Kinase Kinase (kinase + kinase)

Distribution by Scientific Domains

Kinds of Kinase Kinase

  • map kinase kinase
  • mitogen-activated protein kinase kinase
  • protein kinase kinase


  • Selected Abstracts


    Resveratrol inhibits proliferation of human epidermoid carcinoma A431 cells by modulating MEK1 and AP-1 signalling pathways

    EXPERIMENTAL DERMATOLOGY, Issue 7 2006
    Arianna L. Kim
    Abstract:, Resveratrol (trans -3,4,,5-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin found in grapes, and has been shown to inhibit the growth of various types of cancer cells. We investigated the mechanism of the antiproliferative effect of resveratrol in A431-transformed keratinocytes harbouring mutant p53, and show that it is accompanied by G1 cell cycle arrest, which coincides with a marked inhibition of G1 cell cycle regulatory proteins, including cyclins A and D1 and cyclin-dependent kinase (CDK)6 and p53-independent induction of p21WAF1. Cell cycle arrest was also associated with the accumulation of hypophosphorylated Rb and p27KIP1. Resveratrol inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1 > extracellular signal-regulated protein kinase (ERK)1/2 signalling, downregulated c-Jun, and suppressed activating protein (AP)-1 DNA-binding and promoter activity. In addition, the inhibition of MEK1 > ERK1/2 signalling appears to be independent of retinoblastoma protein (pRb) hypophosphorylation in A431 cells, as PD098059 did not suppress pRb phosphorylation. Our results demonstrate that resveratrol affects multiple cellular targets in A431 cells, and that the downregulation of both AP-1 and pRb contributes to its antiproliferative activity in these cells. [source]


    Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells

    FEBS JOURNAL, Issue 8 2004
    In Duk Jung
    Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3K,, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3K, signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3K, and Rac1/Cdc42. [source]


    A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation

    GLIA, Issue 11 2007
    Therese M. Roth
    Abstract The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/,), or null (,/,) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant. © 2007 Wiley-Liss, Inc. [source]


    Signalling events involved in interferon-,-inducible macrophage nitric oxide generation

    IMMUNOLOGY, Issue 4 2003
    Julie Blanchette
    Summary Nitric oxide (NO) produced by macrophages (M,) in response to interferon-, (IFN-,) plays a pivotal role in the control of intracellular pathogens. Current knowledge of the specific biochemical cascades involved in this IFN-,-inducible M, function is still limited. In the present study, we evaluated the participation of various second messengers , Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 1,, MAP kinase kinase (MEK1/2), extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) and nuclear factor kappa B (NF-,B) , in the regulation of NO production by IFN-,-stimulated J774 murine M,. The use of specific signalling inhibitors permitted us to establish that JAK2/STAT1,- and Erk1/Erk2-dependent pathways are the main players in IFN-,-inducible M, NO generation. To determine whether the inhibitory effect was taking place at the pre- and/or post-transcriptional level, we evaluated the effect of each antagonist on inducible nitric oxide synthase (iNOS) gene and protein expression, and on the capacity of IFN-, to induce JAK2, Erk1/Erk2 and STAT1, phosphorylation. All downregulatory effects occurred at the pretranscriptional level, except for NF-,B, which seems to exert its role in NO production through an iNOS-independent event. In addition, electrophoretic mobility shift assay (EMSA) analysis revealed that STAT1, is essential for IFN-,-inducible iNOS expression and NO production, whereas the contribution of NF-,B to this cellular regulation seems to be minimal. Moreover, our data suggest that Erk1/Erk2 are responsible for STAT1, Ser727 residue phosphorylation in IFN-,-stimulated M,, thus contributing to the full activation of STAT1,. Taken together, our results indicate that JAK2, MEK1/2, Erk1/Erk2 and STAT1, are key players in the IFN-,-inducible generation of NO by M,. [source]


    Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells

    INTERNATIONAL ENDODONTIC JOURNAL, Issue 12 2005
    F.-M. Huang
    Abstract Aim, To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. Methodology, The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. Results, The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). Conclusions,Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA. [source]


    Expression of Acid-Sensing Ion Channel 3 (ASIC3) in Nucleus Pulposus Cells of the Intervertebral Disc Is Regulated by p75NTR and ERK Signaling,

    JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2007
    Yoshiyasu Uchiyama
    Abstract Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment. Introduction: The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined. Materials and Methods: Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities. Results: ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3. Conclusions: Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc. [source]


    Osteoclast Differentiation by RANKL Requires NF-,B-Mediated Downregulation of Cyclin-Dependent Kinase 6 (Cdk6),

    JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2004
    Toru Ogasawara
    Abstract This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-,B signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-,B and c- jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the I,B kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-,B pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-,B-mediated downregulation is essential for efficient osteoclast differentiation. [source]


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

    JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6 2007
    Raina Devi Ramnath
    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 NF,B-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-l, (MIP-l,) 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), NF,B and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NF,B 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, NF,B 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 NF,B and AP-1 signalling pathways in mouse pancreatic acini. [source]


    Inhibition of human breast cancer cell (MBA-MD-231) invasion by the Ea4-peptide of rainbow trout pro-IGF-I

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2006
    Sineenat Siri
    Abstract It was shown previously that Ea4-peptide of trout pro-IGF-I exerted mitogenic activity in non-transformed cells and inhibited colony formation in a soft agar medium of established human cancer cells. Here we report that the same peptide inhibits the invasion of human breast cancer cells (MDA-MB-231) through a matrigel membrane in a dose-dependent manner. The expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI1) genes in MDA-MB-231 cells were downregulated by treatment with rtEa4-peptide. The inhibition of expression of these genes in response to rtEa4-peptide treatment was reduced to the control level when inhibitors for c-Jun N-terminal kinase 1/2 (JNK1/2), mitogen activated protein kinase kinase 1/2 (Mek1/2), p38 mitogen activated protein kinase (p38 MAPK), phosphatidylinositol 3-kinase (PI3K), and phosphokinase C (PKC) were used. These results suggest that inhibition of invasion of MDA-MB-231 cells by rtEa4-peptide may be mediated via the suppression of uPA, tPA, and PAI1 gene activities through signal transduction pathways. J. Cell. Biochem. 99: 1363,1373, 2006. © 2006 Wiley-Liss, Inc. [source]


    Noradrenaline enhances the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of PI3K/Akt and the mTOR/S6K pathway

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2007
    Julie Chenal
    Abstract Monocarboxylate transporter 2 (MCT2) expression is up-regulated by noradrenaline (NA) in cultured cortical neurons via a putative but undetermined translational mechanism. Western blot analysis showed that p44/p42 mitogen-activated protein kinase (MAPK) was rapidly and strongly phosphorylated by NA treatment. NA also rapidly induced serine/threonine protein kinase from AKT virus (Akt) phosphorylation but to a lesser extent than p44/p42 MAPK. However, Akt activation persisted over a longer period. Similarly, NA induced a rapid and persistent phosphorylation of mammalian target of rapamycin (mTOR), a kinase implicated in the regulation of translation in the central nervous system. Consistent with activation of the mTOR/S6 kinase pathway, phosphorylation of the ribosomal S6 protein, a component of the translation machinery, could be observed upon treatment with NA. In parallel, it was found that the NA-induced increase in MCT2 protein was almost completely blocked by LY294002 (phosphoinositide 3-kinase inhibitor) as well as by rapamycin (mTOR inhibitor), while mitogen-activated protein kinase kinase and p38 MAPK inhibitors had much smaller effects. Taken together, these data reveal that NA induces an increase in neuronal MCT2 protein expression by a mechanism involving stimulation of phosphoinositide 3-kinase/Akt and translational activation via the mTOR/S6 kinase pathway. Moreover, considering the role of NA in synaptic plasticity, alterations in MCT2 expression as described in this study might represent an adaptation to face energy demands associated with enhanced synaptic transmission. [source]


    Identification of a new functional target of haloperidol metabolite: implications for a receptor-independent role of 3-(4-fluorobenzoyl) propionic acid

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2006
    Hyeon Soo Kim
    Abstract Haloperidol, a dopamine D2 receptor blocker, is a classical neuroleptic drug that elicits extrapyramidal symptoms. Its metabolites include 3-(4-fluorobenzoyl) propionic acid (FBPA) and 4-(4-chlorophenyl)-4-piperidinol (CPHP). Until now, the biological significance of these metabolites has remained largely unknown. Here, we report that the administration of FBPA to mice effected a suppression of locomotor activity and induced catalepsy in a manner similar to that observed with haloperidol, whereas CPHP had no significant effects. Neither of these two metabolites, however, exhibited any ability to bind to the dopamine D2 receptor. FBPA blocked dopamine-induced extracellular signal-regulated kinase 1/2 phosphorylation, and it specifically affected mitogen-activated protein kinase kinase (MEK)1/2 activity in hippocampal HN33 cells. Moreover, FBPA was capable of direct interaction with MEK1/2, and inhibited its activity in vitro. We demonstrated the generation of haloperidol metabolites within haloperidol-treated cells by mass spectrometric analyses. Collectively, our results confirm the biological activity of FBPA, and provide initial clues as to the receptor-independent role of haloperidol. [source]


