Liangyou; Zhao, Yujun; Chen, Zheng

2017-02-01

Potent and selective chemical probes are valuable tools for discovery of novel treatments for human diseases. NF-κB-inducing kinase (NIK) is a key trigger in the development of liver injury and fibrosis. Whether inhibition of NIK activity by chemical probes ameliorates liver inflammation and injury is largely unknown. In this study, a small-molecule inhibitor of NIK, B022, was found to be a potent and selective chemical probe for liver inflammation and injury. B022 inhibited the NIK signaling pathway, including NIK-induced p100-to-p52 processing and inflammatory gene expression, both in vitro and in vivo Furthermore, in vivo administration of B022 protected against not only NIK but also CCl 4 -induced liver inflammation and injury. Our data suggest that inhibition of NIK is a novel strategy for treatment of liver inflammation, oxidative stress, and injury.-Ren, X., Li, X., Jia, L., Chen, D., Hou, H., Rui, L., Zhao, Y., Chen, Z. A small-molecule inhibitor of NF-κB-inducing kinase (NIK) protects liver from toxin-induced inflammation, oxidative stress, and injury. © FASEB.

Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping

PubMed Central

Ben-Shimon, Avraham; Niv, Masha Y.

2011-01-01

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions. PMID:22125489

Activity of adenylyl cyclase and protein kinase A contributes to morphine-induced spinal apoptosis.

PubMed

Lim, Grewo; Wang, Shuxing; Lim, Jeong-Ae; Mao, Jianren

2005-12-02

Our previous study has shown that chronic morphine exposure induces neuronal apoptosis within the spinal cord dorsal horn; however, the mechanisms of morphine-induced apoptosis remain unclear. Here we examined whether adenylyl cyclase (AC) and protein kinase A (PKA) would play a role in this process. Intrathecal morphine regimen (10 microg, twice daily x 7 days) that resulted in antinociceptive tolerance induced spinal apoptosis as revealed by in situ terminal deoxynucleotidyl transferase (TdT)-UTP-biotin nick end labeling (TUNEL). The TUNEL-positive cells were detected primarily in the superficial laminae of the spinal cord dorsal horn, which was associated with an increase in the expression of activated caspase-3 and mitogen-activated protein kinase (MAPK) within the same spinal region. Co-administration of morphine with a broad AC inhibitor (ddA), a PKA inhibitor (H89), or a MAPK inhibitor (PD98059) substantially reduced the number of TUNEL-positive cells, as compared with the morphine alone group. The results indicate that the spinal AC and PKA pathway through intracellular MAPK may be contributory to the cellular mechanisms of morphine-induced apoptosis.

Role of protein kinase C alpha and mitogen-activated protein kinases in endothelin-1-stimulation of cytosolic phospholipase A2 in iris sphincter smooth muscle.

PubMed

Abdel-Latif, A A; Husain, S; Yousufzai, S Y

2000-11-01

We have investigated the roles of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) in the phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in endothelin-1- (ET-1) stimulated cat iris sphincter smooth muscle (CISM) cells. We found that in these cells both PKC and p38 MAP kinases play a critical role in ET-1-induced cPLA, phosphorylation and arachidonic acid (AA) release. Our findings indicate that stimulation of the endothelin-A- (ET(A)) receptor leads to: (1) activation of Gq protein which stimulates phospholipase C to hydrolyze the polyphosphoinositide PIP, into diacylglycerol (DAG) and inositol trisphosphate (IP3), the DAG may then activate PKC to phosphorylate and activate cPLA2; and (2) activation of Gi protein, which, through a series of kinases, leads to the stimulation of p38 MAPK and subsequently to phosphorylation and activation of cPLA2. The ability of the activated ET(A)-receptor, which is coupled to both Gq and Gi proteins, to recruit and activate this complex signal transduction mechanism remains to be clarified.

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

PubMed Central

Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

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. PMID:24475167

The MAP kinase substrate MKS1 is a regulator of plant defense responses

PubMed Central

Andreasson, Erik; Jenkins, Thomas; Brodersen, Peter; Thorgrimsen, Stephan; Petersen, Nikolaj H T; Zhu, Shijiang; Qiu, Jin-Long; Micheelsen, Pernille; Rocher, Anne; Petersen, Morten; Newman, Mari-Anne; Bjørn Nielsen, Henrik; Hirt, Heribert; Somssich, Imre; Mattsson, Ole; Mundy, John

2005-01-01

Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors. PMID:15990873

Chitin-induced and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) phosphorylation-dependent endocytosis of Arabidopsis thaliana LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5 (LYK5).

PubMed

Erwig, Jan; Ghareeb, Hassan; Kopischke, Michaela; Hacke, Ronja; Matei, Alexandra; Petutschnig, Elena; Lipka, Volker

2017-07-01

To detect potential pathogens, plants perceive the fungal polysaccharide chitin through receptor complexes containing lysin motif receptor-like kinases (LysM-RLKs). To investigate the ligand-induced spatial dynamics of chitin receptor components, we studied the subcellular behaviour of two Arabidopsis thaliana LysM-RLKs involved in chitin signalling, CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) and LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5. We performed standard and quantitative confocal laser scanning microscopy on stably transformed A. thaliana plants expressing fluorescently tagged CERK1 and LYK5 from their native promoters. Microscopy approaches were complemented by biochemical analyses in plants and in vitro. Both CERK1 and LYK5 localized to the plasma membrane and showed constitutive endomembrane trafficking. After chitin treatment, however, CERK1 remained at the plasma membrane while LYK5 relocalized into mobile intracellular vesicles. Detailed analyses revealed that chitin perception transiently induced the internalization of LYK5 into late endocytic compartments. Plants that lacked CERK1 or expressed an enzymatically inactive CERK1 variant did not exhibit chitin-induced endocytosis of LYK5. CERK1 could phosphorylate LYK5 in vitro and chitin treatment induced CERK1-dependent phosphorylation of LYK5 in planta. Our results suggest that chitin-induced phosphorylation by CERK1 triggers LYK5 internalization. Thus, our work identifies phosphorylation as a key regulatory step in endocytosis of plant RLKs and also provides evidence for receptor complex dissociation after ligand perception. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Ionizing radiation induces cellular senescence of articular chondrocytes via negative regulation of SIRT1 by p38 kinase.

PubMed

Hong, Eun-Hee; Lee, Su-Jae; Kim, Jae-Sung; Lee, Kee-Ho; Um, Hong-Duck; Kim, Jae-Hong; Kim, Song-Ja; Kim, Jong-Il; Hwang, Sang-Gu

2010-01-08

Radiotherapy is increasingly used in the treatment of joint diseases, but limited information is available on the effects of radiation on cartilage. Here, we characterize the molecular mechanisms leading to cellular senescence in irradiated primary cultured articular chondrocytes. Ionizing radiation (IR) causes activation of ERK, in turn generating intracellular reactive oxygen species (ROS) with induction of senescence-associated beta-galactosidase (SA-beta-gal) activity. ROS activate p38 kinase, which further promotes ROS generation, forming a positive feedback loop to sustain ROS-p38 kinase signaling. The ROS inhibitors, nordihydroguaiaretic acid and GSH, suppress phosphorylation of p38 and cell numbers positive for SA-beta-gal following irradiation. Moreover, inhibition of the ERK and p38 kinase pathways leads to blockage of IR-induced SA-beta-gal activity via reduction of ROS generation. Although JNK is activated by ROS, this pathway is not associated with cellular senescence of chondrocytes. Interestingly, IR triggers down-regulation of SIRT1 protein expression but not the transcript level, indicative of post-transcriptional cleavage of the protein. SIRT1 degradation is markedly blocked by SB203589 or MG132 after IR treatment, suggesting that cleavage occurs as a result of binding with p38 kinase, followed by processing via the 26 S proteasomal degradation pathway. Overexpression or activation of SIRT1 significantly reduces the IR-induced senescence phenotype, whereas inhibition of SIRT1 activity induces senescence. Based on these findings, we propose that IR induces cellular senescence of articular chondrocytes by negative post-translational regulation of SIRT1 via ROS-dependent p38 kinase activation.

Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation.

PubMed

Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

2016-01-01

Rice ( Oryza sativa ) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin E2, RHE: rice hull extract, ROS: reactive oxygen species

Stimulation of spinal dorsal horn β2-adrenergic receptor ameliorates neuropathic mechanical hypersensitivity through a reduction of phosphorylation of microglial p38 MAP kinase and astrocytic c-jun N-terminal kinase.

PubMed

Zhang, Fang Fang; Morioka, Norimitsu; Abe, Hiromi; Fujii, Shiori; Miyauchi, Kazuki; Nakamura, Yoki; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

2016-12-01

The noradrenaline-adrenergic system has a crucial role in controlling nociceptive transduction at the spinal level. While α-adrenergic receptors are known to regulate nociceptive neurotransmitter release at the spinal presynaptic level, it is not entirely clear whether β-adrenergic receptors are involved in controlling pain transduction at the spinal level as well. The current study elucidated a role of β-adrenergic receptors in neuropathic pain in mice following a partial sciatic nerve ligation (PSNL). In addition, the cellular and intracellular signaling cascade induced by β-adrenergic receptors in neuropathic mice was elaborated. Intrathecal injection of isoproterenol (1 nmol), a nonselective β-adrenergic receptor agonist, briefly ameliorated hind paw mechanical hypersensitivity of PSNL mice. Isoproterenol's antinociceptive effect was mediated through β2-adrenergic receptors since pretreatment with ICI118551, a selective β2-adrenergic receptor antagonist, but not with CGP20712A, a selective β1-adrenergic receptor antagonist, significantly attenuated isoproterenol's effect. Furthermore, intrathecal treatment with a selective β2-adrenergic receptor agonist, terbutaline, but not a selective β1-adrenergic receptor agonist, dobutamine, also significantly ameliorated neuropathic pain. Fourteen days after PSNL, increased phosphorylation of both p38 Mitogen-activated protein kinase (MAPK) in microglia and c-jun N-terminal kinase (JNK) in astrocytes of ipsilateral spinal dorsal horn were observed. Phosphorylation of both microglial p38 MAPK and astrocytic JNK were downregulated by stimulation of the β2-adrenergic receptor. Together, these results suggest that spinal β2-adrenergic receptor have an inhibitory role in neuropathic nociceptive transduction at the spinal level through a downregulation of glial activity, perhaps through modulation of MAP kinases phosphorylation. Thus, targeting of β2-adrenergic receptors could be an effective therapeutic strategy

Inhibition of PI3-kinase-Akt pathway enhances dexamethasone-induced apoptosis in a human follicular lymphoma cell line.

