Nitric oxide and cGMP activate the Ras-MAP kinase pathway-stimulating protein tyrosine phosphorylation in rabbit aortic endothelial cells

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dc.contributor.authorOliveira, CJR
dc.contributor.authorSchindler, F.
dc.contributor.authorVentura, A. M.
dc.contributor.authorMorais, M. S.
dc.contributor.authorArai, R. J.
dc.contributor.authorDebbas, V
dc.contributor.authorStern, A.
dc.contributor.authorMonteiro, H. P.
dc.contributor.institutionFundacao Pro Sangue Hemoctr S Paulo
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionNYU
dc.date.accessioned2016-01-24T12:33:59Z
dc.date.available2016-01-24T12:33:59Z
dc.date.issued2003-08-15
dc.description.abstractThe free radical nitric oxide is a very effective signal transducer, stimulating the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. in the present study using rabbit aortic endothelial cells (RAEC), it is demonstrated that the nitric-oxide-generating substances sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and a stable analog of cyclic GMP, 8BrcGMP stimulate p21Ras activity. Tyrosine phosphorylation of cytosolic proteins was stimulated and intracellular production of cGMP was increased, indicating that the NO/cGMP-stimulated tyrosine phosphorylation-dependent signaling pathway is most likely associated with the activation of p21Ras. NO and cGMP-dependent activation of p21Ras result in binding of the oncoprotein to the Ras-binding domain of Raf-1 kinase. Incubation of RAEC with FPT 11, a potent and selective inhibitor of p21Ras, prevented NO-dependent tyrosine phosphorylation. ODQ, a potent inhibitor of the soluble form of guanylyl cyclase, inhibited the signal as well. Conversely, the use of KT5823, a cGMP-dependent protein kinase (PKG) blocker, showed no effect on protein tyrosine phosphorylation. To further establish a role for p2lRas on the NO-stimulated tyrosine phosphorylation-signaling pathway, RAEC were constitutively transfected with a dominant negative mutant of p2lRas, N17Ras. NO and cGMP-stimulated tyrosine phosphorylation were prevented in N17Ras-expressing RAEC exposed to NO donors and 8BrcGMP. the above findings indicate that NO and cGMP stimulation of protein tyrosine phosphorylation requires the participation of fully functional p2lRas. ERK1/2 MAP kinases and their subsequent targets, the transcription factors, lie downstream to Ras, Raf-1 kinase, and MEK. Treatment of both RAEC and mock-transfected RAEC with NO resulted in phosphorylation and activation of ERK1/2. On the other hand, NO did not stimulate phosphorylation of ERK1/2 in N17Ras-expressing RAEC. in addition, PD98059, a MEK inhibitor, prevented overall tyrosine phosphorylation and phosphorylation of ERK1/2. Upstream to Ras ERK1/2 MAP kinases target the EGF receptor. Incubation of RAEC or mock-transfected RAEC with NO donors resulted in activation of the EGF receptor autophosphorylation. PD98059 effectively blocked this activation. EGF receptor autophosphorylation was insensitive to NO stimulation in N17Ras-expressing RAEC. It is concluded that NO and cGMP stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MEK-ERK1/2. Activation of this signaling pathway is connected to NO-stimulated overall tyrosine phosphorylation that also involves the transactivation of the EGF receptor mediated by ERK1/2. (C) 2003 Elsevier Inc.en
dc.description.affiliationFundacao Pro Sangue Hemoctr S Paulo, Dept Biochem, BR-05403000 São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem Mol Biol, São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Inst Ciencias Biomed, Dept Microbiol, São Paulo, Brazil
dc.description.affiliationNYU, Sch Med, Dept Pharmacol, New York, NY USA
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem Mol Biol, São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Inst Ciencias Biomed, Dept Microbiol, São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent381-396
dc.identifierhttp://dx.doi.org/10.1016/S0891-5849(03)00311-3
dc.identifier.citationFree Radical Biology and Medicine. New York: Elsevier B.V., v. 35, n. 4, p. 381-396, 2003.
dc.identifier.doi10.1016/S0891-5849(03)00311-3
dc.identifier.issn0891-5849
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/27364
dc.identifier.wosWOS:000184488400005
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relation.ispartofFree Radical Biology and Medicine
dc.rightsinfo:eu-repo/semantics/restrictedAccess
dc.rights.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dc.subjectfree radicalsen
dc.subjecttyrosine phosphorylationen
dc.subjectp21Rasen
dc.subjectEGF receptoren
dc.subjectnitric oxideen
dc.subjectcGMPen
dc.subjectMAP kinasesen
dc.titleNitric oxide and cGMP activate the Ras-MAP kinase pathway-stimulating protein tyrosine phosphorylation in rabbit aortic endothelial cellsen
dc.typeinfo:eu-repo/semantics/article
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