    Substance P release evoked by capsaicin or potassium from rat cultured dorsal root ganglion neurons is conversely modulated with bradykinin

    JOURNAL OF NEUROCHEMISTRY, Issue 5 2006
    He-Bin Tang
    Abstract To clarify the molecular mechanism of substance P (SP) release from dorsal root ganglion (DRG) neurons, we investigated the involvement of several intracellular effectors in the regulation of SP release evoked by capsaicin, potassium or/and bradykinin. Bradykinin-evoked SP release from cultured adult rat DRG neurons was attenuated by either the mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) or cycloheximide. As the long-term exposure of DRG neurons to bradykinin (3 h) resulted in extracellular signal-regulated kinase (ERK) phosphorylation at an early stage and thereafter induced cyclooxygenase-2 (COX-2) protein expression, which both contribute to the SP release triggered by bradykinin B2 receptor. The long-term exposure of DRG neurons to bradykinin enhanced the SP release by capsaicin, but attenuated that by potassium. Interestingly, the inositol 1,4,5-triphosphate (IP3)-induced calcium release blocker [2-aminoethyl diphenylborinate (2-APB)] not only inhibited the potassium-evoked SP release, but also completely abolished the enhancement of capsaicin-induced SP release by bradykinin from cultured DRG neurons. Together, these findings suggest that the molecular mechanisms of SP release by bradykinin involve the activation of MEK, and also require the de novo protein synthesis of COX-2 in DRG neurons. The IP3 -dependent calcium release could be involved in the processes of the regulation by bradykinin of capsaicin-triggered SP release. [source]


    Regulation of axotomy-induced dopaminergic neuron death and c-Jun phosphorylation by targeted inhibition of cdc42 or mixed lineage kinase

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2006
    Stephen J. Crocker
    Abstract Mechanical transection of the nigrostriatal dopamine pathway at the medial forebrain bundle (MFB) results in the delayed degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We have previously demonstrated that c-Jun activation is an obligate component of neuronal death in this model. Here we identified the small GTPase, cdc42, and mixed lineage kinases (MLKs) as upstream factors regulating neuronal loss and activation of c-Jun following MFB axotomy. Adenovirus-mediated expression of a dominant-negative form of cdc42 in nigral neurons blocked MFB axotomy-induced activation (phosphorylation) of MAP kinase kinase 4 (MKK4) and c-Jun, resulting in attenuation of SNpc neuronal death. Pharmacological inhibition of MLKs, MKK4-activating kinases, significantly reduced the phosphorylation of c-Jun and abrogated dopaminergic neuronal degeneration following MFB axotomy. Taken together, these findings suggest that death of nigral dopaminergic neurons following axotomy can be attenuated by targeting cell signaling events upstream of c-Jun N-terminal mitogen-activated protein kinase/c-Jun. [source]


    Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspects

    JOURNAL OF NEUROCHEMISTRY, Issue 4 2005
    Tiziana Rubino
    Abstract We investigated the role of the Ras/extracellular-regulated kinase (ERK) pathway in the development of tolerance to ,9 -tetrahydrocannabinol (THC)-induced reduction in spontaneous locomotor activity by a genetic (Ras-specific guanine nucleotide exchange factor (Ras-GRF1) knock-out mice) and pharmacological approach. Pre-treatment of wild-type mice with SL327 (50 mg/kg i.p.), a specific inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase of ERK, fully prevented the development of tolerance to THC-induced hypolocomotion. We investigated the impact of the inhibition of ERK activation on the biological processes involved in cannabinoid tolerance (receptor down-regulation and desensitization), by autoradiographic cannabinoid CB1 receptor and cannabinoid-stimulated [35S]GTP,S binding studies in subchronically treated mice (THC, 10 mg/kg s.c., twice a day for 5 days). In the caudate putamen and cerebellum of Ras-GRF1 knock-out mice and SL327 pre-treated wild-type mice, CB1 receptor down-regulation and desensitization did not occur, suggesting that ERK activation might account for CB1 receptor plasticity involved in the development of tolerance to THC hypolocomotor effect. In contrast, the hippocampus and prefrontal cortex showed CB1 receptor adaptations regardless of the genetic or pharmacological inhibition of the ERK pathway, suggesting regional variability in the cellular events underlying the altered CB1 receptor function. These findings suggest that at least in the caudate putamen and cerebellum, the Ras/ERK pathway is essential for triggering the alteration in CB1 receptor function responsible for tolerance to THC-induced hypomotility. [source]


