NF-kappaB-inducing kinase restores defective IkappaB kinase activity and NF-kappaB signaling in intestinal epithelial cells

Cytokine-stimulated IkappaBalpha degradation is impaired in HT-29 and primary intestinal epithelial cells. To gain more insight into the mechanism of this defect, we dissected cytokine-induced NF-kappaB signaling pathway in HT-29 cells. IL-1beta and TNF, alone or in combination with IFNgamma, failed to induce IkappaBalpha or IkappaBbeta degradation in HT-29 cells. Despite similar 125I-IL-1beta binding, HT-29 cells displayed no IRAK degradation, a 75% reduction of IKK activity, and decreased IkappaBalpha phosphorylation, NF-kappaB DNA binding activity and IL-8 mRNA accumulation in response to IL-1beta compared to Caco-2 cells. Selective activation of NF-kappaB pathway by adenoviral delivery of NF-kappaB-inducing kinase (Ad5NIK) or IKKbeta (Ad5IKKbeta) strongly activated IKK activity (>20 fold) in HT-29 cells with concomitant endogenous IkappaBalpha serine 32 phosphorylation and total IkappaBalpha degradation. In addition, NF-kappaB DNA binding activity and IL-8 secretion is higher in Ad5NIK-infected than in IL-1beta-stimulated HT-29 cells. These data show that altered NF-kappaB signaling is associated with impaired stimulation of an upstream IKK activator.     

Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression

Airway smooth muscle constriction leads to the development of compressive stress on bronchial epithelial cells. Normal human bronchial epithelial cells exposed to an apical-to-basal transcellular pressure difference equivalent to the computed stress in the airway during bronchoconstriction demonstrate enhanced phosphorylation of extracellular signal-regulated kinase (ERK). The response is pressure dependent and rapid, with phosphorylation increasing 14-fold in 30 min, and selective, since p38 and c-Jun NH(2)-terminal kinase phosphorylation remains unchanged after pressure application. Transcellular pressure also elicits a ninefold increase in expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF) after 1 h, followed by prominent immunostaining for pro-HB-EGF after 6 h. Inhibition of the ERK pathway with PD-98059 results in a dose-dependent reduction in pressure-induced HB-EGF gene expression. The magnitude of the HB-EGF response to transcellular pressure and tumor necrosis factor (TNF)-alpha (1 ng/ml) is similar, and the combined mechanical and inflammatory stimulus is more effective than either stimulus alone. These results demonstrate that compressive stress is a selective and potent activator of signal transduction and gene expression in bronchial epithelial cells.     

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.     

Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation

Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.     

cAMP-pKA signaling regulates multiple steps of fungal infection cooperatively with Cmk1 MAP kinase in Colletotrichum lagenarium

In Colletotrichum lagenarium, RPK1 encoding the regulatory subunit of PKA is required for pathogenicity. From the rpkl mutant that forms small colonies, we isolated three growth-suppressor mutants. All rpk1-suppressor mutants are nonpathogenic and contain amino acid changes in the PKA catalytic subunit Cpkl. To assess the roles of cyclic AMP (cAMP) signaling in detail, we generated knockout mutants of CPK1 and the adenylate cyclase gene CAC1. The cpk1 and cac1 mutants are nonpathogenic on cucumber. Interestingly, both of the mutants germinated poorly, suggesting involvement of cAMP signaling in germination. Germination defect in the cpk1 and cac1 mutants is partially rescued by incubation of the conidia at lower concentrations. Germinating conidia of the cpk1 and cac1 mutants can form appressoria, but the appressoria formed by them are nonfunctional, like those of the rpk1 mutant. Cytological analysis indicates that the appressoria of the cpk1 mutant contain larger numbers of lipid bodies compared with the wild type, whereas lipid levels in the rpk1 mutants are lower, suggesting cAMP-mediated regulation of lipid metabolism for appressorium functionality. Furthermore, the cpk1 and cacl mutants have a defect in infectious growth in plant. In C. lagenarium, Cmkl mitogen-activated protein kinase (MAPK) regulates germination, appressorium formation, and infectious growth. These results suggest that cAMP signaling controls multiple steps of fungal infection in cooperative regulation with Cmkl MAPK in C. lagenarium.     