PubMed

Nuutinen, Ulla; Postila, Ville; Mättö, Mikko; Eeva, Jonna; Ropponen, Antti; Eray, Mine; Riikonen, Pekka; Pelkonen, Jukka

2006-02-01

Glucocorticoids are commonly used in the treatment of various lymphoid malignancies. In the present study, we show that dexamethasone (Dex) induced depolarization of mitochondrial membrane, release of cytochrome c and DNA fragmentation in a human follicular lymphoma cell line, HF28RA. New protein synthesis was required before Dex-induced mitochondrial changes, and the kinetics of the apoptotic events correlated with the upregulation of the Bim protein. Furthermore, we studied whether specific inhibitors of known survival pathways would potentiate Dex-induced apoptosis. Our results show that inhibition of PKC and ERK pathways had no effect on apoptosis. In contrast, inhibition of PI3-kinase or Akt markedly enhanced Dex-induced apoptosis. The enhancement was seen at the mitochondrial level, and the kinetics of apoptosis was notably accelerated. In addition, inhibition of PI3-kinase did not alter levels of Bax, Bcl-2, Bcl-X(L) or Bim proteins in mitochondria but caused translocation of the pro-apoptotic protein Bad to mitochondria. However, inhibition of PI3-kinase-Akt pathway and subsequent translocation of Bad to mitochondria did not induce apoptosis itself. Based on these results and our current understanding of Bim and Bad action, it seems that both proteins play a synergistic role in this process. Thus, these results indicate that inhibitors of PI3-kinase-Akt pathway might be combined in future with glucocorticoids to improve the treatment of lymphoid malignancies.

Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase Cε-Dependent Mechanism in Neurons

PubMed Central

Björnström, Karin; Turina, Dean; Strid, Tobias; Sundqvist, Tommy; Eintrei, Christina

2014-01-01

Background The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction. Methods In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser729 phosphorylation were detected with Western blot. Results The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser729 phosphorylation, a crucial step in the activation of PKCε. Conclusions Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge. PMID:24828410

Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

PubMed Central

Liu, D; Huang, Y; Bu, D; Liu, A D; Holmberg, L; Jia, Y; Tang, C; Du, J; Jin, H

2014-01-01

The present study was designed to investigate the role of endogenous sulfur dioxide (SO2) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO2 inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO2 suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO2 did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO2 dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO2 simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO2 could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in

Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked behavioral deficits in mice.

PubMed

Rehni, Ashish K; Singh, Nirmal

2007-01-01

The present study has been designed to pharmacologically investigate the role of phosphoinositide 3-kinase in ischemic postconditioning-induced reversal of global cerebral ischemia and reperfusion-induced behavioral dysfunction in mice. Bilateral carotid artery occlusion for 10 min followed by reperfusion for 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in mice. Short-term memory was evaluated using the elevated plus maze test. The inclined beam walking test was employed to assess motor incoordination. Bilateral carotid artery occlusion followed by reperfusion produced impaired short-term memory, motor co-ordination and lateral push response. Three episodes of carotid artery occlusion for a period of 10 s and reperfusion of 10 s (ischemic postconditioning) significantly prevented ischemia-reperfusion-induced behavioral deficit measured in terms of loss of short-term memory, motor coordination and lateral push response. Wortmannin (2 mg/kg, iv), a phosphoinositide 3-kinase inhibitor given 10 min before ischemia attenuated the beneficial effects of ischemic postconditioning. It may be concluded that beneficial effects of ischemic postconditioning on global cerebral ischemia and reperfusion-induced behavioral deficits may involve activation of phosphoinositide 3-kinase-linked pathway.

Osmostress induces autophosphorylation of Hog1 via a C-terminal regulatory region that is conserved in p38α.

PubMed

Maayan, Inbal; Beenstock, Jonah; Marbach, Irit; Tabachnick, Shira; Livnah, Oded; Engelberg, David

2012-01-01

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.

Osmostress Induces Autophosphorylation of Hog1 via a C-Terminal Regulatory Region That Is Conserved in p38α

PubMed Central

Maayan, Inbal; Beenstock, Jonah; Marbach, Irit; Tabachnick, Shira; Livnah, Oded; Engelberg, David

2012-01-01

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it’s removal from p38α abolishes p38α’s autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation. PMID:22984552

KSHV-TK is a tyrosine kinase that disrupts focal adhesions and induces Rho-mediated cell contraction

PubMed Central

Gill, Michael B; Turner, Rachel; Stevenson, Philip G; Way, Michael

2015-01-01

Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV-TK modulates signalling and cell morphology. PMID:25471072

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

PubMed

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

2016-10-18

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

PubMed Central

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

2016-01-01

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

PubMed

Lu, D; Yang, H; Raizada, M K

1996-12-01

Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice.

PubMed

Kim, Sungjin; Yang, Xiangkun; Li, Qianjin; Wu, Meng; Costyn, Leah; Beharry, Zanna; Bartlett, Michael G; Cai, Houjian

2017-11-10

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

PubMed Central

Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

PubMed

Fang, Jung-Da; Lee, Sheau-Ling

2014-07-01

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation. Copyright © 2014. Published by Elsevier B.V.

Age-dependent contribution of Rho kinase in carbachol-induced contraction of human detrusor smooth muscle in vitro

PubMed Central

Kirschstein, Timo; Protzel, Chris; Porath, Katrin; Sellmann, Tina; Köhling, Rüdiger; Hakenberg, Oliver W

2014-01-01

Aim: Activation of muscarinic receptors on the detrusor smooth muscle is followed by contraction, which involves both myosin light chain kinase (MLCK) and Rho kinase (ROCK). The aim of this study was to determine the relative contributions of MLCK and ROCK to carbachol-induced contraction of human detrusor smooth muscle in vitro. Methods: Detrusor smooth muscle strips were prepared from the macroscopically unaffected bladder wall of patients underwent cystectomy. The strips were fixed in an organ bath, and carbachol or KCl-induced isometric contractions were measured by force transducers. Results: Addition of carbachol (0.4-4 μmol/L) into the bath induced concentration-dependent contractions of detrusor specimens, which was completely abolished by atropine (1 μmol/L). Pre-incubation of detrusor specimens with either the MLCK inhibitor ML-9 or the ROCK inhibitors HA1100 and Y-27632 (each at 10 μmol/L) significantly blocked carbachol-induced contractions as compared to the time-control experiments. Moreover, MLCK and ROCK inhibition were equally effective in reducing carbachol-induced contractions. The residual carbachol-induced contractions in the presence of both MLCK and ROCK inhibitors were significantly smaller than the contractions obtained when only one enzyme (either MLCK or ROCK) was inhibited, suggesting an additive effect of the two kinases. Interestingly, ROCK-mediated carbachol-induced contractions were positively correlated to the age of patients (r=o.52, P<0.05). Conclusion: Both MLCK and ROCK contribute to carbachol-induced contractions of human detrusor smooth muscle. ROCK inhibitors may be a new pharmacological approach to modulate human bladder hyperactivity. PMID:24122009

Age-dependent contribution of Rho kinase in carbachol-induced contraction of human detrusor smooth muscle in vitro.

PubMed

Kirschstein, Timo; Protzel, Chris; Porath, Katrin; Sellmann, Tina; Köhling, Rüdiger; Hakenberg, Oliver W

2014-01-01

Activation of muscarinic receptors on the detrusor smooth muscle is followed by contraction, which involves both myosin light chain kinase (MLCK) and Rho kinase (ROCK). The aim of this study was to determine the relative contributions of MLCK and ROCK to carbachol-induced contraction of human detrusor smooth muscle in vitro. Detrusor smooth muscle strips were prepared from the macroscopically unaffected bladder wall of patients underwent cystectomy. The strips were fixed in an organ bath, and carbachol or KCl-induced isometric contractions were measured by force transducers. Addition of carbachol (0.4-4 μmol/L) into the bath induced concentration-dependent contractions of detrusor specimens, which was completely abolished by atropine (1 μmol/L). Pre-incubation of detrusor specimens with either the MLCK inhibitor ML-9 or the ROCK inhibitors HA1100 and Y-27632 (each at 10 μmol/L) significantly blocked carbachol-induced contractions as compared to the time-control experiments. Moreover, MLCK and ROCK inhibition were equally effective in reducing carbachol-induced contractions. The residual carbachol-induced contractions in the presence of both MLCK and ROCK inhibitors were significantly smaller than the contractions obtained when only one enzyme (either MLCK or ROCK) was inhibited, suggesting an additive effect of the two kinases. Interestingly, ROCK-mediated carbachol-induced contractions were positively correlated to the age of patients (r=o.52, P<0.05). Both MLCK and ROCK contribute to carbachol-induced contractions of human detrusor smooth muscle. ROCK inhibitors may be a new pharmacological approach to modulate human bladder hyperactivity.

Ca(2+)-sensitive tyrosine kinase Pyk2/CAK beta-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy.

PubMed

Hirotani, Shinichi; Higuchi, Yoshiharu; Nishida, Kazuhiko; Nakayama, Hiroyuki; Yamaguchi, Osamu; Hikoso, Shungo; Takeda, Toshihiro; Kashiwase, Kazunori; Watanabe, Tetsuya; Asahi, Michio; Taniike, Masayuki; Tsujimoto, Ikuko; Matsumura, Yasushi; Sasaki, Terukatsu; Hori, Masatsugu; Otsu, Kinya

2004-06-01

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Souza, C.F.; Carneiro, A.B.; Silveira, A.B.

2009-12-18

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasitemore » proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.« less

Rutin inhibits B[a]PDE-induced cyclooxygenase-2 expression by targeting EGFR kinase activity.

PubMed

Choi, Seunghwan; Lim, Tae-Gyu; Hwang, Mun Kyung; Kim, Yoon-A; Kim, Jiyoung; Kang, Nam Joo; Jang, Tae Su; Park, Jun-Seong; Yeom, Myeong Hun; Lee, Ki Won

2013-11-15

Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR(-/-)mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin. Copyright © 2013 Elsevier Inc. All rights reserved.

An interaction map of small-molecule kinase inhibitors with anaplastic lymphoma kinase (ALK) mutants in ALK-positive non-small cell lung cancer.

PubMed

Ai, Xinghao; Shen, Shengping; Shen, Lan; Lu, Shun

2015-05-01

Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Suppressive Effects of Pelargonidin on Endothelial Protein C Receptor Shedding via the Inhibition of TACE Activity and MAP Kinases.

PubMed

Kang, Hyejin; Lee, Taeho; Bae, Jong-Sup

2016-01-01

Beyond its role in the activation of protein C, the endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-[Formula: see text] converting enzyme (TACE). Pelargonidin is a well-known red pigment found in plants, and has been reported to have important biological activities that are potentially beneficial to human health. However, little is known about the effects of pelargonidin on EPCR shedding. We investigated this issue by monitoring the effects of pelargonidin on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-[Formula: see text]-, interleukin (IL)-1β-, and cecal ligation and puncture (CLP)-mediated EPCR shedding and by investigating the underlying mechanism of pelargonidin action. Data demonstrate that pelargonidin induced potent inhibition of PMA-, TNF-[Formula: see text]-, IL-1β-, and CLP-induced EPCR shedding by inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. Pelargonidin also inhibited the expression and activity of PMA-induced TACE in endothelial cells. These results demonstrate the potential of pelargonidin as an anti-EPCR shedding reagent against PMA- and CLP-mediated EPCR shedding.

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

PubMed Central

Ramnath, Raina Devi; Sun, Jia; Adhikari, Sharmila; Bhatia, Madhav

2007-01-01

Abstract Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini. PMID:18205703

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

PubMed

Marquis, Miriam; Boulet, Salix; Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

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.

Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras

PubMed Central

van Weering, David H. J.; de Rooij, Johan; Marte, Barbara; Downward, Julian; Bos, Johannes L.; Burgering, Boudewijn M. T.

