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A Redox-triggered Ras-Effector Interaction

RECRUITMENT OF PHOSPHATIDYLINOSITOL 3′-KINASE TO Ras BY REDOX STRESS*
Open AccessPublished:November 06, 1998DOI:https://doi.org/10.1074/jbc.273.45.29923
      Reactive free radical species are known to trigger biochemical events culminating in transcription factor activation and modulation of gene expression. The cytosolic signaling events triggered by free radicals that result in nuclear responses are largely unknown. Here we identify a signaling cascade triggered immediately upon redox activation of Ras. We examined two physiologically relevant models of redox signaling: 1) nitric oxide in human T cells, and 2) advanced glycation end product in rat pheochromocytoma cells. Reactive free radical species generated by nitric oxide donors and the interaction of advanced glycation end product with its receptor led to the recruitment of p85/p110 phosphatidylinositol 3′-kinase (PI3K) to the plasma membrane, where it associated directly with the effector domain of Ras and became activated. Only the p110β and p110δ (but not p110α) catalytic subunits were recruited by redox-activated Ras. Activation of downstream targets of PI3K such as protein kinase B/Akt and mitogen-activated protein kinase was found to be PI3K dependent. Our study demonstrates that nitrosative and oxidative stressors trigger Ras-dependent and PI3K-regulated events in cells and define a biochemical pathway that is triggered by redox signaling.
      AGE
      advanced glycation end product
      NO
      nitric oxide
      PI3K
      phosphotidylinositol 3′-kinase
      SNP
      sodium nitroprusside
      SNAP
      S-nitroso-N-acetylpenicillamine
      ERK
      extracellular signal-regulated kinase.
      Free radical signal transduction is thought to play a crucial role in diverse physiological and pathological processes. These include receptor signaling, such as advanced glycation end product (AGE)1 (
      • Sundaresan M., Yu, Z.-X.
      • Ferrans V.J.
      • Irani K.
      • Finkel T.
      ) and platelet-derived growth factor-mediated signaling in smooth muscle cells, and the regulation of cytoplasmic and nuclear signals, such as stimulation of guanylyl cyclase, ion channels, and transcription factors (
      • Sundaresan M., Yu, Z.-X.
      • Ferrans V.J.
      • Irani K.
      • Finkel T.
      ,
      • Ignarro L.J.
      ,
      • Schrek R.
      • Riueber P.
      • Baeuerle P.A.
      ,
      • Lander H.M.
      ,
      • Yanagihara Y.
      • Basaki Y.
      • Ikizawa K.
      • Kajiwara K.
      ,
      • Yan S.D.
      • Schmidt A.M.
      • Anderson G.M.
      • Zhang J.
      • Brett J.
      • Zou Y.S.
      • Pinsky D.
      • Stern D.
      ,
      • Lander H.M.
      • Tauras J.M.
      • Ogiste J.S.
      • Hori O.
      • Moss R.A.
      • Schmidt A.M.
      ). These events, particularly those regulated by the free radical nitric oxide (NO) and its congeners, result in vasodilation, host defense, synaptic plasticity, and inhibition of smooth muscle cell proliferation (
      • Lander H.M.
      ,
      • Nathan C.
      ,
      • Dinerman J.L.
      • Lowenstein C.J.
      • Synder S.H.
      ,
      • Karupiah G.
      • Xie Q.-W.
      • Buller M.L.
      • Nathan C.
      • Duarte C.
      • MacMicking J.D.
      ,
      • Schuman E.M.
      • Madison D.V.
      ). The mechanisms by which free radicals participate in these processes remain largely unknown. Hence, investigating the various molecular targets that are involved in these processes will play a pivotal role in our mechanistic understanding of redox signaling.
      We have reported a reversible interaction between redox modulators and the monomeric G protein Ras, resulting in Ras activation via GDP/GTP exchange. Redox modification of amino acid residue Cys118of the Ras protein was found to be crucial for its activation (
      • Lander H.M.
      • Milbank A.J.
      • Tauras J.M.
      • Hajjar D.P.
      • Hempstead B.L.
      • Schwartz G.D.
      • Kraemer R.T.
      • Mirza U.A.
      • Chait B.T.
      • Campbell-Burk S.
      • Quilliam L.A.
      ,
      • Lander H.M.
      • Ogiste J.S.
      • Teng K.K.
      • Novogrodsky A.
      ,
      • Lander H.M.
      • Hajjar D.P.
      • Hempstead B.L.
      • Mirza U.A.
      • Chait B.T.
      • Campbell S.
      • Quilliam L.A.
