Activation Of P21ras Research Articles

RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP

BackgroundRas gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling.MethodsThe E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models.ResultsThe technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines.ConclusionRGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

MEK inhibition exerts temporal and myeloid cell-specific effects in the pathogenesis of neurofibromatosis type 1 arteriopathy

Mutations in the NF1 tumor suppressor gene are linked to arteriopathy. Nf1 heterozygosity (Nf1+/–) results in robust neointima formation, similar to humans, and myeloid-restricted Nf1+/– recapitulates this phenotype via MEK-ERK activation. Here we define the contribution of myeloid subpopulations to NF1 arteriopathy. Neutrophils from WT and Nf1+/– mice were functionally assessed in the presence of MEK and farnesylation inhibitors in vitro and neutrophil recruitment to lipopolysaccharide was assessed in WT and Nf1+/– mice. Littermate 12–15 week-old male wildtype and Nf1+/– mice were subjected to carotid artery ligation and provided either a neutrophil depleting antibody (1A8), liposomal clodronate to deplete monocytes/macrophages, or PD0325901 and neointima size was assessed 28 days after injury. Bone marrow transplant experiments assessed monocyte/macrophage mobilization during neointima formation. Nf1+/– neutrophils exhibit enhanced proliferation, migration, and adhesion via p21Ras activation of MEK in vitro and in vivo. Neutrophil depletion suppresses circulating Ly6Clow monocytes and enhances neointima size, while monocyte/macrophage depletion and deletion of CCR2 in bone marrow cells abolish neointima formation in Nf1+/– mice. Taken together, these findings suggest that neurofibromin-MEK-ERK activation in circulating neutrophils and monocytes during arterial remodeling is nuanced and points to important cross-talk between these populations in the pathogenesis of NF1 arteriopathy.

Neurofibromin is a novel regulator of Ras-induced reactive oxygen species production in mice and humans

Neurofibromatosis type 1 (NF1) predisposes individuals to early and debilitating cardiovascular disease. Loss of function mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin, leads to accelerated p21Ras activity and phosphorylation of multiple downstream kinases, including Erk and Akt. Nf1 heterozygous (Nf1+/-) mice develop a robust neointima that mimics human disease. Monocytes/macrophages play a central role in NF1 arterial stenosis as Nf1 mutations in myeloid cells alone are sufficient to reproduce the enhanced neointima observed in Nf1+/- mice. Though the molecular mechanisms underlying NF1 arterial stenosis remain elusive, macrophages are important producers of reactive oxygen species (ROS) and Ras activity directly regulates ROS production. Here, we use compound mutant and lineage-restricted mice to demonstrate that Nf1+/- macrophages produce excessive ROS, which enhance Nf1+/- smooth muscle cell proliferation in vitro and in vivo. Further, use of a specific NADPH oxidase-2 inhibitor to limit ROS production prevents neointima formation in Nf1+/- mice. Finally, mononuclear cells from asymptomatic NF1 patients have increased oxidative DNA damage, an indicator of chronic exposure to oxidative stress. These data provide genetic and pharmacologic evidence that excessive exposure to oxidant species underlie NF1 arterial stenosis and provide a platform for designing novels therapies and interventions.

Metabolic reprogramming and cell fate regulation in alcoholic liver disease.

Alcoholic liver disease (ALD) should be defined as a life-style metabolic disease. Its pathogenesis is driven by altered cell fate of both parenchymal and non-parenchymal liver cell types, contributing to different pathologic spectra. A critical turning point in progression of ALD is chronic alcoholic steatohepatitis (ASH) or alcoholic neutrophilic hepatitis (AH), which markedly predisposes patients to most devastating ALD sequela, cirrhosis and liver cancer. Our research identifies the pivotal roles of unique metabolic reprogramming in M1 activation of hepatic macrophages (HM) and myofibroblastic activation (MF) of hepatic stellate cells (HSC) in the genesis of inflammation and fibrosis, the two key histological features of chronic ASH and neutrophilic AH. For M1 HM activation, heightened proinflammatory iron redox signaling in endosomes or caveosomes results from altered iron metabolism and storage, promoting IKK/NF-kB activation via interactive activation of p21ras, TAK1, and PI3K. For MF cell fate regulation of HSC, activation of the morphogen Wnt pathway caused by the nuclear protein NECDIN or the single-pass trans-membrane protein DLK1, reprograms lipid metabolism via MeCP2-mediated epigenetic repression of the key HSC quiescence gene Ppar-γ. The findings from these studies re-enforce the importance of metabolic reprogramming in cell fate regulation required for the pathogenesis of ALD.