1998-01-01

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events. PMID:9528752

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors.

PubMed

Ratajczak-Wrona, W; Jablonska, E; Garley, M; Jablonski, J; Radziwon, P; Iwaniuk, A

2013-01-01

The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.

Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat.

PubMed

Yang, H; Raizada, M K

1999-04-01

Chronic stimulation of norepinephrine (NE) neuromodulation by angiotensin II (Ang II) involves activation of the Ras-Raf-MAP kinase signal transduction pathway in Wistar Kyoto (WKY) rat brain neurons. This pathway is only partially responsible for this heightened action of Ang II in the spontaneously hypertensive rat (SHR) brain neurons. In this study, we demonstrate that the MAP kinase-independent signaling pathway in the SHR neuron involves activation of PI3-kinase and protein kinase B (PKB/Akt). Ang II stimulated PI3-kinase activity in both WKY and SHR brain neurons and was accompanied by its translocation from the cytoplasmic to the nuclear compartment. Although the magnitude of stimulation by Ang II was comparable, the stimulation was more persistent in the SHR neuron compared with the WKY rat neuron. Inhibition of PI3-kinase had no significant effect in the WKY rat neuron. However, it caused a 40-50% attenuation of the Ang II-induced increase in norepinephrine transporter (NET) and tyrosine hydroxylase (TH) mRNAs and [3H]-NE uptake in the SHR neuron. In contrast, inhibition of MAP kinase completely attenuated Ang II stimulation of NET and TH mRNA levels in the WKY rat neuron, whereas it caused only a 45% decrease in the SHR neuron. However, an additive attenuation was observed when both kinases of the SHR neurons were inhibited. Ang II also stimulated PKB/Akt activity in both WKY and SHR neurons. This stimulation was 30% higher and lasted longer in the SHR neuron compared with the WKY rat neuron. In conclusion, these observations demonstrate an exclusive involvement of PI3-kinase-PKB-dependent signaling pathway in a heightened NE neuromodulatory action of Ang II in the SHR neuron. Thus, this study offers an excellent potential for the development of new therapies for the treatment of centrally mediated hypertension.

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

PubMed

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

PubMed

Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

2016-01-01

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-β Synthesis Induced by Lipopolysaccharide.

PubMed

Saleh, Danish; Najjar, Malek; Zelic, Matija; Shah, Saumil; Nogusa, Shoko; Polykratis, Apostolos; Paczosa, Michelle K; Gough, Peter J; Bertin, John; Whalen, Michael; Fitzgerald, Katherine A; Slavov, Nikolai; Pasparakis, Manolis; Balachandran, Siddharth; Kelliher, Michelle; Mecsas, Joan; Degterev, Alexei

2017-06-01

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-β. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-β production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-β synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-β production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-β production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-β is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella , and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-β during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors. Copyright © 2017 by The American Association of Immunologists, Inc.

[6]-Gingerol Induces Caspase-Dependent Apoptosis and Prevents PMA-Induced Proliferation in Colon Cancer Cells by Inhibiting MAPK/AP-1 Signaling

PubMed Central

Narayanan, Sai Shyam; Nath, Lekshmi R.; Thulasidasan, Arun Kumar T.; Soniya, Eppurathu Vasudevan; Anto, Ruby John

2014-01-01

We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer. PMID:25157570

[6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling.

PubMed

Radhakrishnan, E K; Bava, Smitha V; Narayanan, Sai Shyam; Nath, Lekshmi R; Thulasidasan, Arun Kumar T; Soniya, Eppurathu Vasudevan; Anto, Ruby John

2014-01-01

We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways.

PubMed

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S; Franklin, Christopher C; McCord, Joe M; Day, Brian J

2011-09-15

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. Copyright © 2011 Elsevier Inc. All rights reserved.

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

PubMed

Dent, P; Chow, Y H; Wu, J; Morrison, D K; Jove, R; Sturgill, T W

1994-10-01

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

PubMed Central

Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

Contribution of RhoA kinase and protein kinase C to weak relaxant effect of pinacidil on carbachol-induced contractions in sensitized guinea-pig trachealis.

PubMed

Gok, Sule; Izanli-Paksoy, Ahenk; Vural, Kamil

2009-02-01

The exact mechanisms underlying the weak bronchodilator effect of K(ATP) channel openers on cholinergic stimulations is unknown. The present study was designed to examine the relaxant efect of pinacidil in guinea-pig trachea stimulated with carbachol by the presence of calcium sensitizer inhibitors; HA 1077, a rhoA kinase inhibitor, and chelerythrine, a protein kinase C inhibitor. Adenosine (10 microM) was used as other contractile agent for comparison. Tracheal tissues were isolated from ovalbumin sensitized guineapigs and changes in tension were recorded isometrically. Pinacidil (1-100 muM, cumulatively) and HA 1077 (0.01-30 microM, cumulatively) produced concentration-dependent relaxations in unstimulated tisues. The relaxant response to pinacidil decreased in carbachol contracted tissues, but increased in adenosine-stimulated tissues. Pretreatment of the tissues with HA 1077 (0.1 microM) and chelerythrine (10 microM) increased the pinacidil-induced relaxations by approximately %100 and %40, respectively. Glibenclamide, a KATP channel blocker, partially antagonized the pinacidil response in contracted tissues. Glibenclamide also inhibited the carbachol and adenosine induced contractions. These results suggest that diminish effect of pinacidil may have related to the enhanced calcium sensitization by cholinergic stimulation. Rho kinase inhibitors appear more effective than PKC inhibitors to achieve of this failure.

Human Protein Kinases and Obesity.

PubMed

Engin, Atilla

2017-01-01

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified by the target amino acid in their substrates. Some protein kinases can phosphorylate both serine/threonine, as well as tyrosine residues. This group of kinases has been known as dual specificity kinases. Unlike the dual specificity kinases, a heterogeneous group of protein phosphatases are known as dual-specificity phosphatases. These phosphatases remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases. The protein kinase-phosphoproteins interactions play an important role in obesity . In obesity, the pro- and anti-inflammatory effects of adipokines and cytokines through intracellular signaling pathways mainly involve the nuclear factor kappa B (NF-kappaB) and the c-Jun N-terminal kinase (JNK) systems as well as the inhibitor of kappaB-kinase beta (IKK beta). Impairment of insulin signaling in obesity is largely mediated by the activation of the IKKbeta and the JNK. Furthermore, oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2alpha kinase (PERK) and activating transcription factor-6. The transcriptional regulation of

Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Chin-Fu; Haase, Candice; Deguchi, Shinji

2010-10-22

Research highlights: {yields} Cyclic stretch induces stress fiber disassembly, reassembly and fusion perpendicular to the direction of stretch. {yields} Stress fiber disassembly and reorientation were not induced at low stretch frequency. {yields} Stretch caused actin fiber formation parallel to stretch in distinct locations in cells treated with Rho-kinase and MLCK inhibitors. -- Abstract: Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxialmore » stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor (Y27632) reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.« less

Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

PubMed

Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

2014-12-01

Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

Glucagon-like peptide-1 increases myocardial glucose uptake via p38alpha MAP kinase-mediated, nitric oxide-dependent mechanisms in conscious dogs with dilated cardiomyopathy.

PubMed

Bhashyam, Siva; Fields, Anjali V; Patterson, Brandy; Testani, Jeffrey M; Chen, Li; Shen, You-Tang; Shannon, Richard P

2010-07-01

We have shown that glucagon-like peptide-1 (GLP-1[7-36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1-induced myocardial glucose uptake are unknown. Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7-36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20+/-7 nmol/min/g; DCM+GLP-1: 61+/-12 nmol/min/g; P=0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98+/-2 density units; DCM: 256+/-58 density units; P=0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1-induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38alpha MAP kinase activity (DCM+vehicle: 97+/-22 pmol ATP/mg/min; DCM+GLP-1: 170+/-36 pmol ATP/mg/min; P=0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151+/-13 density units; DCM+GLP-1: 306+/-12 density units; P=0.001), and GLUT-1 translocation (DCM+vehicle: 21+/-3% membrane bound; DCM+GLP-1: 39+/-3% membrane bound; P=0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38alpha MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro-l-arginine. GLP-1 stimulates myocardial glucose uptake through a non-Akt-1-dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

PubMed

Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

2014-10-01

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

Fish oil modulates glycogen synthase kinase-3 signaling pathway in diabetes-induced hippocampal neurons apoptosis.

PubMed

Sun, Li-Juan; Hou, Xiang-Hong; Xue, Sen-Hai; Yan, Feng; Dai, Yu-Jie; Zhao, Chang-Hai; Wang, Feng; Yang, Rui-Hua

2014-07-29

Previous research has demonstrated that diabetes induces learning and memory deficits. However, the mechanism of memory impairment induced by diabetes is poorly understood. Dietary fatty acids, especially polyunsaturated fatty acids, have been shown to enhance learning and memory and prevent memory deficits in various experimental conditions. The present study investigated the effects of fish oil supplementation on the neuron apoptosis in the hippocampus of streptozotocin (STZ)-induced diabetes rats, further explored the effect of fish oil on the phosphorylation of protein kinase B and glycogen synthase kinase-3 beta. The effects of diabetes and fish oil treatment on the spatial learning and memory were also evaluated using the Morris Water Maze. STZ-induced diabetes impaired spatial learning and memory of rats, which was associated with the apoptosis of hippocampal neurons and oxidative stress. Fish oil administration ameliorated cognitive deficit, reduced oxidative stress, increased AKT phosphorylation, decreased GSK-3β phosphorylation, and decreased pro-apoptotic molecules expression, which protected the hippocampal neurons from apoptosis in diabetic rats. These results suggested a potential role for fish oil as an adjuvant therapy for the prevention and treatment of diabetic complications. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of regional liquefaction-induced deformation hazard maps

USGS Publications Warehouse

Rosinski, A.; Knudsen, K.-L.; Wu, J.; Seed, R.B.; Real, C.R.; ,

2004-01-01

This paper describes part of a project to assess the feasibility of producing regional (1:24,000-scale) liquefaction hazard maps that are based-on potential liquefaction-induced deformation. The study area is the central Santa Clara Valley, at the south end of San Francisco Bay in Central California. The information collected and used includes: a) detailed Quaternary geological mapping, b) over 650 geotechnical borings, c) probabilistic earthquake shaking information, and d) ground-water levels. Predictions of strain can be made using either empirical formulations or numerical simulations. In this project lateral spread displacements are estimated and new empirical relations to estimate future volumetric and shear strain are used. Geotechnical boring data to are used to: (a) develop isopach maps showing the thickness of sediment thatis likely to liquefy and deform under earthquake shaking; and (b) assess the variability in engineering properties within and between geologic map units. Preliminary results reveal that late Holocene deposits are likely to experience the greatest liquefaction-induced strains, while Holocene and late Pleistocene deposits are likely to experience significantly less horizontal and vertical strain in future earthquakes. Development of maps based on these analyses is feasible.

An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

NASA Astrophysics Data System (ADS)

Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D'Amelio, Nicola; Gervasio, Francesco Luigi

2016-04-01

Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

Cot, a novel kinase of histone H3, induces cellular transformation through up-regulation of c-fos transcriptional activity.