    Activation of MKK6, an upstream activator of p38, in Alzheimer's disease

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2001
    Xiongwei Zhu
    Mitogen-activated protein kinase (MAPK) p38 has been implicated in the pathogenesis of Alzheimer's disease, but the upstream cascade leading to p38 activation has not been elucidated in the disease. In the present study, we focused on mitogen-activated protein kinase kinase 6 (MKK6), one of the upstream activators of p38 MAPK. We found that MKK6 was not only increased but also specifically associated with granular structures in the susceptible neurons in the hippocampus and cortex of Alzheimer's disease patients, but was only weakly diffuse in the cytoplasm in neurons in control cases. Immunoblot analysis demonstrated a significant increase of MKK6 level in Alzheimer's disease compared with age-matched controls. In this regard, in hippocampal and cortical regions of individuals with Alzheimer's disease, the activated phospho-MKK6 was localized exclusively in association with pathological alterations including neurofibrillary tangles, senile plaques, neuropil threads and granular structures, overlapping with activated p38 MAPK suggesting both a functional and mechanic link. By immunoblot analysis, phospho-MKK6 is also significantly increased in AD compared with control cases. Together, these findings lend further credence to the notion that the p38 MAPK pathway is dysregulated in Alzheimer's disease and also indicates an active role for this pathway in disease pathogenesis. [source]


    The upregulation of heat shock protein 47 in human gingival fibroblasts stimulated with cyclosporine A

    JOURNAL OF PERIODONTAL RESEARCH, Issue 3 2010
    T.-Y. Chang
    Chang T-Y, Tsai C-H, Chang Y-C. The upregulation of heat shock protein 47 in human gingival fibroblasts stimulated with cyclosporine A. J Periodont Res 2010; 45: 317,322. © 2009 John Wiley & Sons A/S Background and Objective:, Heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. Heat shock protein 47 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare Hsp47 expression in normal gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of Hsp47 expression. Material and Methods:, Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of cyclosporine A on the expression of Hsp47 in human gingival fibroblasts. In addition, Aggregatibacter actinomycetemcomitans, interleukin-1, (IL-1,) and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 were added to seek the possible regulatory mechanisms of Hsp47 expression. Results:, A significantly higher percentage of cells positively stained for Hsp47 was noted in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). Expression of Hsp47 was observed mainly in the cytoplasm of fibroblasts, endothelial cells, epithelial cells and inflammatory cells. Expression of Hsp47 was significantly higher in cyclosporine A-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). Cyclosporine A upregulated Hsp47 expression in human gingival fibroblasts in a dose-dependent manner (p < 0.05). The addition of A. actinomycetemcomitans or interlukin-1, significantly increased Hsp47 expression compared with cyclosporine A alone (p < 0.05). The MEK inhibitor U0126 was found to inhibit cyclosporine A-induced Hsp47 expression (p < 0.05). Conclusion:, Expression of Hsp47 is significantly upregulated in cyclosporine A-induced gingival overgrowth specimens, and Hsp47 expression induced by cyclosporine A in fibroblasts may be mediated by the MEK signal transduction pathway. The expression of Hsp47 could be significantly enhanced by A. actinomycetemcomitans and interlukin-1,. [source]


    Signal transduction pathways involved in the stimulation of tissue type plasminogen activator by interleukin-1, and Porphyromonas gingivalis in human osteosarcoma cells

    JOURNAL OF PERIODONTAL RESEARCH, Issue 5 2006
    Yu-Chao Chang
    Background:, Recently, evidences have shown that tissue type plasminogen activator (t-PA) may play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of t-PA in human osteosarcoma cells are not fully understood. Objectives:, The purpose of this study was to investigate the caseinolytic activity in human osteosarcoma cell line U2OS cells stimulated with interleukin-1, (IL-1,) or Porphyromonas gingivalis in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Methods:, IL-1, and the supernatants of P. gingivalis were used to evaluate the caseinolytic activity in U2OS cells by using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the caseinolytic activity. Results:, Casein zymography exhibited a caseinolytic band with a molecular weight of approximately 70 kDa, suggestive of the presence of t-PA. Secretion of t-PA was found to be stimulated with IL-1, and P. gingivalis during a 2-day culture period (p < 0.05). From the results of casein zymography and ELISA, SB203580, U0126, and LY294002 significantly reduced the IL-1, or P. gingivalis -stimulated t-PA production, respectively (p < 0.05). Conclusions:, Our findings demonstrated that IL-1, and P. gingivalis enhance t-PA production in human osteosarcoma cells, and that the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of t-PA. SB203580, U0126, and LY294002 suppress t-PA production and/or activity and may therefore be valuable therapeutics in t-PA-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis. [source]