      ). Ras has been reported to be activated by endogenous NO in endothelial cells and primary cortical cultures and exogenous NO in T cells and PC12 cells (
      • Lander H.M.
      • Ogiste J.S.
      • Teng K.K.
      • Novogrodsky A.
      ,
      • Lander H.M.
      • Ogiste J.S.
      • Pearce S.F.A.
      • Levi R.
      • Novogrodsky A.
      ,
      • Yun H.Y.
      • Gonzalez-Zulueta M.
      • Dawson V.L.
      • Dawson T.M.
      ).
      Activation-induced conformational changes in Ras result in its interaction with various signaling proteins, termed effectors. Ras has multiple effectors and, depending on the stimuli, one or more effector and downstream signals propagated by it are utilized. These effectors include phosphatidylinositol 3′-kinase, Raf-1, protein kinase C-ζ, Ral-GDS family, Rin1, AF6, diacylglycerol kinases, and mitogen-activated protein kinase kinase kinase (
      • Marshall C.J.
      ).
      Here we examined signaling events immediate to redox-activated Ras by identifying Ras effectors. PI3K is an effector of GTP-bound Ras (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Sjolander A.
      • Yamamoto K.
      • Huber B.E.
      • Lapetina E.G.
      ) and is implicated in the regulation of many biological responses including cell survival, mitogenesis, differentiation, the oxidative burst, membrane ruffling, and glucose uptake (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). We have observed that NO induces a dramatic increase in glucose uptake in human peripheral blood mononuclear cells (
      • Lander H.M.
      • Sehajpal P.
      • Levine D.M.
      • Novogrodsky A.
      ). Hence, we examined whether PI3K was regulated by redox signals.
      Class IA PI3Ks are heterodimeric enzymes composed of a 110-kDa catalytic subunit (p110) and a SH2 domain-containing adaptor protein. Mammals have genes for three adaptor subunits (p85α, p85β, and p55γ) and three p110 subunits (p110α, p110β, and p110δ; Refs.
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Vanhaesebroeck B.
      • Welham M.J.
      • Kotani K.
      • Stein R.
      • Warne P.H.
      • Zvelebil M.J.
      • Higashi K.
      • Volinia S.
      • Downward J.
      • Waterfield M.D.
      , and
      • Chantry D.
      • Vojtek A.
      • Kashishian A.
      • Holtzman D.A.
      • Wood C.
      • Gray P.W.
      • Cooper J.A.
      • Hoekstra M.F.
      ). Interaction of the adaptor subunit with phosphorylated tyrosine residues on receptors or membrane-localized docking proteins recruits PI3K to the plasma membrane. In addition, the class IA PI3Ks can also interact with GTP-bound Ras. p110 catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate at the 3′ position of the inositol ring (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). The lipid products have various downstream targets, including the serine-threonine kinase Akt (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Toker A.
      • Cantley L.C.
      ,
      • Downward J.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ) and possibly the extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (
      • Duckworth B.C.
      • Cantley L.C.
      ).

      DISCUSSION

      Post-translational modifications of proteins are crucial events in cell signaling. They include glycosylation, phosphorylation, and redox modifications such as the nitrosylation of cysteine residues. Oxidative and nitrosative agents are known to modulate the functions of proteins by modifying cysteine residues that are strategically located at catalytic or allosteric sites. Some of the proteins whose functions are regulated by modification of cysteine residues are Ras, calcium-dependent potassium channels,N-methyl-d-aspartate receptor, caspases, the mammalian transcription factors nuclear factor κB and activator protein 1, and the bacterial transcription factors OxyR and SoxR (
      • Lander H.M.
      ). Unlike Ras, most redox-sensitive proteins are not involved in generating divergent cellular outcomes. Ras is a key element of various signaling pathways and is implicated in the regulation of proliferation and differentiation by tyrosine kinase and G protein-coupled receptors (
      • Marshall C.J.
      ); thus, we attempted to identify the effectors involved in the signaling cascade propagated by redox-activated Ras.
      In this report, experimental evidence suggests that one of the effectors recruited by redox-activated Ras is PI3K. SNP and AGE-induced an increase in levels of Ras and p85α in the immunoprecipitate complex of anti-p85α antibody and anti-Ras antibody, respectively, suggesting a redox-induced interaction of Ras and PI3K (Fig. 1,A–C). Augmentation of this interaction was observed when cells were pretreated withl-buthionine-(S,R)-sulfoximine, confirming the redox nature of the signal. Of interest is that we observed the Ras-effector complex best using the anti-Ras antibody Y13-259. This antibody is known to bind to the Ras effector region. In contrast, the anti-Ras antibody Y13-238 is a non-neutralizing antibody but did not immunoprecipitate a NO-dependent Ras-PI3K complex. It is possible that Y13-238 binds to a region of Ras at which PI3K binds and is thus neutralizing for the Ras-PI3K interaction.