PubMed

Choi, Hong Seok; Kang, Bong Seok; Shim, Jung-Hyun; Cho, Yong-Yeon; Choi, Bu Young; Bode, Ann M; Dong, Zigang

2008-01-01

Post-translational modification of histones is critical for gene expression, mitosis, cell growth, apoptosis, and cancer development. Thus, finding protein kinases that are responsible for the phosphorylation of histones at critical sites is considered an important step in understanding the process of histone modification. The serine/threonine kinase Cot is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family. We show here that Cot can phosphorylate histone H3 at Ser-10 in vivo and in vitro, and that the phosphorylation of histone H3 at Ser-10 is required for Cot-induced cell transformation. We found that activated Cot is recruited to the c-fos promoter resulting in increased activator protein-1 (AP-1) transactivation. The formation of the Cot-c-fos promoter complex was also apparent when histone H3 was phosphorylated at Ser-10. Furthermore, the use of dominant negative mutants of histone H3 revealed that Cot was required for phosphorylation of histone H3 at Ser-10 to induce neoplastic cell transformation. These results revealed an important function of Cot as a newly discovered histone H3 kinase. Moreover, the transforming ability of Cot results from the coordinated activation of histone H3, which ultimately converges on the regulation of the transcriptional activity of the c-fos promoter, followed by AP-1 transactivation activity.

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

PubMed

Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

1999-03-01

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

PubMed

Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

2016-09-01

Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana, E-mail: knarayana@hsc.edu.kw

Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13–15 weeks; n = 6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5 mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicularmore » levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (P < 0.05). Resveratrol also recovered diabetes-induced increases in JNK signaling pathway proteins, namely, ASK1 (apoptosis signal-regulating kinase 1), JNKs (46 and 54 kDa isoforms) and p-JNK to normal control levels (P < 0.05). Interestingly, the expression of a down-stream target of ASK1, MKK4 (mitogen-activated protein kinase kinase 4) and its phosphorylated form (p-MKK4) did not change in experimental groups. Resveratrol inhibited diabetes-induced increases in AP-1 (activator protein-1) components, c-Jun and ATF2 (activating transcription factor 2), but not their phosphorylated forms, to normal control levels (P < 0.05). Further, Resveratrol inhibited diabetes-induced increase in cleaved-caspase-3 to normal control levels. In conclusion, Resveratrol alleviates diabetes-induced apoptosis in testis by modulating oxidative stress, JNK signaling pathway and caspase-3 activities, but not by inhibiting hyperglycemia, in rats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. - Highlights: • Resveratrol up

Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy.

PubMed

Chen, Cheng; Gong, Wenyan; Li, Changzheng; Xiong, Fengxiao; Wang, Shaogui; Huang, Junying; Wang, Yu; Chen, Zhiquan; Chen, Qiuhong; Liu, Peiqing; Lan, Tian; Huang, Heqing

2017-10-03

Activation of sphingosine kinase 1 (SphK1) signaling pathway mediates fibronectin (FN) upregulation in glomerular mesangial cells (GMCs) under high glucose (HG) condition. However, the roles of SphK1 in advanced glycation end products (AGEs)-induced DN have not been elucidated. Here we show that AGEs upregulated FN and SphK1 and SphK1 activity. Inhibition of SphK1 signaling attenuated AGEs-induced FN synthesis in GMCs. Inhibition of AGE receptor (RAGE) signaling reduced the upregulation of FN and SphK1 and SphK1 activity in GMCs induced by AGEs. Treatment of aminoguanidine ameliorates the renal injury and fibrosis in STZ-induced diabetic mice and attenuated SphK1 expression and activity in diabetic mouse kidneys. The renal injury and fibrosis in diabetic SphK1 -/- mice was significantly attenuated than WT mice. Furthermore, AGEs upregulated SphK1 by reducing its degradation and prolonging its half-life. SphK1 mediates AGEs-induced FN synthesis in GMCs and diabetic mice under hyperglycemic condition .

Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy

PubMed Central

Chen, Cheng; Gong, Wenyan; Li, Changzheng; Xiong, Fengxiao; Wang, Shaogui; Huang, Junying; Wang, Yu; Chen, Zhiquan; Chen, Qiuhong; Liu, Peiqing; Lan, Tian; Huang, Heqing

2017-01-01

Activation of sphingosine kinase 1 (SphK1) signaling pathway mediates fibronectin (FN) upregulation in glomerular mesangial cells (GMCs) under high glucose (HG) condition. However, the roles of SphK1 in advanced glycation end products (AGEs)-induced DN have not been elucidated. Here we show that AGEs upregulated FN and SphK1 and SphK1 activity. Inhibition of SphK1 signaling attenuated AGEs-induced FN synthesis in GMCs. Inhibition of AGE receptor (RAGE) signaling reduced the upregulation of FN and SphK1 and SphK1 activity in GMCs induced by AGEs. Treatment of aminoguanidine ameliorates the renal injury and fibrosis in STZ-induced diabetic mice and attenuated SphK1 expression and activity in diabetic mouse kidneys. The renal injury and fibrosis in diabetic SphK1-/- mice was significantly attenuated than WT mice. Furthermore, AGEs upregulated SphK1 by reducing its degradation and prolonging its half-life. Conclusion: SphK1 mediates AGEs-induced FN synthesis in GMCs and diabetic mice under hyperglycemic condition. PMID:29108256

Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jixia; Malakhova, Margarita; Mottamal, Madhusoodanan

Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific bindingmore » of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G{sub 2}-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-{kappa}B during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.« less

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

PubMed

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal H5N1 infection.

PubMed

Börgeling, Yvonne; Schmolke, Mirco; Viemann, Dorothee; Nordhoff, Carolin; Roth, Johannes; Ludwig, Stephan

2014-01-03

Highly pathogenic avian influenza viruses (HPAIV) induce severe inflammation in poultry and men. One characteristic of HPAIV infections is the induction of a cytokine burst that strongly contributes to viral pathogenicity. This cell-intrinsic hypercytokinemia seems to involve hyperinduction of p38 mitogen-activated protein kinase. Here we investigate the role of p38 MAPK signaling in the antiviral response against HPAIV in mice as well as in human endothelial cells, the latter being a primary source of cytokines during systemic infections. Global gene expression profiling of HPAIV-infected endothelial cells in the presence of the p38-specific inhibitor SB 202190 revealed that inhibition of p38 MAPK leads to reduced expression of IFNβ and other cytokines after H5N1 and H7N7 infection. More than 90% of all virus-induced genes were either partially or fully dependent on p38 signaling. Moreover, promoter analysis confirmed a direct impact of p38 on the IFNβ promoter activity. Furthermore, upon treatment with IFN or conditioned media from HPAIV-infected cells, p38 controls interferon-stimulated gene expression by coregulating STAT1 by phosphorylation at serine 727. In vivo inhibition of p38 MAPK greatly diminishes virus-induced cytokine expression concomitant with reduced viral titers, thereby protecting mice from lethal infection. These observations show that p38 MAPK acts on two levels of the antiviral IFN response. Initially the kinase regulates IFN induction and, at a later stage, p38 controls IFN signaling and thereby expression of IFN-stimulated genes. Thus, inhibition of MAP kinase p38 may be an antiviral strategy that protects mice from lethal influenza by suppressing excessive cytokine expression.

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels inmore » breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy.« less

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells.

PubMed

Chen, Lisa S; Redkar, Sanjeev; Bearss, David; Wierda, William G; Gandhi, Varsha

2009-11-05

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

PubMed Central

Chen, Lisa S.; Redkar, Sanjeev; Bearss, David; Wierda, William G.

2009-01-01

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-XL, XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction. PMID:19734450

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction.

PubMed

Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

2013-12-15

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. © 2013.

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

PubMed

Noh, Hae Sook; Hah, Young-Sool; Zada, Sahib; Ha, Ji Hye; Sim, Gyujin; Hwang, Jin Seok; Lai, Trang Huyen; Nguyen, Huynh Quoc; Park, Jae-Yong; Kim, Hyun Joon; Byun, June-Ho; Hahm, Jong Ryeal; Kang, Kee Ryeon; Kim, Deok Ryong

2016-11-01

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

[INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

PubMed

Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

2015-01-01

To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only

Deceleration of arginine kinase refolding by induced helical structures.

PubMed

Li, Hai-Long; Zhou, Sheng-Mei; Park, Daeui; Jeong, Hyoung Oh; Chung, Hae Young; Yang, Jun-Mo; Meng, Fan-Guo; Hu, Wei-Jiang

2012-04-01

Arginine kinase (AK) is a key metabolic enzyme for keeping energy balance in invertebrates. Therefore, regulation of the enzymatic activity and the folding studies of AK from the various invertebrates have been the focus of investigation. We studied the effects of helical structures by using hexafluoroisopropanol (HFIP) on AK folding. Folding kinetic studies showed that the folding rates of the urea-denatured AKs were significantly decelerated after being induced in various concentrations of HFIP. AK lost its activity completely at concentrations greater than 60%. The results indicated that the HFIP-induced helical structures in the denatured state play a negative role in protein folding, and the helical structures induced in 5% (v/v) HFIP act as the most effective barrier against AK taking its native structure. The computational docking simulations (binding energies for -2.19 kcal/mol for AutoDock4.2 and -20.47 kcal/mol for Dock6.3) suggested that HFIP interacts with the several important residues that are predicted by both programs. The excessively pre-organized helical structures not only hampered the folding process, but also ultimately brought about changes in the three-dimensional conformation and biological function of AK.

SCO2 induces p53-mediated apoptosis by Thr845 phosphorylation of ASK-1 and dissociation of the ASK-1-Trx complex.

PubMed

Madan, Esha; Gogna, Rajan; Kuppusamy, Periannan; Bhatt, Madan; Mahdi, Abbas Ali; Pati, Uttam

2013-04-01

p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr(845) residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching "on" an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS-ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

DNA-hypomethylating agent, 5'-azacytidine, induces cyclooxygenase-2 expression via the PI3-kinase/Akt and extracellular signal-regulated kinase-1/2 pathways in human HT1080 fibrosarcoma cells.

PubMed

Yu, Seon-Mi; Kim, Song-Ja

2015-10-01

The cytosine analogue 5'-azacytidine (5'-aza) induces DNA hypomethylation by inhibiting DNA methyltransferase. In clinical trials, 5'-aza is widely used in epigenetic anticancer treatments. Accumulated evidence shows that cyclooxygenase-2 (COX-2) is overexpressed in various cancers, indicating that it may play a critical role in carcinogenesis. However, few studies have been performed to explore the molecular mechanism underlying the increased COX-2 expression. Therefore, we tested the hypothesis that 5'-aza regulates COX-2 expression and prostaglandin E2 (PGE2) production. The human fibrosarcoma cell line HT1080, was treated with various concentrations of 5'-aza for different time periods. Protein expressions of COX-2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), pAkt, Akt, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (pERK) were determined using western blot analysis, and COX-2 mRNA expression was determined using RT-PCR. PGE2 production was evaluated using the PGE2 assay kit. The localization and expression of COX-2 were determined using immunofluorescence staining. Treatment with 5'-aza induces protein and mRNA expression of COX-2. We also observed that 5'-aza-induced COX-2 expression and PGE2 production were inhibited by S-adenosylmethionine (SAM), a methyl donor. Treatment with 5'-aza phosphorylates PI3-kinase/Akt and ERK-1/2; inhibition of these pathways by LY294002, an inhibitor of PI3-kinase/Akt, or PD98059, an inhibitor of ERK-1/2, respectively, prevents 5'-aza-induced COX-2 expression and PGE2 production. Overall, these observations indicate that the hypomethylating agent 5'-aza modulates COX-2 expression via the PI3-kinase/Akt and ERK-1/2 pathways in human HT1080 fibrosarcoma cells.