    Regulation of type I plasminogen activator inhibitor in human gingival fibroblasts with cyclosporine A

    ORAL DISEASES, Issue 4 2010
    Y-C Ho
    Oral Diseases (2010) 16, 396,401 Objectives:, Cyclosporine A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth. Type I plasminogen activator inhibitor (PAI-1) has shown to play an important role in CsA-induced gingival overgrowth. However, little is known about whether factors can modulate CsA-induced PAI-1 expression. Methods:, Cytotoxicity, reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to investigate the effects of Human gingival fibroblasts (HGFs) exposed to CsA. In addition, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, interlukin-1,, tumor necrosis factor-,, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, signal-regulated protein kinase (ERK) inhibitor PD98059 and cell-permeable glutathione precursor N -acetyl- L -cysteine (NAC) were added to test how they modulated the effects of CsA-induced PAI-1 expression. Results:, The concentration of CsA higher than 500 ng ml,1 demonstrated cytotoxicity to HGFs (P < 0.05). Periodontal pathogens as well as proinflammatory cytokines were found to increase the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Pharmacological agents NAC, U0126, and PD98059 were found to decrease the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Conclusions:, Cyclosporine A (CsA) may predispose to gingival overgrowth under inflammatory environments. The regulation of PAI-1 expression induced by CsA might be critically related with the intracellular glutathione and the ERK-MAPK pathway. [source]


    Effect of Polymorphisms in Four Candidate Genes for Fertility on Litter Size in a German Pig Line

    REPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2010
    A Spötter
    Contents We carried out an SNP discovery project in pigs for candidate genes playing potentially important roles in embryonic development. Using eight pigs one each from eight breeds (Meishan, Mangalitza, Duroc, Pietrain, German Landrace, Hampshire, Husum Red Pied, German Large White), 36 SNPs were identified in intronic sequences of 21 porcine candidate genes based on sequencing of PCR products. The primer pairs were designed using porcine EST sequences allowing amplification of introns. These SNPs were tested for their association with the number of piglets born alive in German Large White sows using a discordant approach. Significant effects (p < 0.001 and p < 0.05, respectively) of intronic SNPs on litter size were found for four genes: mitogen-activated protein kinase kinase kinase 3 (MAP3K3), vascular endothelial growth factor receptor (KDR), erbb2 interacting protein (ERBB2IP) and peroxisome proliferator-activated receptor delta (PPARD). These SNPs can be further tested in upcoming association studies for their influence on litter size in different breeds using larger sample sizes. [source]


    Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings

    THE PLANT JOURNAL, Issue 5 2002
    Daisuke Matsuoka
    Summary The mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes. MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clarified. Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that fits both of the plant- and animal-type motifs. We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro. The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme. The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins. In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate specificity preferring ATMPK4 to ATMPK3, at least in vitro. Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change significantly. These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis. [source]


    c-Jun NH2 -terminal kinase-dependent fas activation contributes to etoposide-induced apoptosis in p53-mutated prostate cancer cells

    THE PROSTATE, Issue 4 2003
    Keiji Shimada
    Abstract Background The death receptor, Fas, has recently been demonstrated to contribute the chemotherapeutic agents-induced apoptosis, however, the detail mechanisms have yet to be fully understood, especially in prostate cancer cells. Methods PC-3 and DU145 stably transfected with dominant negative form of Fas-associated death domain (FADD) or specific kinase of c-Jun NH2 -terminal kinase (JNK) (mitogen-activated protein kinase kinase, MKK7) were selected in the presence of hygromycin B (Hyg B). Cell viability was examined by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphonyl)- 2H-tetrazolium, inner salt (MTS) assay or flowcytometric analysis using green fluorescent protein (GFP). Apoptosis was examined by DNA ladder, Western blotting analysis of cleaved caspases, or morphological analysis. The expression of Fas and JNK activation were investigated by Western blotting/flowcytometric analysis and in vitro kinase assay, respectively. Results Stimulation with etoposide significantly up-regulated Fas, and the death-inducing signaling complex (DISC) was formed in PC-3 and DU145. Stable transfection with dominant-negative FADD inhibited etoposide-induced apoptosis. In addition, stable transfection with dominant-negative MKK7, by which JNK activation was inhibited, canceled both the up-regulation of Fas and the formation of DISC by etoposide. Re-introduction of wild type p53 into PC-3 and DU145 completely suppressed these inhibitory effects. Conclusions These results suggest that, in p53-mutated prostate cancer, JNK-initiated Fas-mediated apoptotic signals may play an important role in chemosensitivity. Prostate 55: 265,280, 2003. © 2003 Wiley-Liss, Inc. [source]


