Activation of mitogen-activated protein kinase and NF-kappaB pathways by a Kaposi's sarcoma-associated herpesvirus K15 membrane protein

The K15 gene of Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus 8) consists of eight alternatively spliced exons and has been predicted to encode membrane proteins with a variable number of transmembrane regions and a common C-terminal cytoplasmic domain with putative binding sites for SH2 and SH3 domains, as well as for tumor necrosis factor receptor-associated factors. These features are reminiscent of the latent membrane proteins LMP-1 and LMP2A of Epstein-Barr virus and, more distantly, of the STP, Tip, and Tio proteins of the related gamma(2)-herpesviruses herpesvirus saimiri and herpesvirus ateles. These viral membrane proteins can activate a number of intracellular signaling pathways. We have therefore examined the abilities of different K15-encoded proteins to initiate intracellular signaling. We found that a 45-kDa K15 protein derived from all eight K15 exons and containing 12 predicted transmembrane domains in addition to the cytoplasmic domain activated the Ras/mitogen-activated protein kinase (MAPK) and NF-kappaB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-kappaB pathways required phosphorylation of tyrosine residue 481 within a putative SH2-binding site (YEEVL). This motif was phosphorylated by the tyrosine kinases Src, Lck, Yes, Hck, and Fyn. The region containing the YEEVL motif interacted with tumor necrosis factor receptor-associated factor 2 (TRAF-2), and a dominant negative TRAF-2 mutant inhibited the K15-mediated activation of the Ras/MAPK pathway, suggesting the involvement of TRAF-2 in the initiation of these signaling routes. In contrast, several smaller K15 protein isoforms activated these pathways only weakly. All of the K15 isoforms tested were, however, localized in lipid rafts, suggesting that incorporation into lipid rafts is not sufficient to initiate signaling. Additional regions of K15, located presumably in exons 2 to 5, may therefore contribute to the activation of these pathways. These findings illustrate that the 45-kDa K15 protein engages pathways similar to LMP1, LMP2A, STP, Tip, and Tio but combines functional features that are separated between LMP1 and LMP2A or STP and Tip.     

A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.     

CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogen-activated protein kinase pathways

Clinical and basic science data support an integral role of calcitonin gene-related peptide (CGRP) in the pathophysiology of temporomandibular joint disorders. Recently, we have shown that CGRP can stimulate the synthesis and release of nitric oxide (NO) from trigeminal ganglion glial cells. The goal of this study was to determine the role of mitogen-activated protein kinase (MAPK) signaling pathways in CGRP regulation of iNOS expression and NO release from cultured trigeminal ganglion glial cells from Sprague–Dawley rats. CGRP treatment for 2 h significantly increased activity of the MAPK reporter genes, Elk, ATF-2, and CHOP. In addition, CGRP increased nuclear staining for the active forms of the MAPKs: extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38. This stimulatory event was not observed in cultures pre-treated with the CGRP receptor antagonist peptide CGRP_8–37. Similarly, pre-treatment with selective MAPK inhibitors repressed increases in reporter gene activity as well as CGRP-induced increases in iNOS expression and NO release mediated by MAPKs. In addition, over-expression of MAPK kinase 1 (MEK1), MEK3, MEK6, and MEK kinase significantly increased iNOS expression and NO production in glial cells. Results from our study provide evidence that CGRP binding to its receptor can stimulate iNOS gene expression via activation of MAPK pathways in trigeminal ganglion glial cells.     

Stimulation of mitogen-activated protein kinase by G protein-coupled alpha(2)-adrenergic receptors does not require agonist-elicited endocytosis.

Agonist-elicited receptor sequestration is strikingly different for the alpha(2A)- versus alpha(2B)-adrenergic receptor (alpha(2)-AR) subtypes; the alpha(2B)-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the alpha(2A)-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648-31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999) J. Neurosci. 19, 56-63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K(+)-depleted medium eliminated alpha(2B)-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via G(i)-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.     

Relations between the mitogen-activated protein kinase and the cAMP-dependent protein kinase pathways: comradeship and hostility

Inter- and intracellular communications and responses to environmental changes are pivotal for the orchestrated and harmonious operation of multi-cellular organisms. These well-tuned functions in living organisms are mediated by the action of signal transduction pathways, which are responsible for receiving a signal, transmitting and amplifying it, and eliciting the appropriate cellular responses. Mammalian cells posses numerous signal transduction pathways that, rather than acting in solitude, interconnect with each other, a phenomenon referred to as cross-talk. This allows cells to regulate the distribution, duration, intensity and specificity of the response. The cAMP/cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascades modulate common processes in the cell and multiple levels of cross-talk between these signalling pathways have been described. The first- and best-characterized interconnections are the PKA-dependent inhibition of the MAPKs ERK1/2 mediated by RAF-1, and PKA-induced activation of ERK1/2 interceded through B-RAF. Recently, novel interactions between components of these pathways and new mechanisms for cross-talk have been elucidated. This review discusses both known and novel interactions between compounds of the cAMP/PKA and MAPKs signalling pathways in mammalian cells.     

Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.     