Tyrosine kinase inhibitors suppress prostaglandin F2alpha-induced phosphoinositide hydrolysis, Ca2+ elevation and contraction in iris sphincter smooth muscle.

PubMed

Yousufzai, S Y; Abdel-Latif, A A

1998-11-06

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells.

PubMed

Ma, Yingyu; Yu, Wei-Dong; Kong, Rui-Xian; Trump, Donald L; Johnson, Candace S

2006-08-15

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.

Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-{beta}1 up-regulation in proximal tubular epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Won Seok; Chang, Jai Won; Han, Nam Jeong

The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. Highmore » glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.« less

Never in mitosis gene A related kinase-6 attenuates pressure overload-induced activation of the protein kinase B pathway and cardiac hypertrophy.

PubMed

Bian, Zhouyan; Liao, Haihan; Zhang, Yan; Wu, Qingqing; Zhou, Heng; Yang, Zheng; Fu, Jinrong; Wang, Teng; Yan, Ling; Shen, Difei; Li, Hongliang; Tang, Qizhu

2014-01-01

Cardiac hypertrophy appears to be a specialized form of cellular growth that involves the proliferation control and cell cycle regulation. NIMA (never in mitosis, gene A)-related kinase-6 (Nek6) is a cell cycle regulatory gene that could induce centriole duplication, and control cell proliferation and survival. However, the exact effect of Nek6 on cardiac hypertrophy has not yet been reported. In the present study, the loss- and gain-of-function experiments were performed in Nek6 gene-deficient (Nek6-/-) mice and Nek6 overexpressing H9c2 cells to clarify whether Nek6 which promotes the cell cycle also mediates cardiac hypertrophy. Cardiac hypertrophy was induced by transthoracic aorta constriction (TAC) and then evaluated by echocardiography, pathological and molecular analyses in vivo. We got novel findings that the absence of Nek6 promoted cardiac hypertrophy, fibrosis and cardiac dysfunction, which were accompanied by a significant activation of the protein kinase B (Akt) signaling in an experimental model of TAC. Consistent with this, the overexpression of Nek6 prevented hypertrophy in H9c2 cells induced by angiotonin II and inhibited Akt signaling in vitro. In conclusion, our results demonstrate that the cell cycle regulatory gene Nek6 is also a critical signaling molecule that helps prevent cardiac hypertrophy and inhibits the Akt signaling pathway.

Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Sharma, Vidhu; Young, Lisa; Cavadas, Miguel; Owen, Kate

2016-03-21

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "COT drives resistance to RAF inhibition through MAPK pathway reactivation" by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Efficacy and gastrointestinal tolerability of ML3403, a selective inhibitor of p38 MAP kinase and CBS-3595, a dual inhibitor of p38 MAP kinase and phosphodiesterase 4 in CFA-induced arthritis in rats.

PubMed

Koch, Diana A; Silva, Rodrigo B M; de Souza, Alessandra H; Leite, Carlos E; Nicoletti, Natália F; Campos, Maria M; Laufer, Stefan; Morrone, Fernanda B

2014-03-01

Mitogen-activated protein kinase (MAPK) p38 inhibitors have entered the clinical phase, although many of them have failed due to high toxicity and lack of efficacy. In the present study we compared the effects of the selective p38 inhibitor ML3403 and the dual p38-PDE4 inhibitor CBS-3595, on inflammatory and nociceptive parameters in a model of polyarthritis in rats. Male Wistar rats (180-200 g) were used for the complete Freund's adjuvant (CFA)-induced arthritis model and they were evaluated at 14-21 days. We also analysed the effects of these pharmacological tools on liver and gastrointestinal toxicity and on cytokine levels. Repeated CBS-3595 (3 mg/kg) or ML3403 (10 mg/kg) administration produced significant anti-inflammatory actions in the chronic arthritis model induced by CFA. CBS-3595 and ML3403 treatment also markedly reduced the production of the proinflammatory cytokine IL-6 in the paw tissue, whereas it widely increased the levels of the anti-inflammatory cytokine IL-10. Moreover, CBS-3595 produced partial anti-allodynic effects in the CFA model at 4 and 8 days after treatment. Notably, ML3403 and CBS-3595 did not show marked signs of hepatoxicity, as supported by unaltered histological observations in the liver sections. Finally, both compounds were safe in the gastrointestinal tract, according to evaluation of intestinal biopsies. CBS-3595 displayed a superior profile regarding its anti-inflammatory effects. Thus p38 MAPK/PDE4 blocking might well constitute a relevant strategy for the treatment of RA.

Crucial role of rho-kinase in pressure overload-induced right ventricular hypertrophy and dysfunction in mice.

PubMed

Ikeda, Shohei; Satoh, Kimio; Kikuchi, Nobuhiro; Miyata, Satoshi; Suzuki, Kota; Omura, Junichi; Shimizu, Toru; Kobayashi, Kenta; Kobayashi, Kazuto; Fukumoto, Yoshihiro; Sakata, Yasuhiko; Shimokawa, Hiroaki

2014-06-01

Right ventricular (RV) failure is the leading cause of death in various cardiopulmonary diseases, including pulmonary hypertension. It is generally considered that the RV is vulnerable to pressure overload as compared with the left ventricle (LV). However, as compared with LV failure, the molecular mechanisms of RV failure are poorly understood, and hence therapeutic targets of the disorder remain to be elucidated. Thus, we aimed to identify molecular therapeutic targets for RV failure in a mouse model of pressure overload. To induce pressure overload to respective ventricles, we performed pulmonary artery constriction or transverse aortic constriction in mice. We first performed microarray analysis and found that the molecules related to RhoA/Rho-kinase and integrin pathways were significantly upregulated in the RV with pulmonary artery constriction compared with the LV with transverse aortic constriction. Then, we examined the responses of both ventricles to chronic pressure overload in vivo. We demonstrated that compared with transverse aortic constriction, pulmonary artery constriction caused greater extents of mortality, Rho-kinase expression (especially ROCK2 isoform), and oxidative stress in pressure-overloaded RV, reflecting the weakness of the RV in response to pressure overload. Furthermore, mice with myocardial-specific overexpression of dominant-negative Rho-kinase showed resistance to pressure overload-induced hypertrophy and dysfunction associated with reduced oxidative stress. Finally, dominant-negative Rho-kinase mice showed a significantly improved long-term survival in both pulmonary artery constriction and transverse aortic constriction as compared with littermate controls. These results indicate that the Rho-kinase pathway plays a crucial role in RV hypertrophy and dysfunction, suggesting that the pathway is a novel therapeutic target of RV failure in humans. © 2014 American Heart Association, Inc.

ERK1 and ERK2 Map Kinases: Specific Roles or Functional Redundancy?

PubMed Central

Buscà, Roser; Pouysségur, Jacques; Lenormand, Philippe

2016-01-01

The MAP kinase signaling cascade Ras/Raf/MEK/ERK has been involved in a large variety of cellular and physiological processes that are crucial for life. Many pathological situations have been associated to this pathway. More than one isoform has been described at each level of the cascade. In this review we devoted our attention to ERK1 and ERK2, which are the effector kinases of the pathway. Whether ERK1 and ERK2 specify functional differences or are in contrast functionally redundant, constitutes an ongoing debate despite the huge amount of studies performed to date. In this review we compiled data on ERK1 vs. ERK2 gene structures, protein sequences, expression levels, structural and molecular mechanisms of activation and substrate recognition. We have also attempted to perform a rigorous analysis of studies regarding the individual roles of ERK1 and ERK2 by the means of morpholinos, siRNA, and shRNA silencing as well as gene disruption or gene replacement in mice. Finally, we comment on a recent study of gene and protein evolution of ERK isoforms as a distinct approach to address the same question. Our review permits the evaluation of the relevance of published studies in the field especially when measurements of global ERK activation are taken into account. Our analysis favors the hypothesis of ERK1 and ERK2 exhibiting functional redundancy and points to the concept of the global ERK quantity, and not isoform specificity, as being the essential determinant to achieve ERK function. PMID:27376062

A Phosphatidylinositol 3-kinase-regulated Akt-independent signaling promotes cigarette smoke-induced FRA-1 expression.

PubMed

Zhang, Qin; Adiseshaiah, Pavan; Kalvakolanu, Dhananjaya V; Reddy, Sekhar P

2006-04-14

The FRA-1 proto-oncogene is overexpressed in a variety of human tumors and is known to up-regulate the expression of genes involved in tumor progression and invasion. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is also known to regulate these cellular processes. More importantly, respiratory toxicants and carcinogens activate both the PI3K-Akt pathway and FRA-1 expression in human bronchial epithelial (HBE) cells. In this study we investigated a potential link between the PI3K-Akt pathway and the cigarette smoke (CS)-stimulated epidermal growth factor receptor-mediated FRA-1 induction in non-oncogenic HBE cells. Treatment of cells with LY294002, an inhibitor of the PI3K-Akt pathway, completely blocked CS-induced FRA-1 expression. Surprisingly pharmacological inhibition of Akt had no significant effect on CS-induced FRA-1 expression. Likewise the inhibition of protein kinase C zeta, which is a known downstream effector of PI3K, did not alter FRA-1 expression. We found that the PI3K through p21-activated kinase 1 regulates FRA-1 proto-oncogene induction by CS and the subsequent activation of the Elk1 and cAMP-response element-binding protein transcription factors that are bound to the promoter in HBE cells.

The role of glycogen synthase kinase 3 beta in brain injury induced by myocardial ischemia/reperfusion injury in a rat model of diabetes mellitus.

PubMed

Zhao, Bo; Gao, Wen-Wei; Liu, Ya-Jing; Jiang, Meng; Liu, Lian; Yuan, Quan; Hou, Jia-Bao; Xia, Zhong-Yuan

2017-10-01

Myocardial ischemia/reperfusion injury can lead to severe brain injury. Glycogen synthase kinase 3 beta is known to be involved in myo-cardial ischemia/reperfusion injury and diabetes mellitus. However, the precise role of glycogen synthase kinase 3 beta in myocardial ischemia/reperfusion injury-induced brain injury is unclear. In this study, we observed the effects of glycogen synthase kinase 3 beta on brain injury induced by myocardial ischemia/reperfusion injury in diabetic rats. Rat models of diabetes mellitus were generated via intraperitoneal injection of streptozotocin. Models of myocardial ischemia/reperfusion injury were generated by occluding the anterior descending branch of the left coronary artery. Post-conditioning comprised three cycles of ischemia/reperfusion. Immunohistochemical staining and western blot assays demonstrated that after 48 hours of reperfusion, the structure of the brain was seriously damaged in the experimental rats compared with normal controls. Expression of Bax, interleukin-6, interleukin-8, terminal deoxynucleotidyl transferase dUTP nick end labeling, and cleaved caspase-3 in the brain was significantly increased, while expression of Bcl-2, interleukin-10, and phospho-glycogen synthase kinase 3 beta was decreased. Diabetes mellitus can aggravate inflammatory reactions and apoptosis. Ischemic post-conditioning with glycogen synthase kinase 3 beta inhibitor lithium chloride can effectively reverse these changes. Our results showed that myocardial ischemic post-conditioning attenuated myocardial ischemia/reperfusion injury-induced brain injury by activating glyco-gen synthase kinase 3 beta. According to these results, glycogen synthase kinase 3 beta appears to be an important factor in brain injury induced by myocardial ischemia/reperfusion injury.