    In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes

    ARTHRITIS & RHEUMATISM, Issue 6 2003
    Jean-Pierre Pelletier
    Objective The primary aim of this study was to investigate, using an experimental rabbit model of osteoarthritis (OA), the effect of a selective mitogen-activated protein kinase kinase 1/2 (MEK-1/2) inhibitor, PD 198306, on the development of structural changes. Additional aims were to assess the effects of the inhibitor on levels of phosphorylated extracellular signal,regulated kinase 1/2 (phospho,ERK-1/2) and matrix metalloproteinase 1 (MMP-1; collagenase 1) in OA chondrocytes. Methods After surgical sectioning of the anterior cruciate ligament of the right knee joint, rabbits with OA were separated into 3 experimental groups: oral treatment with placebo or with PD 198306 at a therapeutic concentration of 10 mg/kg/day or 30 mg/kg/day. Each treatment started immediately after surgery. The animals were killed 8 weeks after surgery. Macroscopic and histologic studies were performed on the cartilage and synovial membrane. The levels of phospho,ERK-1/2 and MMP-1 in OA cartilage chondrocytes were evaluated by immunohistochemistry. Normal, untreated rabbits were used as controls. Results OA rabbits treated with the highest dosage of MEK-1/2 inhibitor showed decreases in the surface area (size) of cartilage macroscopic lesions (P < 0.002) and in osteophyte width on the lateral condyles (P = 0.05). Histologically, the severity of synovial inflammation (villous hyperplasia) was also reduced (P < 0.02). In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in the superficial layer stained positive for phospho,ERK-1/2 and MMP-1 compared with normal controls. Rabbits treated with the highest dosage of PD 198306 demonstrated a significant and dose-dependent reduction in the level of phospho,ERK-1/2 and a lower level of MMP-1. Conclusion This study demonstrates that, in vivo, PD 198306, a selective inhibitor of MEK-1/2, can partially decrease the development of some of the structural changes in experimental OA. This effect was associated with a reduction in the level of phospho,ERK-1/2 in OA chondrocytes, which probably explains the action of the drug. [source]


    Effect of inhibitors of mitogen-activated protein kinase kinase on ,1B -adrenoceptor phosphorylation

    AUTONOMIC & AUTACOID PHARMACOLOGY, Issue 1-2 2009
    R. Alcántara-Hernández
    Summary 1,Mitogen-activated protein kinases mediate hormone/neurotransmitter action on proliferation and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on ,1B -adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2,-amino-3,-methoxyflavone) and UO126 [1,4-(diamino-2,3-dicyano/1,4-bis-(2-aminophenylthio)-butadiene], increased ,1B -adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid. 2,Staurosporine (IC50 , 0.8 nm) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC50 , 200 nm) (a selective protein kinase C inhibitor) inhibited PD98059-induced ,1B -adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or serine/threonine protein phosphatases. 3,Coimmunoprecipitation studies showed a constant association of total extracellular signal-regulated kinase 2 (ERK2) with ,1B -adrenoceptors. Association of phospho-ERK 1/2 to ,1B -adrenoceptors increased not only in response to agonist but also in response to agents that increase ,1B -adrenoceptor and ERK1/2 phosphorylation [such as endothelin-1, phorbol 12-myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect. 4,Our data show that blockade of MEK activity results in increased ,1B -adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor,ERK1/2 interaction. [source]