Oncolytic Virotherapy for Thyroid Cancer: Will it Translate to the Clinic?

Observations that viruses can induce tumor regression date back to the beginning of the 20th century [1] and the use of viruses for cancer treatment has been theorized since then ; however only in the late 1990s advancement in viral manipulation technologies together with a better understanding of the molecular alterations in cancer cells allowed the development of oncolytic viruses (virotherapy). So far, dozen of viruses have been tested for their oncolytic activity. Oncolytic viruses (OVs) have several potential benefits as anticancer therapeutics: tumorselective replication, amplification of the input dose, efficacy independent of resistance to current anticancer therapies. OVs do not contribute to the generation of neoplastic cell clones or treatment resistance [2]. To generate viruses able to selectively replicate in neoplastic cells, different viral strain and several strategies have been exploited [3]. Viral strains displaying a natural oncotropism have also been tested [2]. The clinical trials performed in the last two decades have demonstrated the safety of OVs and substantial antineoplastic effects. The best clinically evaluated replication-selective oncolytic viruses to date are the mutant adenoviruses dl1520, ONYX-015 and H101 (Oncorine; Shanghai Sunway Biotech). These mutants bear a E1B55K gene deletion, enabling the mutants to replicate only in cells with functional inactivation of p53. E1B55K viral gene is required for other important functions and its deletion greatly impairs viral replication [4], explaining the modest activity as single agent reported in clinical trials. However, dl1520 and H101 were shown to enhance the effects of chemotherapeutic drugs and on the base of these results, H101 was licensed in China for the treatment of head and neck cancers in combination with cisplatin and 5-FU [5,6]. The majority of thyroid cancers are well differentiated papillary or follicular carcinomas with a good cure rate and an excellent prognosis. In contrast, anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers with a short survival time. Multimodality therapy (surgery, chemotherapy and radiotherapy) has shown a limited success and although ATC accounts for only 2% of thyroid carcinoma, it contributes up to 50% of thyroid cancer deaths [7]. Not surprisingly, the studies with OVs have been focused to the treatment of ATC. The first studies testing selective replicating adenoviruses were performed by our group using dl1520 mutant alone or in combination with paclitaxlel, doxorubicin [8] and radiotherapy [9]. Lovastatin, a cholesterol-lowering drug also acting as an inhibitor of p21 ras activity was used by us in conjunction with dl1520 to improve viral cytotoxicity. The combination significantly reduced the growth of ATC xenografts [10]. It is worth noting that p53 inactivation frequency in ATCs is about 50%, with a strong limitation of the therapeutic range for dl1520. Moreover, the studies were conducted in a variety of long established ATC cells lines, some of them successively identified as derived from other tumors. Despite these limitations, the studies with dl1520 have shown that OVs hold a potential benefit for the treatment of ATC. Abbosh et al. have used an oncolytic adenovirus selectively replicating in cells with an active Wnt/β-catenin pathway showing an oncolytic activity and a prolonged survival of animals bearing ATC xenografts [11], confirming the efficacy of the approach. Due to the attenuated replication potential and the limited fraction of ATCs with a nonfunctional p53 pathway, we evaluated dl922–947, a more effective second generation oncolytic adenovirus. dl922–947 bears a deletion of 24bp in E1A-Conserved Region 2 (CR2) [12].

Caveosomal Oxidative Stress Causes Src-p21ras Activation and Lysine 63 TRAF6 Protein Polyubiquitination in Iron-induced M1 Hepatic Macrophage Activation

Proinflammatory M1 activation of hepatic macrophages (HM) is critical in pathogenesis of hepatitis, but its mechanisms are still elusive. Our earlier work demonstrates the role of ferrous iron (Fe(2+)) as a pathogen-associated molecular pattern-independent agonist for activation of IκB kinase (IKK) and NF-κB in HM via activation and interaction of p21(ras), transforming growth factor β-activated kinase-1 (TAK1), and phosphatidylinositol 3-kinase (PI3K) in caveosomes. However, iron-induced signaling upstream of these kinases is not known. Here we show that Fe(2+) induces generation of superoxide anion (O(2)()) in endosomes, reduces protein-tyrosine phosphatase (PTP) activity, and activates Src at 2∼10 min of Fe(2+) addition to rat primary HM culture. Superoxide dismutase (SOD) blocks O(2)() generation, PTP inhibition, and Src activation. Fe(2+)-induced p21(ras) activity is abrogated with the Src inhibitor PP2 and SOD. Fe(2+) stimulates Lys(63)-linked polyubiquitination (polyUb) of TRAF6 in caveosomes, and a dominant negative K63R mutant of ubiquitin or SOD prevents iron-induced TRAF6 polyUb and TAK1 activation. These results demonstrate that Fe(2+)-generated O(2)() mediates p21(ras) and TAK1 activation via PTP inhibition and Lys(63)-polyUb of TRAF6 in caveosomes for proinflammatory M1 activation in HM.

