Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxygen radicals attack guanine bases in DNA but they also attack cytoplasmic GTP forming 8-oxoGTP. The presence of 8-oxoGTP in cytoplasm is evidenced by the fact that cells contain MutT/MTH1 which hydrolyze 8-oxoGTP into 8-oxoGMP. In this study, the interaction between 8-oxoGTP and Ras, a small GTP-binding protein, was tested in vitro, and the action of 8-oxoGTP was compared to that of GTP. When purified Ras was treated with 8-oxoGTPgammaS, Ras was activated, as indicated by the enhanced binding of Ras with Raf-1. GTPgammaS also activated Ras but 8-oxoGTPgammaS had a much more potent effect. In lysates of human embryo kidney 293 cells, 8-oxoGTPgammaS activated not only Ras but also the downstream effectors of the Ras-ERK pathway, i.e., Raf-1 and ERK1/2. In contrast to Ras, other small GTP-binding proteins, Rac1 and Cdc42, were inactivated by 8-oxoGTPgammaS, whereas both of these proteins were activated by GTPgammaS, indicating that the biological natures of 8-oxoGTP and GTP differ. These results suggest the possibility that 8-oxoGTP is not a simple by-product but a functional molecule transmitting an oxidative signal to small GTP-binding proteins like Ras.
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PMID:In vitro evidence for the recognition of 8-oxoGTP by Ras, a small GTP-binding protein. 1562 68

Histone deacetylase (HDAC) inhibitors are expected to be effective for refractory cancer because their mechanism of action differs from that of conventional antineoplastic agents. In this study, we examined the effect of the HDAC inhibitor FK228 on malignant melanoma, as well as its molecular mechanisms. FK228 was highly effective against melanoma compared with other commonly used drugs. By comparing the gene expression profiles of melanoma cells and normal melanocytes, we defined a subset of genes specifically upregulated in melanoma cells by FK228, which included Rap1, a small GTP-binding protein of the Ras family. The expression of Rap1 mRNA and protein increased in FK228-treated melanoma cells in both a dose- and a time-dependent manner. A decrease in the phosphorylation of c-Raf, MEK1/2, and ERK1/2 was accompanied by an increase in Rap1 expression in both FK228-treated and Rap1-overexpressing cells. Inhibition of Rap1 upregulation by small interfering RNA (siRNA) abrogated the induction of apoptosis and suppression of ERK1/2 phosphorylation in FK228-treated melanoma cells. These results indicate that the cytotoxic effects of FK228 are mediated via the upregulation of Rap1. Furthermore, we found that Rap1 was overexpressed and formed a complex with B-Raf in melanoma cell lines with a V599E mutation of B-Raf. The siRNA-mediated abrogation of Rap1 overexpression increased the viability of these cells, suggesting that Rap1 is also an endogenous regulator of Ras-MAP kinase signaling in melanomas.
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PMID:Histone deacetylase inhibitor FK228 suppresses the Ras-MAP kinase signaling pathway by upregulating Rap1 and induces apoptosis in malignant melanoma. 1618 4

Rab3A, a member of the Rab3 small GTP-binding protein (G protein) family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of a GDP/GTP exchange protein (GEP). We have previously purified a GEP from rat brain with lipid-modified Rab3A as a substrate. Purified Rab3 GEP is active on all the Rab3 subfamily members including Rab3A, -3B, -3C, and -3D. Purified Rab3 GEP is active on the lipid-modified form, but not on the lipid-unmodified form. Purified Rab3 GEP is inactive on Rab3A complexed with Rab GDI. The recombinant protein is prepared from the Rab3 GEP-expressed Spodoptera frugiperda cells (Sf9 cells). The properties of recombinant Rab3 GEP, including the requirement for lipid modifications of Rab3A, the substrate specificity, and the sensitivity to Rab GDI, are similar to those of purified Rab3 GEP. Overexpression of Rab3 GEP inhibits Ca(2+)-dependent exocytosis from PC12 cells. On the other hand, Rab3 GEP is identical to a protein named DENN/MADD: differentially expressed in normal versus neoplastic (DENN)/mitogen-activated protein kinase-activating death domain (MADD). Here, we describe the purification method for recombinant Rab3 GEP from Sf9 cells and the functional properties of Rab3 GEP in Ca(2+)-dependent exocytosis by use of the human growth hormone coexpression assay system of PC12 cells.
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PMID:Purification and properties of Rab3 GEP (DENN/MADD). 1647 92