MAP kinase signaling during pollen development

The stereotypical events of pollen grain maturation and its coordinated development with other flower tissues requires the interplay of different signalling pathways in order to ensure efficient fertilisation and, eventually, seed set. In recent years evidence has accumulated that members of the mitogen-activated protein kinase (MAP kinase) family are expressed in pollen and may function as regulators of both pollen development and germination. In this review we describe what is known about MAP kinases in pollen and discuss their possible function(s) in pollen biology. Received: 14 December 2000 / Accepted: 6 June 2001     

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

A MAP kinase kinase interacts with SymRK and regulates nodule organogenesis in Lotus japonicus

The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis.     

MAP kinase signaling in diverse effects of ethanol

Chronic ethanol abuse is associated with liver injury, neurotoxicity, hypertension, cardiomyopathy, modulation of immune responses and increased risk for cancer, whereas moderate alcohol consumption exerts protective effect on coronary heart disease. However, the signal transduction mechanisms underlying these processes are not well understood. Emerging evidences highlight a central role for mitogen activated protein kinase (MAPK) family in several of these effects of ethanol. MAPK signaling cascade plays an essential role in the initiation of cellular processes such as proliferation, differentiation, development, apoptosis, stress and inflammatory responses. Modulation of MAPK signaling pathway by ethanol is distinctive, depending on the cell type; acute or chronic; normal or transformed cell phenotype and on the type of agonist stimulating the MAPK. Acute exposure to ethanol results in modest activation of p42/44 MAPK in hepatocytes, astrocytes, and vascular smooth muscle cells. Acute ethanol exposure also results in potentiation or prolonged activation of p42/44MAPK in an agonist selective manner. Acute ethanol treatment also inhibits serum stimulated p42/44 MAPK activation and DNA synthesis in vascular smooth muscle cells. Chronic ethanol treatment causes decreased activation of p42/44 MAPK and inhibition of growth factor stimulated p42/44 MAPK activation and these effects of ethanol are correlated to suppression of DNA synthesis, impaired synaptic plasticity and neurotoxicity. In contrast, chronic ethanol treatment causes potentiation of endotoxin stimulated p42/44 MAPK and p38 MAPK signaling in Kupffer cells leading to increased synthesis of tumor necrosis factor. Acute exposure to ethanol activates pro-apoptotic JNK pathway and anti-apoptotic p42/44 MAPK pathway. Apoptosis caused by chronic ethanol treatment may be due to ethanol potentiation of TNF induced activation of p38 MAPK. Ethanol induced activation of MAPK signaling is also involved in collagen expression in stellate cells. Ethanol did not potentiate serum stimulated or Gi-protein dependent activation of p42/44 MAPK in normal hepatocytes but did so in embryonic liver cells and transformed hepatocytes leading to enhanced DNA synthesis. Ethanol has a 'triangular effect' on MAPK that involve direct effects of ethanol, its metabolically derived mediators and oxidative stress. Acetaldehyde, phosphatidylethanol, fatty acid ethyl ester and oxidative stress, mediate some of the effects seen after ethanol alone whereas ethanol modulation of agonist stimulated MAPK signaling appears to be mediated by phosphatidylethanol. Nuclear MAPKs are also affected by ethanol. Ethanol modulation of nuclear p42/44 MAPK occurs by both nuclear translocation of p42/44 MAPK and its activation in the nucleus. Of interest is the observation that ethanol caused selective acetylation of Lys 9 of histone 3 in the hepatocyte nucleus. It is plausible that ethanol modulation of cross talk between phosphorylation and acetylations of histone may regulate chromatin remodeling. Taken together, these recent developments place MAPK in a pivotal position in relation to cellular actions of ethanol. Furthermore, they offer promising insights into the specificity of ethanol effects and pharmacological modulation of MAPK signaling. Such molecular signaling approaches have the potential to provide mechanism-based therapy for the management of deleterious effects of ethanol or for exploiting its beneficial effects.     

Redox signaling and the MAP kinase pathways

The mitogen-activated protein (MAP) kinases are a large family of proline-directed, serine/threonine kinases that require tyrosine and threonine phosphorylation of a TxY motif in the activation loop for activation through a phosphorylation cascade involving a MAPKKK, MAPKK and MAPK, often referred to as the MAP kinase module. Three separate such modules have been identified, based on the TxY motif of the MAP kinase and the dual-specificity kinases that strictly phosphorylate their specific TxY sequence. They are the extracellular signal regulated kinases (ERKs), c-jun N-terminal kinases (JNKs) and p38 MAPKs. The ERKs are mainly associated with proliferation and differentiation while the JNKs and p38MAP kinases regulate responses to cellular stresses. Redox homeostasis is critical for proper cellular function. While reactive oxygen species (ROS) and oxidative stress have been implicated in injury, a rapidly growing literature suggests that a transient increase in ROS levels is an important mediator of proliferation and results in activation of various signaling molecules and pathways, among which the MAP kinases. This review will summarize the role of ROS in MAP kinase activation in various systems, including in macrophages, cells of myeloid origin that play an essential role in inflammation and express a multi-component NADPH oxidase that catalyzes the receptor-regulated production of ROS.     