Sodium phenylbutyrate controls neuroinflammatory and antioxidant activities and protects dopaminergic neurons in mouse models of Parkinson's disease.

Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21rac, but not p21ras, attenuated the production of ROS from activated microglia. Inhibition of both p21ras and p21rac activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21ras and p21rac in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. Oral administration of NaPB reduced nigral activation of p21ras and p21rac, protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders.

Constitutively enhanced p21Ras activity promotes dynamic extracellular matix establishment

Constitutively enhanced p21Ras activity promotes dynamic extracellular matix establishment

Simvastatin Inhibits the Activation of p21rasand Prevents the Loss of Dopaminergic Neurons in a Mouse Model of Parkinson's Disease

Parkinson's disease (PD) is second only to Alzheimer's disease as the most common devastating human neurodegenerative disorder. Despite intense investigation, no interdictive therapy is available for PD. We investigated whether simvastatin, a Food and Drug Administration-approved cholesterol-lowering drug, could protect against nigrostriatal degeneration after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication to model PD in mice. First, MPP(+) induced the activation of p21(ras) and nuclear factor-kappaB (NF-kappaB) in mouse microglial cells. Inhibition of MPP(+)-induced activation of NF-kappaB by Deltap21(ras), a dominant-negative mutant of p21(ras), supported the involvement of p21(ras) in MPP(+)-induced microglial activation of NF-kappaB. Interestingly, simvastatin attenuated activation of both p21(ras) and NF-kappaB in MPP(+)-stimulated microglial cells. Consistently, we found a very rapid activation of p21(ras) in vivo in the substantia nigra pars compacta of MPTP-intoxicated mice. However, after oral administration, simvastatin entered into the nigra, reduced nigral activation of p21(ras), attenuated nigral activation of NF-kappaB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Similarly, pravastatin, another cholesterol-lowering drug, suppressed microglial inflammatory responses and protected dopaminergic neurons in MPTP-intoxicated mice, but at levels less than simvastatin. Furthermore, both the statins administered 2 d after initiation of the disease were still capable of inhibiting the demise of dopaminergic neurons and concomitant loss of neurotransmitters, suggesting that statins are capable of slowing down the progression of neuronal loss in the MPTP mouse model. Therefore, we conclude that statins may be of therapeutic benefit for PD patients.

Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive, Reduces Microglial and Astroglial Inflammatory Responses

Upon activation, microglia and astrocytes produce a number of proinflammatory molecules that participate in the pathophysiology of several neurodegenerative disorders. This study explores the anti-inflammatory property of cinnamon metabolite sodium benzoate (NaB) in microglia and astrocytes. NaB, but not sodium formate, was found to inhibit LPS-induced expression of inducible NO synthase (iNOS), proinflammatory cytokines (TNF-alpha and IL-1beta) and surface markers (CD11b, CD11c, and CD68) in mouse microglia. Similarly, NaB also inhibited fibrillar amyloid beta (Abeta)-, prion peptide-, double-stranded RNA (polyinosinic-polycytidylic acid)-, HIV-1 Tat-, 1-methyl-4-phenylpyridinium(+)-, IL-1beta-, and IL-12 p40(2)-induced microglial expression of iNOS. In addition to microglia, NaB also suppressed the expression of iNOS in mouse peritoneal macrophages and primary human astrocytes. Inhibition of NF-kappaB activation by NaB suggests that NaB exerts its anti-inflammatory effect through the inhibition of NF-kappaB. Although NaB reduced the level of cholesterol in vivo in mice, reversal of the inhibitory effect of NaB on iNOS expression, and NF-kappaB activation by hydroxymethylglutaryl-CoA, mevalonate, and farnesyl pyrophosphate, but not cholesterol and ubiquinone, suggests that depletion of intermediates, but not end products, of the mevalonate pathway is involved in the anti-inflammatory effect of NaB. Furthermore, we demonstrate that an inhibitor of p21(ras) farnesyl protein transferase suppressed the expression of iNOS, that activation of p21(ras) alone was sufficient to induce the expression of iNOS, and that NaB suppressed the activation of p21(ras) in microglia. These results highlight a novel anti-inflammatory role of NaB via modulation of the mevalonate pathway and p21(ras).