    Activation of protein kinase B by the A1 -adenosine receptor in DDT1MF-2 cells

    BRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000
    Renée Germack
    In this study the effect of insulin and A1 -adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT1MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser473. Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT1MF-2 cells (EC50 5±1 pM). The selective A1 -adenosine receptor agonist N6 -cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in PKB phosphorylation in DDT1MF-2 cells (EC50 1.3±0.5 nM). CPA-mediated increases in PKB phosphorylation were antagonized by the A1 -adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent KD value of 2.3 nM. Pre-treatment of DDT1MF-2 cells with pertussis toxin (PTX, 100 ng ml,1 for 16 h), to block Gi/Go -dependent pathways, abolished CPA (1 ,M) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC50 10.3±0.6 nM) and LY 294002 (IC50 10.3±1.2 ,M) attenuated the phosphorylation of PKB elicited by CPA (1 ,M) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 ,M) also blocked responses to insulin (100 nM). Removal of extracellular Ca2+ and chelation of intracellular Ca2+ with BAPTA had no significant effect on CPA-induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31-8220; 10 ,M), tyrosine kinase (genistein; 100 ,M), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 ,M) and p38 MAPK (SB 203580; 20 ,M) had no significant effect on CPA-induced PKB phosphorylation. In conclusion, these data demonstrate that A1 -adenosine receptor stimulation in DDT1MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway. British Journal of Pharmacology (2000) 130, 867,874; doi:10.1038/sj.bjp.0703396 [source]


    Inhibitory effect of c-Met mutants on the formation of branching tubules by a porcine aortic endothelial cell line

    CANCER SCIENCE, Issue 12 2006
    Marino Maemura
    The association of hepatocyte growth factor (HGF) with its high-affinity receptor (c-Met) has been shown to induce mitogenesis, motogenesis and morphogenesis in a variety of cell types. Various point mutations in c-Met have been identified in hereditary and sporadic papillary renal carcinomas as well as in other carcinomas. In the present study, we examined the effects of c-Met point mutations on the morphology of a porcine aortic endothelial (PAE) cell line. When cultured in three-dimensional collagen gel, PAE cells formed branching tubule structures, and HGF treatment caused breakdown of the structures and induced a scattered morphology. The exogenous expression of c-Met point mutants inhibited the formation of tubules. HGF treatment induced the formation of tubules by PAE cells expressing some c-Met mutants, but it induced the scattering of PAE cells expressing other c-Met mutants. The presence of a low concentration of a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor cancelled the inhibitory effect of the c-Met point mutations on the formation of tubules. These results suggest that c-Met point mutations affect the extracellular signal-regulated kinase (ERK) signaling required for the formation of tubules by PAE cells, and HGF binding changes the conformation of c-Met mutants, leading to the different signals required for formation of tubules and cell scattering. (Cancer Sci 2006; 97: 1343,1350) [source]


    Induction of insulin-like growth factor-I by interleukin-17F in bronchial epithelial cells

    CLINICAL & EXPERIMENTAL ALLERGY, Issue 7 2010
    M. Kawaguchi
    Summary Cite this as: M. Kawaguchi, J. Fujita, F. Kokubu, G. Ohara, S-K Huang, S. Matsukura, Y. Ishii, M. Adachi, H. Satoh and N. Hizawa, Clinical & Experimental Allergy, 2010 (40) 1036,1043. Background Increased expression of IL-17F has been noted in the airway of asthmatic patients, but its role in asthma has not been fully elucidated. Insulin-like growth factor-I (IGF-I) is known to be involved in airway remodelling and inflammation, while its regulatory mechanisms remain to be defined. Objective To further clarify the biological function of IL-17F, we investigated whether IL-17F is able to regulate the expression of IGF-I in bronchial epithelial cells. Methods Bronchial epithelial cells were stimulated with IL-17F in the presence or absence of T-helper type 2 cytokines. Various kinase inhibitors were added to the culture to identify the key signalling events leading to the expression of IGF-I, in conjunction with the use of short interfering RNAs (siRNAs) targeting mitogen- and stress-activated protein kinase (MSK) 1, p90 ribosomal S6 kinase (p90RSK), and cyclic AMP response element-binding protein (CREB). Results IL-17F significantly induced IGF-I gene and protein expression, and co-stimulation with IL-4 and IL-13 augmented its production. MAP kinase kinase (MEK) inhibitors and the Raf1 kinase inhibitor significantly inhibited IGF-I production, and the combination of PD98059 and Raf1 kinase inhibitor showed further inhibition. Overexpression of Raf1 and Ras dominant-negative mutants inhibited its expression. MSK1 inhibitors significantly blocked IL17F-induced IGF-I expression. Moreover, transfection of the siRNAs targeting MSK1, p90RSK, and CREB blocked its expression. Conclusions In bronchial epithelial cells, IL-17F is able to induce the expression of IGF-I via the Raf1,MEK1/2,ERK1/2,MSK1/p90RSK,CREB pathway in vitro. [source]


    Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica, in Virus-free and Virus-infected Isogenic Isolates