A novel MAP kinase regulates flagellar length in Chlamydomonas

Little is known about the molecular basis of organelle size control in eukaryotes. Cells of the biflagellate alga Chlamydomonas reinhardtii actively maintain their flagella at a precise length. Chlamydomonas mutants that lose control of flagellar length have been isolated and used to demonstrate that a dynamic process keeps flagella at an appropriate length. To date, none of the proteins required for flagellar length control have been identified in any eukaryotic organism. Here, we show that a novel MAP kinase is crucial to enforcing wild-type flagellar length in C. reinhardtii. Null mutants of LF4 [2], a gene encoding a protein with extensive amino acid sequence identity to a mammalian MAP kinase of unknown function, MOK [3], are unable to regulate the length of their flagella. The LF4 protein (LF4p) is localized to the flagella, and in vitro enzyme assays confirm that the protein is a MAP kinase. The long-flagella phenotype of lf4 cells is rescued by transformation with the cloned LF4 gene. The demonstration that a novel MAP kinase helps enforce flagellar length control indicates that a previously unidentified signal transduction pathway controls organelle size in C. reinhardtii.     

Opposing actions of TGFbeta and MAP kinase signaling in undifferentiated hen granulosa cells

The present studies were conducted to establish interactions between transforming growth factor (TGF)-beta and the epidermal growth factor (EGF) family members, TGFalpha and betacellulin (BTC), relative to proliferation and differentiation of granulosa cells in hen ovarian follicles. Results presented demonstrate expression of TGFbeta isoforms, plus TGFalpha, BTC, and ErbB receptors in prehierarchal follicles, thus establishing the potential for autocrine/paracrine signaling and cross-talk within granulosa cells at the onset of differentiation. Treatment with TGFalpha or BTC increases levels of TGFbeta1 mRNA in undifferentiated granulosa cells, while the selective inhibitor of mitogen activated protein kinase signaling, U0126, reverses these effects. Moreover, TGFbeta1 attenuates c-myc mRNA expression and granulosa cell proliferation, while TGFalpha blocks both these inhibitory effects. Collectively, these data provide evidence that EGF family ligands regulate both the expression and biological actions of TGFbeta1 in hen granulosa cells, and indicate that the timely interaction of these opposing factors is an important modulator of both granulosa cell proliferation and differentiation.     

Tyrosine kinase Syk associates with toll-like receptor 4 and regulates signaling in human monocytic cells

Toll-like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP-1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP-1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP-1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin-1 (IL1) receptor-associated kinase 1 (IRAK-1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS-mediated release of cytokines IL6, IL1beta, tumor necrosis factor-alpha, neither on chemokines macrophage inhibitory protein (MIP)1alpha, MIP1beta, monocyte chemoattractant protein -1, IL8, Groalpha and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS-mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement.     

Control of MAP kinase signaling to the nucleus

MAP kinase (MAPK) signaling is among central signaling pathways that regulate cell proliferation, cell differentiation and apoptosis. As MAPK should transmit extracellular signals to proper regions or compartments in cells, controlling subcellular localization of MAPK is important for regulating fidelity and specificity of MAPK signaling. The ERK1/2-type of MAPK is the best characterized member of the MAPK family. In response to extracellular stimulus, ERK1/2 translocates from the cytoplasm to the nucleus by passing through the nuclear pore by several independent mechanisms. Sef (similar expression to fgf genes), a transmembrane protein, has been shown to be a regulator of subcellular distribution of ERK1/2. Sef binds to activated MEK1/2, the specific activator of ERK1/2, and tethers the activated MEK1/2/activated ERK1/2 complex to the Golgi apparatus and the plasma membrane. Thus, Sef blocks ERK1/2 signaling to the nucleus and allows signaling to the cytoplasm. Here we review recent findings on spatial regulation of MAPK, especially on nucleocytoplasmic trafficking of ERK1/2.     

Phospholipase activity of phospholipase C-gamma1 is required for nerve growth factor-regulated MAP kinase signaling cascade in PC12 cells

Phospholipase C-gamma1 (PLC-gamma1) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. Here, we report that the phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, we show that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs.