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  • The TGFβ receptor-interacting protein km23-1/DYNLRB1 plays an adaptor role in TGFβ1 autoinduction via its association with Ras. 22637579

    We have previously elucidated the signaling events that are required for TGFβ1 autoinduction (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). Further, we have reported that the TGFβ receptor (TβR)-interacting protein km23-1 plays an important role in TGFβ signal transduction (Jin, Q., Ding, W., and Mulder, K. M. (2007) J. Biol. Chem. 282, 19122-19132). Here we examined the role of km23-1 in TGFβ1 autoinduction in TGFβ-sensitive epithelial cells. siRNA blockade of km23-1 reduced TGFβ1 mRNA expression, as well as DNA binding and transcriptional activation of the relevant activator protein-1 site in the human TGFβ1 promoter. Further, knockdown of km23-1 inhibited TGFβ-mediated activation of ERK and JNK, phosphorylation of c-Jun, and transactivation of the c-Jun promoter. Sucrose gradient analyses indicate that km23-1 was present in lipid rafts together with Ras and TβRII after TGFβ treatment. Immunoprecipitation/blot analyses revealed the formation of a TGFβ-inducible complex between Ras and km23-1 in vivo within minutes of TGFβ addition. Moreover, we demonstrate for the first time that km23-1 is required for Ras activation by TGFβ. Our results indicate that km23-1 is required for TGFβ1 autoinduction through Smad2-independent Ras/ERK/JNK pathways. More importantly, our findings demonstrate that km23-1 functions as a critical adaptor coupling TβR activation to activation of Ras effector pathways downstream.
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  • Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells. 14670956

    We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.
    Document Type:
    Reference
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  • Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery. 11278851

    Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.
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    Reference
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  • Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity. 19001441

    Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87Delta) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87Delta cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87Delta cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Delta cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, beta-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Delta cells with a decrease in G(1) and increase in S and G(2) fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Delta cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor.
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    Reference
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  • Macrophage migration inhibitory factor up-regulates matrix metalloproteinase-9 and -13 in rat osteoblasts. Relevance to intracellular signaling pathways. 11751895

    Neutral matrix metalloproteinases (MMPs) play an important role in bone matrix degradation accompanied by bone remodeling. We herein show for the first time that macrophage migration inhibitory factor (MIF) up-regulates MMP-13 (collagenase-3) mRNA of rat calvaria-derived osteoblasts. The mRNA up-regulation was seen at 3 h in response to MIF (10 microg/ml), reached the maximum level at 6-12 h, and returned to the basal level at 36 h. MMP-13 mRNA up-regulation was preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1 and MMP-9 (92-kDa type IV collagenase) were also up-regulated, but to a lesser extent. The MMP-13 mRNA up-regulation was significantly suppressed by genistein, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Similarly, a selective mitogen-activated protein kinase (MAPK) kinase (MEK)1/2 inhibitor (PD98059) and c-jun/activator protein (AP)-1 inhibitor (curcumin) suppressed MMP-13 mRNA up-regulation induced by MIF. The mRNA levels of c-jun and c-fos in response to MIF were also inhibited by PD98059. Consistent with these results, MIF stimulated phosphorylation of tyrosine, autophosphorylation of Src, activation of Ras, activation of extracellular signal-regulated kinases (ERK) 1/2, a MAPK, but not c-Jun N-terminal kinase or p38, and phosphorylation of c-Jun. Osteoblasts obtained from calvariae of newborn JunAA mice, defective in phosphorylation of c-Jun, or newborn c-Fos knockout (Fos -/- ) mice, showed much less induction of MMP-13 with the addition of MIF than osteoblasts obtained from wild-type or littermate control mice. Taken together, these results suggest that MIF increases the MMP-13 mRNA level of rat osteoblasts via the Src-related tyrosine kinase-, Ras-, ERK1/2-, and AP-1-dependent pathway.
    Document Type:
    Reference
    Product Catalog Number:
    17-218
    Product Catalog Name:
    Ras Activation Assay Kit

  • Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein. 16018961

    Accurate measurement of activity of wild-type K-ras protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-workers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transferase/raf-1 ras binding domain (GST-RBD) fusion protein attached to glutathione beads to isolate activated ras protein. We systematically optimized the method before use on lung tissues. Changing the GST-RBD protein induction temperature from the original 37 to 30 degrees C produced a consistently greater yield of fusion protein. To improve stability of the GST-RBD beads so as to perform large-scale experiments, 0.1% NaN(3) was added. NaN(3)-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST-RBD beads with two commercial assay kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST-RBD affinity method to recover activated K-ras greatly increased the yield of fusion protein, prolonged the useful life of GST-RBD beads to at least 24 days, and enhanced detection sensitivity.
    Document Type:
    Reference
    Product Catalog Number:
    17-218
    Product Catalog Name:
    Ras Activation Assay Kit

  • Activated K-RAS increases polyamine uptake in human colon cancer cells through modulation of caveolar endocytosis. 18176934

    Endocytic pathways have been implicated in polyamine transport in mammalian cells, but specific mechanisms have not been described. We have shown that expression of a dominant negative (DN) form of the GTPase Dynamin, but not Eps15, diminished polyamine uptake in colon cancer cells indicating a caveolar and nonclathrin uptake mode. Polyamines co-sediment with lipid raft/caveolin-1 rich fractions, of the plasma membrane in a sucrose density gradient. Knock down of caveolin-1 significantly increased polyamine uptake. Conversely, ectopic expression of this protein resulted in diminished polyamine uptake. We also found that presence of an activated K-RAS oncogene significantly increased polyamine uptake by colon cancer cells. This effect is through an increase in caveolin-1 phosphorylation at tyrosine residue 14. Caveolin-1 is a negative regulator of caveolar endocytosis and phosphorylation in a K-RAS dependent manner leads to an increase in caveolar endocytosis. In cells expressing wild type K-RAS, addition of exogenous uPA was sufficient to stimulate caveolar endocytosis of polyamines. This effect was abrogated by the addition of a SRC kinase inhibitor. These data indicate that polyamine transport follows a dynamin-dependent and clathrin-independent endocytic uptake route, and this route is positively regulated by the oncogenic expression of K-RAS in a caveolin-1 dependent manner.
    Document Type:
    Reference
    Product Catalog Number:
    17-218
    Product Catalog Name:
    Ras Activation Assay Kit