Absence of Tec Family Kinases Interleukin-2 Inducible T cell Kinase (Itk) and Bruton's Tyrosine Kinase (Btk) Severely Impairs FcϵRI-dependent Mast Cell Responses*

PubMed Central

Iyer, Archana S.; Morales, J. Luis; Huang, Weishan; Ojo, Folake; Ning, Gang; Wills, Elizabeth; Baines, Joel D.; August, Avery

2011-01-01

Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcϵRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcϵRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcϵRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcϵRI-induced cytokine secretion. PMID:21212279

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis.

PubMed

Okuno, S; Kitani, T; Fujisawa, H

2001-10-01

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.

Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle

PubMed Central

Brault, Jeffrey J.; Pizzimenti, Natalie M.; Dentel, John N.; Wiseman, Robert W.

2013-01-01

Muscle contractions strongly activate p38 MAP kinases, but the precise contraction-associated sarcoplasmic event(s) (e.g. force production, energetic demands and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N-benzyl-p-toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer’s solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze-clamped for metabolite and western blot analysis. BTS and BTS+CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant 7-fold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS+CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non-stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction-induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/βisoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform while other factors are involved in activating the α/β isoforms, and also may explain how contractions differentially activate signaling pathways. PMID:23296747

The Atypical MAP Kinase SWIP-13/ERK8 Regulates Dopamine Transporters through a Rho-Dependent Mechanism

PubMed Central

Bermingham, Daniel P.; Snider, Sam L.; Miller, David M.

2017-01-01

The neurotransmitter dopamine (DA) regulates multiple behaviors across phylogeny, with disrupted DA signaling in humans associated with addiction, attention-deficit/ hyperactivity disorder, schizophrenia, and Parkinson's disease. The DA transporter (DAT) imposes spatial and temporal limits on DA action, and provides for presynaptic DA recycling to replenish neurotransmitter pools. Molecular mechanisms that regulate DAT expression, trafficking, and function, particularly in vivo, remain poorly understood, though recent studies have implicated rho-linked pathways in psychostimulant action. To identify genes that dictate the ability of DAT to sustain normal levels of DA clearance, we pursued a forward genetic screen in Caenorhabditis elegans based on the phenotype swimming-induced paralysis (Swip), a paralytic behavior observed in hermaphrodite worms with loss-of-function dat-1 mutations. Here, we report the identity of swip-13, which encodes a highly conserved ortholog of the human atypical MAP kinase ERK8. We present evidence that SWIP-13 acts presynaptically to insure adequate levels of surface DAT expression and DA clearance. Moreover, we provide in vitro and in vivo evidence supporting a conserved pathway involving SWIP-13/ERK8 activation of Rho GTPases that dictates DAT surface expression and function. SIGNIFICANCE STATEMENT Signaling by the neurotransmitter dopamine (DA) is tightly regulated by the DA transporter (DAT), insuring efficient DA clearance after release. Molecular networks that regulate DAT are poorly understood, particularly in vivo. Using a forward genetic screen in the nematode Caenorhabditis elegans, we implicate the atypical mitogen activated protein kinase, SWIP-13, in DAT regulation. Moreover, we provide in vitro and in vivo evidence that SWIP-13, as well as its human counterpart ERK8, regulate DAT surface availability via the activation of Rho proteins. Our findings implicate a novel pathway that regulates DA synaptic availability and that

Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain.

PubMed

Miyake, Zenshi; Takekawa, Mutsuhiro; Ge, Qingyuan; Saito, Haruo

2007-04-01

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45alpha/beta/gamma). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.

The Btk-dependent PIP5K1γ lipid kinase activation by Fas counteracts FasL-induced cell death.

PubMed

Rossin, Aurélie; Lounnas, Nadia; Durivault, Jérôme; Miloro, Giorgia; Gagnoux-Palacios, Laurent; Hueber, Anne-Odile

2017-11-01

The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P 2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.

Cot/Tpl-2 protein kinase as a target for the treatment of inflammatory disease.

PubMed

George, D; Salmeron, A

2009-01-01

Cot/Tpl-2/MAP3K8 is a serine/threonine protein kinase that is essential for lipopolysaccharide (LPS)-induced activation of the MEK/ERK pathway in macrophages as demonstrated in Cot/Tpl-2-deficient mice. Cot/Tpl-2 kinase activation plays an integral role in the production of pro-inflammatory cytokines such as TNF and IL-1beta in this immune cell type. Elevated levels of these cytokines have been clinically implicated as mediators of a number of autoimmune diseases, in particular, the pain and joint destruction of rheumatoid arthritis. By inference, pharmaceutical agents that inhibit Cot/Tpl-2 kinase have the potential to be novel and effective therapies for the treatment of these diseases. This review will describe the physiological regulation and importance of Cot/Tpl-2 in inflammation as well as the landscape of small molecules that have been reported as Cot/Tpl-2 inhibitors.

Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia.

PubMed

Ding, Li; Liou, Geou-Yarh; Schmitt, Daniel M; Storz, Peter; Zhang, Jin-San; Billadeau, Daniel D

2017-09-01

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras G12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The gene for creatine kinase, mitochondrial 2 (sarcomeric; CKMT2), maps to chromosome 5q13. 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richard, I.; Devaud, C.; Cherif, D.

1993-10-01

YAC clones for the creatine kinase, mitochrondial 2 (sarcomeric; CKMT2), gene were isolated. One of these YACs was localized on chromosome 5q13.3 by fluorescence in situ hybridization. A polymorphic dinucleotide repeat (heterozygosity 0.77) was identified within the seventh intron of the CKMT2 gene. Genotyping of CEPH families allowed positioning of CKMT2 on the multipoint map of chromosome 5 between D5S424 and D5S428, distal to spinal muscular atrophy (SMA) (5q12-q14). 8 refs., 1 fig., 2 tabs.

Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway

PubMed Central

Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D.; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn

2017-01-01

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted. PMID:28107519

Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway.

PubMed

Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn; Mai, Chun-Wai

2017-01-01

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted.

Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

NASA Astrophysics Data System (ADS)

Yang, Kun; Rong, Wei; Qi, Lin; Li, Jiarui; Wei, Xuening; Zhang, Zengyan

2013-10-01

Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

PubMed Central

Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

2015-01-01

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

PubMed Central

Li, Dechang; Liu, Ming S.; Ji, Baohua

2015-01-01

Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems. PMID:26244746

Design, Synthesis, and Biological Evaluation of Novel Type I1/2 p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the R-Spine.

PubMed

Walter, Niklas M; Wentsch, Heike K; Bührmann, Mike; Bauer, Silke M; Döring, Eva; Mayer-Wrangowski, Svenja; Sievers-Engler, Adrian; Willemsen-Seegers, Nicole; Zaman, Guido; Buijsman, Rogier; Lämmerhofer, Michael; Rauh, Daniel; Laufer, Stefan A

2017-10-12

We recently reported 1a (skepinone-L) as a type I p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, as a type I inhibitor, it is entirely ATP-competitive and shows just a moderate residence time. Thus, the scope was to develop a new class of advanced compounds maintaining the structural binding features of skepinone-L scaffold like inducing a glycine flip at the hinge region and occupying both hydrophobic regions I and II. Extending this scaffold with suitable residues resulted in an interference with the kinase's R-Spine. By synthesizing 69 compounds, we could significantly prolong the target residence time with one example to 3663 s, along with an excellent selectivity score of 0.006 and an outstanding potency of 1.0 nM. This new binding mode was validated by cocrystallization, showing all binding interactions typifying type I 1 / 2 binding. Moreover, microsomal studies showed convenient metabolic stability of the most potent, herein reported representatives.

o,p'-DDT induces cyclooxygenase-2 gene expression in murine macrophages: Role of AP-1 and CRE promoter elements and PI3-kinase/Akt/MAPK signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Eun Hee; Kim, Ji Young; Kim, Hyung-Kyun

Dichlorodiphenyltrichloroethane (DDT) has been used as an insecticide to prevent the devastation of malaria in tropical zones. However, many reports suggest that DDT may act as an endocrine disruptor and may have possible carcinogenic effects. Cyclooxygenase-2 (COX-2) acts as a link between inflammation and carcinogenesis through its involvement in tumor promotion. In the present study, we examined the effect of o,p'-DDT on COX-2 gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Exposure to o,p'-DDT markedly enhanced the production of prostaglandin E{sub 2} (PGE{sub 2}), a major COX-2 metabolite, in murine macrophages. Furthermore, o,p'-DDTmore » dose-dependently increased the levels of COX-2 protein and mRNA. Transfection with human COX-2 promoter construct, electrophoretic mobility shift assays and DNA-affinity protein-binding assay experiments revealed that o,p'-DDT activated the activator protein 1 (AP-1) and cyclic AMP response element (CRE) sites, but not the NF-{kappa}B site. Phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by the o,p'-DDT-induced AP-1 and CRE activation. These results demonstrate that o,p'-DDT induced COX-2 expression via AP-1 and CRE activation through the PI3-K/Akt/ERK, JNK, and p38 MAP kinase pathways. These findings provide further insight into the signal transduction pathways involved in the carcinogenic effects of o,p'-DDT.« less

A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

PubMed Central

Escobedo, Galileo; Soldevila, Gloria; Ortega-Pierres, Guadalupe; Chávez-Ríos, Jesús Ramsés; Nava, Karen; Fonseca-Liñán, Rocío; López-Griego, Lorena; Hallal-Calleros, Claudia; Ostoa-Saloma, Pedro; Morales-Montor, Jorge

2010-01-01

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design. PMID:20145710

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

PubMed

D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

2016-04-01

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

Mutation that blocks ATP binding creates a pseudokinase stabilizing the scaffolding function of kinase suppressor of Ras, CRAF and BRAF.