G protein-mediated mitogen-activated protein kinase activation by two dopamine D2 receptors

Two isoforms of dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of 29 amino acids specific to D2L within the putative third intracellular loop of the receptor, which appears to be important in selectivity for G-protein coupling. We have generated D2L- and D2S-expressing Chinese hamster ovary (CHO) cells, and regulation of the mitogen-activated protein kinase (MAPK) pathway was examined in these cells. Both D2L and D2S mediated a rapid and transient activation of MAPK with dominant activation of p42-kDa MAPK. Pertussis toxin treatment completely abrogated stimulation of MAPK mediated by D2L and D2S, demonstrating that both receptors couple to pertussis toxin-sensitive G proteins in this signaling. Stimulation of MAPK mediated by both D2L and D2S receptor was markedly attenuated by coexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct), which selectively inhibits Gbetagamma-mediated signal transduction. Further analysis of D2L- and D2S-mediated MAPK activation demonstrated that D2L-mediated MAPK activation was not significantly affected by PKC depletion or partially affected by genistein. In contrast, D2S-mediated MAPK activation was potentially inhibited by PKC depletion and genistein was capable of completely inhibiting D2S-mediated MAPK activation. Together, these results suggest that D2L- and D2S-mediated MAPK activation is predominantly Gbetagamma subunit-mediated signaling and that protein kinase C and tyrosine phosphorylations are involved in these signaling pathways.     

Activation of members of the mitogen-activated protein kinase family by glucose in endothelial cells

To better understand the molecular mechanisms for hyperglycemia-induced proatherogenic changes in endothelial cells, the effect of high glucose on activation of members of the mitogen-activated protein kinase (MAPK) family, including c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)-1, -2, and -5, and p38 kinase, was examined in bovine pulmonary artery endothelial cells (PAEC). Glucose, fructose, and raffinose induced a concentration-dependent decrease in PAEC growth. Addition of 25 mM glucose, fructose, or raffinose to normal growth medium stimulated an approximately twofold increase in JNK1 activity that was maximal after 24 h, whereas only glucose markedly increased ERK5 activity. Neither ERK1/2 nor p38 kinase activity was increased by glucose, fructose, or raffinose. The antioxidant N-acetylcysteine partially abrogated the glucose-induced increase in ERK5 activity but had no effect on the increase in JNK1 activity. In contrast, azaserine, which prevents increased flux through the hexosamine pathway, decreased glucose-induced JNK1 activity but had no effect on fructose- or raffinose-induced JNK1 activity. Consistent with this finding, glucosamine stimulated a 2.4-fold increase in JNK1 activity and reproduced the inhibitory effect of glucose on PAEC growth. In summary, glucose activates different members of the MAPK family in PAEC via distinct mechanisms. Moreover, the correlation between the ability of different sugars to activate JNK1 and inhibit cell growth suggests that activation of this signaling pathway may contribute to the growth inhibitory effect of glucose in endothelial cells.     

Regulation of stress-responsive mitogen-activated protein (MAP) kinase pathways by TAO2

Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways. Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38. Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6. Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances.     

Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.     

Graded mitogen-activated protein kinase activity precedes switch-like c-Fos induction in mammalian cells

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-or-nothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.     

Cyclosporine A-induced prostacyclin release is maintained by extracellular calcium in rat aortic endothelial cells: role of protein kinase C.

Chronic treatment with the immunosuppressive drug Cyclosporine A (CsA) is associated with increased intracellular calcium in vascular smooth muscle cells, which may activate phospholipase A2. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in CsA-induced prostacyclin (PGI2) release. CsA (10(-9) M) produced a significant increase in PGI2 release. CsA-induced PGI2 release were inhibited 80-85% by 10(-9) M, and 99-100% by 10(-6) M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), staurosporine or 1-(5-isoquinolinesulfonyl)piperazine. Pretreatment with (10(-9) M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced PGI2 release. Conversely, pretreatment with (10(-9) M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium, mediates CsA-induced PGI2 release in rat aortic endothelial cells.     

TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways

TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex.     

E-selectin-dependent signaling via the mitogen-activated protein kinase pathway in vascular endothelial cells

E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1beta-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.     

Endothelins regulate arachidonic acid release and mitogen-activated protein kinase activity in Schwann cells

Immortalized rat Schwann cells (iSC) express endothelin (ET) receptors coupled to inhibition of adenylyl cyclase and stimulation of phospholipase C (PLC). These effects precede phenotypic changes and increased DNA synthesis. We have investigated the role of ETs in the regulation of arachidonic acid (AA) release and mitogen-activated protein kinases (MAPKs). Both ET-1 and ET-3 increased AA release in iSC. This effect was sensitive to the phospholipase A(2) (PLA(2)) inhibitors E:-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H:-pyran-2-one and arachidonyl-trifluoromethyl ketone but was insensitive to inhibitors of PLC or phospholipase D-dependent diacylglycerol generation. ET-1-dependent AA release was also unaffected by removal of extracellular Ca(2+) and blocking the concomitant elevation in [Ca(2+)](i), consistent with participation of a Ca(2+)-independent PLA(2). Treatment of iSC with ETs also resulted in activation of extracellular signal-regulated kinase, c-Jun-NH(2)-terminal kinase (JNK), and p38 MAPK. A cause-effect relationship between agonist-dependent AA release and stimulation of MAPKs, but not the opposite, was suggested by activation of JNK by exogenous AA and by the observation that inhibition of MAPK kinase or p38 MAPK was inconsequential to ET-1-induced AA release. Similar effects of ETs on AA release and MAPK activity were observed in cultures expanded from primary SC and in iSC. Regulation of these effectors may mediate the control of proliferation and differentiation of SC by ETs during peripheral nerve development and regeneration.