The TLR agonists, flagellin (FLG) and lipopolysaccharide (LPS) stimulate functional activation and cytokine gene expression via the extracellular signal regulated kinase 1/2 (ERK1/2) MAP kinase cascade. However, the upstream mechanisms of these signaling events remain unknown. In mammals, the small GTP-binding protein Ras mediates ERK1/2 activation through activation of downstream effectors Raf-1-MEK1/2-ERK1/2 in response to a variety of stimuli. It is not clear whether this classic Ras cascade plays a role in TLR signaling in avian cells. In the present study, we investigated the role of Ras in FLG- and LPS-mediated signaling in ERK activation in chicken heterophils. Treatment of heterophils with LPS caused a rapid (within 5min) activation of Ras-GTP. The role of Ras activation in LPS-induced stimulation of ERK1/2 was corroborated when the specific Ras inhibitor, FTI-277, inhibited ERK1/2 activation. The classic Ras-mediated pathway of ERK1/2 activation by LPS was confirmed when the specific Raf-1 inhibitor, GW 5074, and the MEK1/2 inhibitor, U0126, both reduced ERK activation by 51-60%. Of more interest was that treatment of the heterophils with FLG did not activate Ras-GTP. Likewise, neither FTI-277 nor GW 5074 had any effect on FLG-mediated activation of ERK1/2. Another small GTPase, Rap1, has been shown to play a role in mammalian neutrophil function. Using a Rap1-GTP pull-down assay, we found that FLG stimulation, but not LPS, of avian heterophils induced a rapid and transient Rap1 activation. Rap1 has been shown to activate the ERK1/2 via a different Raf family member B-Raf whose downstream effector is MEK1/2. We show here that FLG stimulation of heterophils induces the phosphorylation of Rap1. The FLG induction of the Rap1-->B-Raf-->MEK1/2-->ERK1/2 cascade was confirmed by the reduction of ERK1/2 activation by the specific Rap1 inhibitor (GGTI-298) and U0126. The results demonstrate that for the first time that the small GTPase Ras family is involved in TLR signaling of avian heterophils with the TLR agonists LPS (Ras) and FLG (Rap1) inducing differential signaling cascades to activate the downstream ERK MAP kinase.
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PMID:Flagellin and lipopolysaccharide stimulate the MEK-ERK signaling pathway in chicken heterophils through differential activation of the small GTPases, Ras and Rap1. 1704 53

This study shows that the small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is an important regulator of tumor growth and metastasis. Using spontaneous melanoma tumor growth assays and experimental metastasis assays in nude mice, we show that sustained activation of ARF6 reduces tumor mass growth but significantly enhances the invasive capacity of tumor cells. In contrast, mice injected with tumor cells expressing a dominantly inhibitory ARF6 mutant exhibited a lower incidence and degree of invasion and lung metastasis compared with control animals. Effects on tumor growth correlate with reduced cell proliferation capacity and are linked at least in part to alterations in mitotic progression induced by defective ARF6 cycling. Furthermore, phospho-ERK levels in subcultured cells from ARF6(GTP) and ARF6(GDP) tumor explants correlate with invasive capacity. ARF6-induced extracellular signal-regulated kinase (ERK) signaling leads to Rac1 activation to promote invadopodia formation and cell invasion. These findings document an intricate role for ARF6 and the regulation of ERK activation in orchestrating mechanisms underlying melanoma growth, invasion, and metastases.
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PMID:ADP-ribosylation factor 6 regulates tumorigenic and invasive properties in vivo. 1927 88

The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.
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PMID:The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4. 1964 50

The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in gamma-radiation-induced apoptosis. However, it is unclear how JNK activation and gamma-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by gamma-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. gamma-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the gamma-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to gamma-radiation.
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PMID:Increase of RhoB in gamma-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells. 1999 57

The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.
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PMID:Decoupling of activation and effector binding underlies ARF6 priming of fast endocytic recycling. 2143 24

The Ras-related small GTP-binding protein RhoB is rapidly induced in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in Jurkat cells.
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PMID:The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter. 2156 67

Activation of the Ras small GTP-binding protein is necessary for normal T cell development and function. However, it is unknown which Ras GTPase-activating proteins (RasGAPs) inactivate Ras in T cells. We used a T cell-specific RASA1-deficient mouse model to investigate the role of the p120 RasGAP (RASA1) in T cells. Death of CD4(+)CD8(+) double-positive thymocytes was increased in RASA1-deficient mice. Despite this finding, on an MHC class II-restricted TCR transgenic background, evidence was obtained for increased positive selection of thymocytes associated with augmented activation of the Ras-MAPK pathway. In the periphery, RASA1 was found to be dispensable as a regulator of Ras-MAPK activation and T cell functional responses induced by full agonist peptides. However, numbers of naive T cells were substantially reduced in RASA1-deficient mice. Loss of naive T cells in the absence of RASA1 could be attributed in part to impaired responsiveness to the IL-7 prosurvival cytokine. These findings reveal an important role for RASA1 as a regulator of double-positive survival and positive selection in the thymus as well as naive T cell survival in the periphery.
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PMID:A role for p120 RasGAP in thymocyte positive selection and survival of naive T cells. 2164 95


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