A novel in vivo assay for the analysis of protein-protein interaction.
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The Ras Recruitment System (RRS) is a method for identification and isolation of protein-protein interaction. The method is based on translocation of cytoplasmic mammalian Ras protein to the inner leaflet of the plasma membrane through protein-protein interaction. The system is studied in a temperature-sensitive yeast strain where the yeast Ras guanyl nucleotide exchange factor is inactive at 36 degrees C. Protein-protein interaction results in cell growth at the restrictive temperature. We developed a gene reporter assay for the analysis of protein-protein interaction in mammalian cells. Ras activation in mammalian cells induces the mitogen-activated kinase cascade (MAPK), which can be monitored using Ras-dependent reporter genes. This greatly extends the usefulness of the system and provides a novel assay for protein-protein interaction in mammalian cells. (+info)
Airway patterning: A paradigm for restricted signalling.
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Intercellular signalling is limited by the range of cell responsiveness, often mediated by repressors. A recently identified repressor, Sprouty, inhibits MAP kinase signalling in flies, mice and humans and has a conserved function in patterning the airways of these divergent species. (+info)
Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.
(3/9108)
We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases. (+info)
TANK potentiates tumor necrosis factor receptor-associated factor-mediated c-Jun N-terminal kinase/stress-activated protein kinase activation through the germinal center kinase pathway.
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Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), in TRAF2-mediated NF-kappaB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-kappaB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions. (+info)
Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cytokine- or stress-activated signal transduction pathways.
(5/9108)
A hallmark of inflammation is the burst-like formation of certain proteins, initiated by cellular stress and proinflammatory cytokines like interleukin 1 (IL-1) and tumor necrosis factor, stimuli which simultaneously activate different mitogen-activated protein (MAP) kinases and NF-kappaB. Cooperation of these signaling pathways to induce formation of IL-8, a prototype chemokine which causes leukocyte migration and activation, was investigated by expressing active and inactive forms of protein kinases. Constitutively active MAP kinase kinase 7 (MKK7), an activator of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, induced IL-8 synthesis and transcription from a minimal IL-8 promoter. Furthermore, MKK7 synergized in both effects with NF-kappaB-inducing kinase (NIK). Activation of the IL-8 promoter by either of the kinases required functional NF-kappaB and AP-1 sites. While NIK and MKK7 did not affect degradation of IL-8 mRNA, an active form of MKK6, which selectively activates p38 MAP kinase, induced marked stabilization of the transcript and further increased IL-8 protein formation induced by NIK plus MKK7. Consistently, the MAP kinase kinase kinase MEKK1, which can activate NF-kappaB, SAPK/JNK, and p38 MAP kinases, most potently induced IL-8 formation. These results provide evidence that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation. (+info)
Cas mediates transcriptional activation of the serum response element by Src.
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The Src substrate p130(Cas) is a docking protein containing an SH3 domain, a substrate domain that contains multiple consensus SH2 binding sites, and a Src binding region. We have examined the possibility that Cas plays a role in the transcriptional activation of immediate early genes (IEGs) by v-Src. Transcriptional activation of IEGs by v-Src occurs through distinct transcriptional control elements such as the serum response element (SRE). An SRE transcriptional reporter was used to study the ability of Cas to mediate Src-induced SRE activation. Coexpression of v-Src and Cas led to a threefold increase in SRE-dependent transcription over the level induced by v-Src alone. Cas-dependent activation of the SRE was dependent on the kinase activity of v-Src and the Src binding region of Cas. Signaling to the SRE is promoted by a serine-rich region within Cas and inhibited by the Cas SH3 domain. Cas-dependent SRE activation was accompanied by an increase in the level of active Ras and in the activity of the mitogen-activated protein kinase (MAPK) Erk2; these changes were blocked by coexpression of dominant-negative mutants of the adapter protein Grb2. SRE activation was abrogated by coexpression of dominant-negative mutants of Ras, MAPK kinase (Mek1), and Grb2. Coexpression of Cas with v-Src enhanced the association of Grb2 with the adapter protein Shc and the protein tyrosine phosphatase Shp-2; coexpression of Shc or Shp-2 mutants significantly reduced SRE activation by Cas and v-Src. Cas-induced Grb2 association with Shp-2 and Shc may account for the Cas-dependent activation of the Ras/Mek/Erk pathway and SRE-dependent transcription. 14-3-3 proteins may also play a role in Cas-mediated signaling to the SRE. Overexpression of Cas was found to modestly enhance epidermal growth factor (EGF)-induced activation of the SRE. A Cas mutant lacking the Src binding region did not potentiate the EGF response, suggesting that Cas enhances EGF signaling by binding to endogenous cellular Src or another Src family member. These observations implicate Cas as a mediator of Src-induced transcriptional activation. (+info)
Phosphorylation of cytosolic phospholipase A2 in platelets is mediated by multiple stress-activated protein kinase pathways.
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Stress-activated protein kinases (SAPKs) are stimulated by cell damaging agents as well as by physiological receptor agonists. In this study we show that human platelets contain the isoforms SAPK2a, SAPK2b, SAPK3 and SAPK4 as determined by immunoblotting with specific antibodies. All four kinases were activated in thrombin-stimulated platelets whereas only SAPK2a and SAPK2b were significantly stimulated by collagen. All four isoforms were able to phosphorylate wild-type human cPLA2 in vitro, although to different extents, but not cPLA2 mutants that had Ser505 replaced by alanine. Phosphorylation at Ser505 was confirmed by phosphopeptide mapping using microbore HPLC. SAPK2a and 42-kDa mitogen-activated protein kinase incorporated similar levels of phosphate into cPLA2 relative to the ability of each kinase to stimulate phosphorylation of myelin basic protein. SAPK2b and SAPK4 incorporated less phosphate, and cPLA2 was a poor substrate for SAPK3. The inhibitor of SAPK2a and SAPK2b, SB 202190, completely blocked collagen-induced phosphorylation of cPLA2 at its two phosphorylation sites in vivo, Ser505 and Ser727. We have also reported previously that SB 202190 partially ( approximately 50%) blocks phosphorylation at both sites and to a similar extent in thrombin-stimulated platelets. Inhibition of phosphorylation resulted in a two- to threefold shift to the right in the concentration response curves for arachidonic acid release from thrombin- and collagen-stimulated platelets. Our data suggest that cPLA2 is a substrate for several SAPK cascades and that phosphorylation of cPLA2 augments arachidonic acid release. (+info)
The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading.
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Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGF(TM)). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGF(TM) results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGF(TM) in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGF(TM)HA/Myc). HB-EGF(TM)HA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGF(TM)HA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGF(TM) shedding were identified as follows. (i) HB-EGF(TM)HA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGF(TM)HA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGF(TM) cleavage. (ii) Activation of an inducible Raf-1 kinase, DeltaRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGF(TM)HA/Myc within 20-40 min. (iii) Serum induced MAP kinase activation and HB-EGF(TM)HA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGF(TM)HA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading. (+info)