    JOURNAL OF PHYTOPATHOLOGY, Issue 6 2010
    Laura Rostagno
    Abstract Cpkk1 and Cpkk2 are two previously characterized Mitogen-activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT-PCR products resulting from the amplification of purified mRNA. TaqMan® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT-Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth. [source]


    Insights into the anthrax lethal factor,substrate interaction and selectivity using docking and molecular dynamics simulations

    PROTEIN SCIENCE, Issue 8 2009
    Georgios A. Dalkas
    Abstract The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn-dependent, highly specific metalloprotease with a molecular mass of ,90 kDa that cleaves most isoforms of the family of mitogen-activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK-based synthetic peptide provided structure-activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF-MEK/MKK interaction along the catalytic channel up to a distance of 20 Å from the zinc atom. This residue-specific view of the enzyme-substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot-spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction. [source]


    Expression of the MAPK kinases MKK-4 and MKK-7 in rheumatoid arthritis and their role as key regulators of JNK

    ARTHRITIS & RHEUMATISM, Issue 9 2003
    Monisha Sundarrajan
    Objective The mitogen-activated protein (MAP) kinase JNK is a key regulator of interleukin-1 (IL-1),induced collagenase gene expression and joint destruction in arthritis. Two upstream kinases, MKK-4 and MKK-7, have been identified as potential activators of JNK. However, the role of MAP kinase kinases (MAPKKs) and their functional organization within fibroblast-like synoviocytes (FLS) have not been defined. We therefore evaluated the interactions between the various MAP kinase components and determined their subcellular localization. Methods MKKs were identified by immunohistochemistry of rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. Western blotting was used to determine the expression of FLS. Immunoprecipitation experiments using antibodies specific for MKK-4, MKK-7, and JNK were performed. Phosphospecific antibodies and immunohistochemistry were used to evaluate the activation state of synovial MKK-4 and MKK-7. Confocal microscopy was used to determine the subcellular location of the kinases. Results Immunohistochemistry studies demonstrated abundant MKK-4 and MKK-7 in RA and OA synovium, but the levels of phosphorylated kinases were significantly higher in RA synovium. MKK-4 and MKK-7 were constitutively expressed by cultured RA and OA FLS, and IL-1 stimulation resulted in rapid phosphorylation of both kinases. JNK was detected in MKK-4 and MKK-7 immunoprecipitates. Furthermore, MKK-4 coprecipitated with MKK-7 and vice versa, indicating that the 3 kinases form a stable complex in FLS. Confocal microscopy confirmed that JNK, MKK-4, and MKK-7 colocalized in the cytoplasm, with JNK migrating to the nucleus after IL-1 stimulation. The signal complex containing MKK-4, MKK-7, and JNK was functionally active and able to phosphorylate c-Jun after IL-1 stimulation of FLS. Conclusion These studies demonstrate that JNK, MKK-4, and MKK-7 form an active signaling complex in FLS. This novel JNK signalsome is activated in response to IL-1 and migrates to the nucleus. The JNK signalsome represents a new target for therapeutic interventions designed to prevent joint destruction. [source]


    Anthrax lethal toxin promotes dephosphorylation of TTP and formation of processing bodies

    CELLULAR MICROBIOLOGY, Issue 4 2010
    Edith M. C. Chow
    Summary Anthrax lethal toxin (LeTx) is composed of protective antigen (PA) and lethal factor (LF) , PA is the receptor-binding moiety and LF is a protease that cleaves mitogen-activated protein kinase kinases (MAPKKs). LeTx subverts the immune response to Bacillus anthracis in several ways, such as downregulating interleukin-8 (IL-8) by increasing the rate of IL-8 mRNA degradation. Many transcripts are regulated through cis -acting elements that bind proteins that either impede or promote degradation. Some of these RNA-binding proteins are regulated by MAPKs and previous work has demonstrated that interfering with MAPK signalling decreases the half-life of IL-8 mRNA. Here, we have localized a segment within the IL-8 3, untranslated region responsible for LeTx-induced transcript destabilization and show that this is caused by inhibition of the p38, ERK and JNK pathways. TTP, an RNA-binding protein involved in IL-8 mRNA decay, became hypophosphorylated in LeTx-treated cells and knock-down of TTP prevented LeTx from destabilizing the IL-8 transcript. Cells that were treated with LeTx exhibited increased localization of TTP to Processing bodies, which are structures that accumulate transcripts targeted for degradation. We furthermore observed that LeTx promoted the formation of Processing bodies, revealing a link between the toxin and a major mRNA decay pathway. [source]