PubMed

Hu, Jiancheng; Yu, Haiyang; Kornev, Alexandr P; Zhao, Jianping; Filbert, Erin L; Taylor, Susan S; Shaw, Andrey S

2011-04-12

Because mutations in RAS and BRAF represent the most common mutations found in human tumors, identification of inhibitors has been a major goal. Surprisingly, new oncogenic BRAF specific inhibitors inhibit cells transformed with mutated BRAF but paradoxically stimulate the growth of cells transformed with RAS. Here, we show that the mechanism for activation is via drug-induced dimer formation between CRAF and kinase suppressor of Ras (KSR)1. To understand the function of KSR1, we generated a KSR1 mutant that cannot bind ATP but stabilizes the closed, active conformation of KSR1. Molecular modeling suggested that the mutant stabilizes the two hydrophobic spines critical for the closed active conformation. We, therefore, could use the mutant to discriminate between the scaffold versus kinase functions of KSR1. The KSR1 mutant bound constitutively to RAF and mitogen-activated protein kinase kinase (MEK) but could not reconstitute activity suggesting that the catalytic activity of KSR1 is required for its function. Analogous mutations in BRAF and CRAF allowed us to test the generality of the model. The mutation induced changes consistent with the active, closed conformation of both kinases and confirmed that BRAF functions distinctly from CRAF in the MAP kinase pathway. Not only does this work suggest that KSR1 may function as a kinase, we anticipate that the mutation that we generated may be broadly applicable to stabilize the closed conformation of other kinases many of which may also form dimers.

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

PubMed

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Calcium homeostasis and protein kinase/phosphatase balance participate in nicotine-induced memory improvement in passive avoidance task in mice.

PubMed

Michalak, Agnieszka; Biala, Grazyna

2017-01-15

Long-term potentiation (LTP) and long-term depression (LTD) depend on specific postsynaptic Ca 2+ /calmodulin concentration. LTP results from Ca 2+ influx through the activated NMDA receptors or voltage-gated calcium channels (VGCCs) and is linked with activation of protein kinases including mitogen-activated protein kinase (MAPK). Weaker synaptic stimulation, as a result of low Ca 2+ influx, leads to activation of Ca 2+ /calmodulin-dependent phosphatase (calcineurin - CaN) and triggers LTD. Interestingly, both memory formation and drug addiction share similar neuroplastic changes. Nicotine, which is one of the most common addictive drugs, manifests its memory effects through nicotinic acetylcholine receptors (nAChRs). Because nAChRs may also gate Ca 2+ , it is suggested that calcium signaling pathways are involved in nicotine-induced memory effects. Within the scope of the study was to evaluate the importance of calcium homeostasis and protein kinase/phosphatase balance in nicotine-induced short- and long-term memory effects. To assess memory function in mice passive avoidance test was used. The presented results confirm that acute nicotine (0.1mg/kg) improves short- and long-term memory. Pretreatment with L-type VGCC blockers (amlodipine, nicardipine verapamil) increased nicotine-induced memory improvement in the context of short- and long-term memory. Pretreatment with FK-506 (a potent CaN inhibitor) enhanced short- but not long-term memory effects of nicotine, while SL-327 (a selective MAPK/ERK kinase inhibitor) attenuated both nicotine-induced short- and long-term memory improvement. Acute nicotine enhances both types of memory via L-type VGCC blockade and via ERK1/2 activation. Only short- but not long-term memory enhancement induced by nicotine is dependent on CaN inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

PubMed Central

Hong, S W; Jon, J H; Kwak, J M; Nam, H G

1997-01-01

A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

Cyclin B Proteolysis and the Cyclin-dependent Kinase Inhibitor rum1p Are Required for Pheromone-induced G1 Arrest in Fission Yeast

PubMed Central

Stern, Bodo; Nurse, Paul

1998-01-01

The blocking of G1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p. We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin–associated cdc2p kinase activity and for phermone-induced G1 arrest. The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G1 arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis. We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome. In contrast, the cig2p–cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p. We suggest that pheromone signaling inhibits the cig2p–cdc2p kinase, bringing about a transient G1 arrest. As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p–cdc2p kinase; this is necessary to maintain G1 arrest. We have also shown that pheromone-induced transcription occurs only in G1 and is independent of rum1p. PMID:9614176

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

PubMed

Labbe, Benjamin D; Kristich, Christopher J

2017-11-01

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis

PubMed Central

Labbe, Benjamin D.

2017-01-01

ABSTRACT Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes. Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo. Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo. IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical

Formononetin-induced apoptosis of human prostate cancer cells through ERK1/2 mitogen-activated protein kinase inactivation.

PubMed

Ye, Y; Hou, R; Chen, J; Mo, L; Zhang, J; Huang, Y; Mo, Z

2012-04-01

Formononetin is a main active component of red clover plants (Trifolium pratense L.), and is considered as a phytoestrogen. Our previous studies demonstrated that formononetin caused cell cycle arrest at the G0/G1 phase by inactivating insulin-like growth factor 1(IGF1)/IGF1R-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in MCF-7 cells. In the present study, we investigated the molecular mechanisms involved in the effect of formononetin on prostate cancer cells. Our results suggested that higher concentrations of formononetin inhibited the proliferation of prostate cancer cells (LNCaP and PC-3), while the most striking effect was observed in LNCaP cells. We further found that formononetin inactivated extracellular signal-regulated kinase1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in a dose-dependent manner, which resulted in increased the expression levels of BCL2-associated X (Bax) mRNA and protein, and induced apoptosis in LNCaP cells. Thus, we concluded that the induced apoptosis effect of formononetin on human prostate cancer cells was related to ERK1/2 MAPK-Bax pathway. Considering that red clover plants were widely used clinically, our results provided the foundation for future development of different concentrations formononetin for treatment of prostate cancer. © Georg Thieme Verlag KG Stuttgart · New York.

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

PubMed

Mou, Linkai; Cui, Tongwei; Liu, Weiguang; Zhang, Hong; Cai, Zhanxiu; Lu, Shaoyong; Gao, Guojun

2017-05-01

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity. © 2016 John Wiley & Sons A/S.

Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

PubMed

Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A

2013-07-26

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

Acquisition of Contextual Discrimination Involves the Appearance of a RAS-GRF1/p38 Mitogen-activated Protein (MAP) Kinase-mediated Signaling Pathway That Promotes Long Term Potentiation (LTP)*

PubMed Central

Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A.

2013-01-01

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway. PMID:23766509

The involvement of protein kinase C-ε in isoflurane induced preconditioning of human embryonic stem cell--derived Nkx2.5(+) cardiac progenitor cells.

PubMed

Song, In-Ae; Oh, Ah-Young; Kim, Jin-Hee; Choi, Young-Min; Jeon, Young-Tae; Ryu, Jung-Hee; Hwang, Jung-Won

2016-02-20

Anesthetic preconditioning can improve survival of cardiac progenitor cells exposed to oxidative stress. We investigated the role of protein kinase C and isoform protein kinase C-ε in isoflurane-induced preconditioning of cardiac progenitor cells exposed to oxidative stress. Cardiac progenitor cells were obtained from undifferentiated human embryonic stem cells. Immunostaining with anti-Nkx2.5 was used to confirm the differentiated cardiac progenitor cells. Oxidative stress was induced by H2O2 and FeSO4. For anesthetic preconditioning, cardiac progenitor cells were exposed to 0.25, 0.5, and 1.0 mM of isoflurane. PMA and chelerythrine were used for protein kinase C activation and inhibition, while εψRACK and εV1-2 were used for protein kinase C -ε activation and inhibition, respectively. Isoflurane-preconditioning decreased the death rate of Cardiac progenitor cells exposed to oxidative stress (death rates isoflurane 0.5 mM 12.7 ± 9.3%, 1.0 mM 12.0 ± 7.7% vs. control 31.4 ± 10.2%). Inhibitors of both protein kinase C and protein kinase C -ε abolished the preconditioning effect of isoflurane 0.5 mM (death rates 27.6 ± 13.5% and 25.9 ± 8.7% respectively), and activators of both protein kinase C and protein kinase C - ε had protective effects from oxidative stress (death rates 16.0 ± 3.2% and 10.6 ± 3.8% respectively). Both PKC and PKC-ε are involved in isoflurane-induced preconditioning of human embryonic stem cells -derived Nkx2.5(+) Cardiac progenitor cells under oxidative stress.

Effect of Tamoxifen and Brain-Penetrant Protein Kinase C and c-Jun N-Terminal Kinase Inhibitors on Tolerance to Opioid-Induced Respiratory Depression in Mice.

PubMed

Withey, Sarah L; Hill, Rob; Lyndon, Abigail; Dewey, William L; Kelly, Eamonn; Henderson, Graeme

2017-04-01

Respiratory depression is the major cause of death in opioid overdose. We have previously shown that prolonged treatment of mice with morphine induces profound tolerance to the respiratory-depressant effects of the drug (Hill et al., 2016). The aim of the present study was to investigate whether tolerance to opioid-induced respiratory depression is mediated by protein kinase C (PKC) and/or c-Jun N-terminal kinase (JNK). We found that although mice treated for up to 6 days with morphine developed tolerance, as measured by the reduced responsiveness to an acute challenge dose of morphine, administration of the brain-penetrant PKC inhibitors tamoxifen and calphostin C restored the ability of acute morphine to produce respiratory depression in morphine-treated mice. Importantly, reversal of opioid tolerance was dependent on the nature of the opioid ligand used to induce tolerance, as these PKC inhibitors did not reverse tolerance induced by prolonged treatment of mice with methadone nor did they reverse the protection to acute morphine-induced respiratory depression afforded by prolonged treatment with buprenorphine. We found no evidence for the involvement of JNK in morphine-induced tolerance to respiratory depression. These results indicate that PKC represents a major mechanism underlying morphine tolerance, that the mechanism of opioid tolerance to respiratory depression is ligand-dependent, and that coadministration of drugs with PKC-inhibitory activity and morphine (as well as heroin, largely metabolized to morphine in the body) may render individuals more susceptible to overdose death by reversing tolerance to the effects of morphine. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Effect of Tamoxifen and Brain-Penetrant Protein Kinase C and c-Jun N-Terminal Kinase Inhibitors on Tolerance to Opioid-Induced Respiratory Depression in Mice

PubMed Central

Withey, Sarah L.; Hill, Rob; Lyndon, Abigail; Dewey, William L.; Kelly, Eamonn

2017-01-01

Respiratory depression is the major cause of death in opioid overdose. We have previously shown that prolonged treatment of mice with morphine induces profound tolerance to the respiratory-depressant effects of the drug (Hill et al., 2016). The aim of the present study was to investigate whether tolerance to opioid-induced respiratory depression is mediated by protein kinase C (PKC) and/or c-Jun N-terminal kinase (JNK). We found that although mice treated for up to 6 days with morphine developed tolerance, as measured by the reduced responsiveness to an acute challenge dose of morphine, administration of the brain-penetrant PKC inhibitors tamoxifen and calphostin C restored the ability of acute morphine to produce respiratory depression in morphine-treated mice. Importantly, reversal of opioid tolerance was dependent on the nature of the opioid ligand used to induce tolerance, as these PKC inhibitors did not reverse tolerance induced by prolonged treatment of mice with methadone nor did they reverse the protection to acute morphine-induced respiratory depression afforded by prolonged treatment with buprenorphine. We found no evidence for the involvement of JNK in morphine-induced tolerance to respiratory depression. These results indicate that PKC represents a major mechanism underlying morphine tolerance, that the mechanism of opioid tolerance to respiratory depression is ligand-dependent, and that coadministration of drugs with PKC-inhibitory activity and morphine (as well as heroin, largely metabolized to morphine in the body) may render individuals more susceptible to overdose death by reversing tolerance to the effects of morphine. PMID:28130265

Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

ERIC Educational Resources Information Center

Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

2016-01-01

In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

Phosphorylation-induced changes in the energetic frustration in human Tank binding kinase 1.