Constitutively enhanced p21Ras activity amplifies dendritic remodeling of hippocampal neurons during physical activity

In the present study we show that overexpression of constitutively active Ras amplifies the dendritic remodeling observed when animals were allowed to be physically active. The monomeric G-protein Ras is a key molecular trigger of distinct signal transduction pathways that play an important role in proper functioning of neurons. Our previous studies on Ras-transgenic synRas mice have demonstrated a considerable impact of Ras on dendritic growth, extension and synaptic connectivity of neurons. Voluntary access to a running wheel resulted in enlargement of hippocampal pyramidal cell dendrites in wild-type mice as expected. However, constitutively elevated Ras activity further enhanced dendritic growth and branching especially of apical arbors. The resultant dendritic surface gain was paralleled by a significant increase in dendritic spine density. Since Ras is crucially involved in signaling and cascades of neurotrophins that are elevated after physical activity, these results strongly suggest an important role of Ras in dendritic dynamics during induced neuronal remodeling.

Pertussis toxin blocks growth factor receptor signalling by attenuating p21ras activity

Pertussis toxin blocks growth factor receptor signalling by attenuating p21ras activity

Role of Glutaredoxin-Mediated ProteinS-Glutathionylation in Cellular Nitroglycerin Tolerance

We hypothesize that nitroglycerin (NTG) causes direct oxidation of multiple cellular sulfhydryl (SH) proteins and that manipulation of SH redox status affects NTG tolerance. In LLC-PK1 cells, we found that nitrate tolerance, as indicated by cGMP accumulation toward NTG, was accompanied by increased protein [(35)S]cysteine incorporation, significant S-glutathionylation of multiple proteins, and decreased metabolic activity of several SH-sensitive enzymes, including creatine kinase, xanthine oxidoreductase, and glutaredoxin (GRX). Cells overexpressing GRX exhibited reduced cellular protein S-glutathionylation (PSSG) and absence of NTG tolerance, whereas those with silenced GRX showed increased extent of NTG-induced tolerance. Incubation of LLC-PK1 cells with oxidized glutathione led to several major observations associated with nitrate tolerance, namely, reduced cGMP accumulation, PSSG formation, superoxide accumulation, and the attenuation of these events by vitamin C. Aortic S-glutathionylated proteins increased approximately 3-fold in rats made tolerant in vivo to NTG and showed significant negative correlation with vascular responsiveness ex vivo. NTG incubation in EA.hy926 endothelial cells and LLC-PK1 cells led to increased S-glutathionylation and activity of p21(ras), a known mediator of cellular signaling. These results indicate that the hallmark events of NTG tolerance, such as reduced bioactivation and redox signaling, are associated with GRX-dependent protein deglutathionylation.

Constitutively enhanced p21Ras activity amplifies dendritic remodeling of hippocampal neurons during physical activity

Constitutively enhanced p21Ras activity amplifies dendritic remodeling of hippocampal neurons during physical activity

Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21ras and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21ras -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21ras activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21ras activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55−/− primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21ras becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21ras-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion.

T lymphocyte activation signals.

Activation of T lymphocytes results in immediate biochemical changes including increases in intracellular calcium levels, activation of protein kinase C (PKC) and changes in tyrosine phosphorylation. In T cells recent studies have indicated that activation of the guanine nucleotide-binding proteins p21ras is mediated by PKC, which suggests that the p21ras proteins may regulate intracellular signalling events downstream of PKC. The p21ras proteins can be activated in T cells by signals generated by triggering of the T cell antigen receptor (TCR), the CD2 antigen and the interleukin 2 receptor. Experiments using a PKC pseudosubstrate inhibitor indicate that PKC does not mediate TCR-induced activation of p21ras. These results imply that an alternative signal transduction pathway not involving PKC can regulate the activity of p21ras proteins in T cells.

Immunomodulator AS101 potentiates apoptosis on Ha-Ras transformed cells which correlates with suppression of a survival protein Akt (48.20)

Abstract The Ras family is the most widely mutated group of human proto-oncogene. Previously, we found an organic Tellurium compound, AS101, which exhibits immunomodulatory activity and strong anti-tumor effects. Whether these properties can be exploited for cancer therapy was examined using v-Ha-Ras transformed and V-mos transformed fibroblasts cells. In this study AS101 appeared to selectively block ~90 % of the proliferative growth of v-Ha-Ras transformed cells, while not affecting the growth of V-mos transformed cells. Furthermore, the proliferative growth inhibiting effect of AS101 was prevented when the activity of p21Ras was blocked by expression of a dominant negative mutant 116y of the v-Ha-Ras or by treating Ha-Ras transformed cells with a farnesyltransferase inhibitor. The anti-proliferative effect of AS101 was accompanied by morphological changes typical of apoptosis i.e.: membrane blebbing, condensation of nuclear chromatin, and formation of apoptotic bodies. AS101-induced apoptosis of v-Ha-Ras transformed cells correlated with suppression of Akt protein expression, activation of caspases, and induction of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Thus, our results suggest that AS101 can be used to effectively treat tumors which, are induced due to an oncogenic mutation in p21Ras encoding gene and to selectively block oncogenic function of mutated Ha-Ras in human tumors.

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotope-coded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative posttranslational thiol modifications of p21ras caused by peroxynitrite (ONOO −) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased after exposure to GSSG, but not to ONOO −. The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO − showed that ICAT labeling of Cys 118 was decreased by 47%, whereas Cys 80 was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys 118 by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras.

S-Glutathiolation of p21ras by Peroxynitrite Mediates Endothelial Insulin Resistance Caused by Oxidized Low-Density Lipoprotein

To understand the mechanism by which oxidants are linked to insulin resistance, bovine aortic endothelial cells were exposed to oxidized low-density lipoproteins (oxLDL) or peroxynitrite. OxLDL transiently increased phosphorylation of Erk and Akt within 5 minutes, but 60 minutes later, resulted in decreased insulin-induced Akt phosphorylation. OxLDL promoted a 2- to 5-fold increase in oxidant generation as measured by dihydrorhodamine or dihydroethidium oxidation that was ascribed to peroxynitrite. Exogenous peroxynitrite (25 to 100 micromol/L) or oxidized glutathione mimicked the effects of oxLDL. OxLDL increased the S-glutathiolation of p21ras, and adenoviral transfection with either a mutant p21ras (C118S) lacking the predominant site of S-glutathiolation or a dominant-negative mutant restored insulin-induced Akt phosphorylation. The requirement for oxidant-mediated S-glutathiolation and activation of p21ras in mediating insulin resistance was further implicated by showing that insulin signaling was restored by Mek inhibitors or by overexpression of glutaredoxin-1. Furthermore, oxLDL increased Erk-dependent phosphorylation of insulin receptor substrate-1 serine-616 that was prevented by inhibiting oxidant generation, Erk activation, or by the p21ras C118S mutant. This study provides direct evidence for a novel molecular mechanism by which oxidants can induce insulin resistance via S-glutathiolation of p21ras and Erk-dependent inhibition of insulin signaling.

Involvement of Protein Kinase Cα (PKCα) in the Early Action of Angiotensin II Type 2 (AT2) Effects on Neurite Outgrowth in NG108–15 Cells: AT2-Receptor Inhibits PKCα and p21ras Activity

The aim of the present study was to investigate whether protein kinase C (PKC) isoforms may be among the putative candidates implicated in the primary effects of the Ang II type 2 (AT2) receptor. Western blot analyses revealed the presence of PKC alpha,epsilon, iota, and zeta in NG108-15 cells. After a 3-d treatment with 3 nm Gö6976, a specific inhibitor of classical PKC isoforms, cells were characterized by the presence of one elongated process similar to that observed after treatment with Ang II or with CGP42112, a selective AT2 receptor agonist. Similar findings were observed in cells expressing a dominant-negative mutant of PKC alpha (K368A). Inhibition of PKC alpha in NG108-15 cells also decreased cell number and proliferation. In conditions of acute stimulation, Ang II induced a time-dependent and transient inhibition of PKC alpha activity, as well as a decrease in PKC alpha levels associated with the membrane. Treatment of cells with Gö6976 was also found to inhibit p21(ras) (between 1-10 min) but stimulated Rap1 activity (1-5 min) in a time-course similar to that of Ang II. Incubation of NG108-15 cells with Gö6976 (3 nm) inhibited basal p42/p44(mapk) phosphorylation, but failed to interfere with its activation by the AT(2) receptor, indicating that inhibition of PKC alpha is not directly involved in the Rap1-MEK-p42/p44(mapk) cascade. Taken together, these results indicate that PKC alpha is a primary target of the AT2 receptor. Inhibition of PKC alpha leads to a decrease in both p21(ras) activity and cell proliferation, which may facilitate AT2 receptor signaling through p42/p44(mapk), thereby leading to neurite outgrowth.