PubMed

Husain, Shahrukh; Kumar, Vijay; Hassan, Md Imtaiyaz

2018-07-14

Tank binding kinase 1 (TBK-1) plays an important role in immunity, inflammation, autophagy, cell growth and proliferation. Nevertheless, a key molecular and structural detail of TBK-1 phosphorylation and activation has been largely unknown. Here we investigated the energy landscape of phosphorylated (active) and unphosphorylated (inactive) forms of human TBK-1 to characterize the interplay between phosphorylation and local frustration. By employing the algorithm equipped with energy function and implemented in Frustratometer web-server (http://www.frustratometer.tk), we quantify the role of frustration in the activation of TBK-1. Accordingly, the conformational changes were observed in phosphoregulated active and inactive TBK-1. Substantial changes in frustration, flexibility and interatomic motions were observed among different forms of TBK-1. Structurally rigid kinase domain constitutes a minimally frustrated hub in the core of the catalytic domain, and highly frustrated clusters mainly at the C-lobe might enable the conformational transitions during activation. Also, a large network of highly frustrated interactions is found in the SDD domain of TBK-1 involved in protein-protein interactions and dimerization. The contact maps of the activation loop and α-C helix of kinase domain showed significant changes upon phosphorylation. Cross correlation analysis indicate that both intra and inter subunit correlated motions increases with phosphorylation of TBK-1. Phosphorylation thus introduces subtle changes in long-range contacts that might lead to significant conformational change of TBK-1. Copyright © 2018 Elsevier Ltd. All rights reserved.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death

PubMed Central

2012-01-01

Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73

Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death.

PubMed

Chang, Alice Y W

2012-11-17

Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at

Activation of MTK1/MEKK4 by GADD45 through Induced N-C Dissociation and Dimerization-Mediated trans Autophosphorylation of the MTK1 Kinase Domain▿ †

PubMed Central

Miyake, Zenshi; Takekawa, Mutsuhiro; Ge, Qingyuan; Saito, Haruo

2007-01-01

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45α/β/γ). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45. PMID:17242196

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala

2010-08-27

Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an eventmore » that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.« less

Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

PubMed

Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

2015-05-13

Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

Calcium/calmodulin‐dependent kinase 2 mediates Epac‐induced spontaneous transient outward currents in rat vascular smooth muscle

PubMed Central

Humphries, Edward S. A.; Kamishima, Tomoko; Quayle, John M.

2017-01-01

Key points The Ca2+ and redox‐sensing enzyme Ca2+/calmodulin‐dependent kinase 2 (CaMKII) is a crucial and well‐established signalling molecule in the heart and brain.In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII.The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent Ca2+ influx. How Epac activates STOCs is unknown.In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII.To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. Abstract Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)‐sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+‐activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage‐dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′, 5‐cyclic monophosphate‐AM (8‐pCPT‐AM) induces autophosphorylation (activation) of calcium/calmodulin‐dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8‐pCPT‐AM‐induced increases in STOC activity. Epac‐induced CaMKII activation is probably initiated by inositol 1,4,5‐trisphosphate (IP3)�

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

PubMed Central

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2013-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

Chrysophanic acid reduces testosterone-induced benign prostatic hyperplasia in rats by suppressing 5α-reductase and extracellular signal-regulated kinase

PubMed Central

Kim, Hye-Lin; Jung, Yunu; Kang, JongWook; Jeong, Mi-Young; Sethi, Gautam; Ahn, Kwang Seok; Um, Jae-Young

2017-01-01

Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in male population, of which incidence increases gradually with age. In this study, we investigated the effect of chrysophanic acid (CA) on BPH. BPH was induced by a 4-week injection of testosterone propionate (TP). Four weeks of further injection with vehicle, TP, TP + CA, TP + finasteride was carried on. In the CA treatment group, the prostate weight was reduced and the TP-induced histological changes were restored as the normal control group. CA treatment suppressed the TP-elevated prostate specific antigen (PSA) expression. In addition, 5α-reductase, a crucial factor in BPH development, was suppressed to the normal level close to the control group by CA treatment. The elevated expressions of androgen receptor (AR), estrogen receptor α and steroid receptor coactivator 1 by TP administration were also inhibited in the CA group when compared to the TP-induced BPH group. Then we evaluated the changes in three major factors of the mitogen-activated protein kinase chain during prostatic hyperplasia; extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). While ERK was elevated in the process of BPH, JNK and p38 was not changed. This up-regulated ERK was also reduced as normal by CA treatment. Further in vitro studies with RWPE-1 cells confirmed TP-induced proliferation and elevated AR, PSA and p-ERK were all reduced by CA treatment. Overall, these results suggest a potential pharmaceutical feature of CA in the treatment of BPH. PMID:27880726

Inhibition of the AMP-activated protein kinase-α2 accentuates agonist-induced vascular smooth muscle contraction and high blood pressure in mice.

PubMed

Wang, Shuangxi; Liang, Bin; Viollet, Benoit; Zou, Ming-Hui

2011-05-01

The aim of the present study was to determine the effects and molecular mechanisms by which AMP-activated protein kinase (AMPK) regulates smooth muscle contraction and blood pressure in mice. In cultured human vascular smooth muscle cells, we observed that activation of AMPK by 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited agonist-induced phosphorylation of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1). Conversely, AMPK inhibition with pharmacological or genetic means potentiated agonist-induced the phosphorylation of MLC and MYPT1, whereas it inhibited both Ras homolog gene family member A and Rho-associated kinase activity. In addition, AMPK activation or Rho-associated kinase inhibition with Y27632 abolished agonist-induced phosphorylation of MLC and MYPT1. Gene silencing of p190-guanosine triphosphatase-activating protein abolished the effects of AMPK activation on MLC, MYPT1, and Ras homolog gene family member A in human smooth muscle cells. Ex vivo analyses revealed that agonist-induced contractions of the mesenteric artery and aortas were stronger in both AMPKα1(-/-) and AMPKα2(-/-) knockout mice than in wild-type mice. Inhibition of Rho-associated kinase with Y27632 normalized agonist-induced contractions of AMPKα1(-/-) and AMPKα2(-/-) vessels. AMPKα2(-/-) mice had higher blood pressure along with decreased serine phosphorylation of p190-guanosine triphosphatase-activating protein. Finally, inhibition of the Ras homolog gene family member A/Rho-associated kinase pathway with Y27632, which suppressed MYPT1 and MLC phosphorylation, lowered blood pressure in AMPKα2(-/-) mice. In conclusion, AMPK decreases vascular smooth muscle cell contractility by inhibiting p190-GTP-activating protein-dependent Ras homolog gene family member A activation, indicating that AMPK may be a new therapeutic target in lowering high blood pressure.

Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, C.; Okabayashi, Y.; Williams, J.

CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact TSP-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the CaS ionophore ionomycin had no effect on either parameter. Recently, insulin has been shownmore » to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in US -glycerophosphate buffer and assayed for the kinase using el-TSP ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither CaS or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase.« less

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

PubMed

Hardie, William D; Davidson, Cynthia; Ikegami, Machiko; Leikauf, George D; Le Cras, Timothy D; Prestridge, Adrienne; Whitsett, Jeffrey A; Korfhagen, Thomas R

2008-06-01

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

n-Propyl gallate suppresses lipopolysaccharide-induced inducible nitric oxide synthase activation through protein kinase Cδ-mediated up-regulation of heme oxygenase-1 in RAW264.7 macrophages.

PubMed

Jeon, Wookwang; Park, Seong Ji; Kim, Byung-Chul

2017-04-15

n-Propyl gallate is a synthetic phenolic antioxidant with potential anti-inflammatory effects. However, the underlying mechanism remains largely unknown. In the present study, we showed that n-propyl gallate increases the expression and activity of the heme oxygenase-1 (HO-1), a stress-inducible protein with potent anti-inflammatory activity, in RAW264.7 macrophages. The inhibition of the HO-1 activity by treatment with zinc (II) protoporphyrin IX (ZnPP) or by knockdown of the HO-1 expression with small interference RNA significantly reversed the inhibitory effect of n-Propyl gallate on activations of nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS). An additional mechanism study using inhibitors of signaling kinases revealed the involvement of protein kinase Cδ (PKCδ) in the expression of HO-1 induced by n-Propyl gallate. Consistent with these results, n-Propyl gallate increased the intracellular levels of phosphorylated PKCδ in concentration- and time-dependent manners. The inhibitory effects of n-Propyl gallate on LPS-induced iNOS expression and nitric oxide production were also significantly attenuated by pretreatment with the PKCδ inhibitor, rottlerin, or by transfection with PKCδ (K376R), a kinase-inactive form of PKCδ. Taken together, these findings provide the first evidence that n-Propyl gallate exerts its anti-inflammatory effect through PKCδ-mediated up-regulation of HO-1 in macrophages. Copyright © 2017 Elsevier B.V. All rights reserved.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

PubMed Central

Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

CIKS, a connection to IκB kinase and stress-activated protein kinase

PubMed Central

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

PubMed

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Feifei; Jiang, Yinan; Zheng, Qiping

Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involvedmore » in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.« less

Signaling through protein kinases and transcriptional regulators in Candida albicans.

PubMed

Dhillon, Navneet K; Sharma, Sadhna; Khuller, G K

2003-01-01

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. Several signaling pathways that regulate morphogenesis have been identified, including various transcription factors that either activate or repress hypha-specific genes. Two well-characterized pathways include the MAP kinase cascade and cAMP-dependent protein kinase pathway that regulate the transcription factors Cph1p and Efg1p, respectively. cAMP also appears to interplay with other second messengers: Ca2+, inositol tri-phosphates in regulating yeast-hyphal transition. Other, less-characterized pathways include two component histidine kinases, cyclin-dependent kinase pathway, and condition specific pathways such as pH and embedded growth conditions. Nrg1 and Rfg1 function as transcriptional repressors of hyphal genes via recruitment of Tup1 co-repressor complex. Different upstream signals converge into a common downstream output during hyphal switch. The levels of expression of several genes have been shown to be associated with hyphal morphogenesis rather than with a specific hypha-inducing condition. Hyphal development is also linked to the expression of a range of other virulence factors. This review explains the relative contribution of multiple pathways that could be used by Candida albican cells to sense subtle differences in the growth conditions of its native host environment.

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-kinase Inhibitors

PubMed Central

Marlowe, Timothy A.; Lenzo, Felicia L.; Figel, Sheila A.; Grapes, Abigail T.; Cance, William G.

2016-01-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms which drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTKs) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK’s critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. Additionally, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: 1) the rapid phosphorylation and activation of RTK signaling pathways in RTKHigh cells and 2) the long-term acquisition of RTKs novel to the parental cell line in RTKLow cells. Finally, HER2+ cancer cells displayed resistance to FAK-kinase inhibition in 3D–growth assays using a HER2 isogenic system and HER2+ cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. PMID:27638858

Cardiotrophin-1 Induces Matrix Metalloproteinase-1 in Human Aortic Endothelial Cells

PubMed Central

Tokito, Akinori; Jougasaki, Michihisa; Ichiki, Tomoko; Hamasaki, Shuichi

2013-01-01