      Intriguingly, we observed a selective association of the catalytic subunit of PI3K. NO donors increased p110β and p110δ levels in co-immunoprecipitates of Ras with unaltered levels of p110α. The fact that phorbol ester and ionomycin can recruit all three catalytic isoforms of PI3K rules out the possibility that different affinities for each of the p110 isoforms for Ras in the co-immunoprecipitate was responsible for the apparent selectivity. The reason for such a differential interaction of p110 isoforms with Ras is unclear. It is in line, however, with the fact that the Ras-binding domain of the p110 catalytic subunits is the most divergent region in these otherwise highly homologous molecules. This may allow for a variety of interactions with small GTP-binding proteins (
      • Vanhaesebroeck B.
      • Welham M.J.
      • Kotani K.
      • Stein R.
      • Warne P.H.
      • Zvelebil M.J.
      • Higashi K.
      • Volinia S.
      • Downward J.
      • Waterfield M.D.
      ). It is worthy of mention that p110α is a proto-oncogene involved in transformation (
      • Chang H.W.
      • Aoki M.
      • Fruman D.
      • Auger K.R.
      • Bellacosa A.
      • Tsichlis P.N.
      • Cantley L.C.
      • Vogt P.K.
      ), and although NO activates Ras, it does not have any mitogenic effect (
      • Lander H.M.
      • Sehajpal P.
      • Levine D.M.
      • Novogrodsky A.
      ). The functional role of the p110β and p110δ isoforms is not yet clear. However, it would be consistent with our studies if these subunits played a role in the regulation of transcription factor activation.
      Our earlier studies identified Cys118 on Ras as a molecular target of reactive free radicals (
      • Lander H.M.
      • Milbank A.J.
      • Tauras J.M.
      • Hajjar D.P.
      • Hempstead B.L.
      • Schwartz G.D.
      • Kraemer R.T.
      • Mirza U.A.
      • Chait B.T.
      • Campbell-Burk S.
      • Quilliam L.A.
      ,
      • Lander H.M.
      • Hajjar D.P.
      • Hempstead B.L.
      • Mirza U.A.
      • Chait B.T.
      • Campbell S.
      • Quilliam L.A.
      ). To test our hypothesis that the binding of free radical species to Cys118 activates Ras and initiates signal transduction by recruiting PI3K, we created a mutant of Ras in which Cys118 was mutated to a serine residue (RasC118S; Ref.
      • Lander H.M.
      • Hajjar D.P.
      • Hempstead B.L.
      • Mirza U.A.
      • Chait B.T.
      • Campbell S.
      • Quilliam L.A.
      ). Jurkat cells overexpressing RasC118S exhibited normal Ras-dependent signaling when stimulated with the classical Ras activators, phorbol ester plus ionomycin, indicating that the mutation in Ras is specific for the NO-binding site (Fig. 3 B). This mutant cell line was unable to trigger an association between Ras and PI3K in response to NO donors. Moreover, overexpressed RasC118S exhibited dominant negative characteristics, because it did not allow any interaction between basal wild type Ras and PI3K (Fig. 3 A). Overexpression of two effector domain mutants of Ras also abrogated the association between Ras and PI3K, indicating that PI3K is indeed recruited to the classical effector domain of Ras by NO (Fig. 3 A). Thus, NO mediates signaling through Cys118 of Ras and triggers the binding of the p110β and p110δ catalytic subunits along with that of adaptor subunit p85α to the effector region of Ras.
      The SNP-induced increase in lipid kinase activity of PI3K suggested that the physical interaction between Ras and PI3K was biologically significant (Fig. 4 A). The increase in lipid kinase activity was similar in immunoprecipitates of both anti-p85α and anti-Ras antibodies, indicating that the stimulation in the kinase activity was due to a redox-activated Ras and PI3K interaction (Fig. 4 B). The SNP-induced increase in lipid kinase activity was not observed in the cell line overexpressing RasC118S, again confirming a requirement for a NO-Ras interaction to stimulate this signaling pathway (Fig. 4 C).
      Akt kinase is a very important downstream target of PI3K. SNP and AGE both activated Akt kinase and recruited it to the membrane. The PI3K inhibitor, wortmannin, completely abrogated activation by SNP and AGE (Fig. 4 D). SNP also activated ERK1/2 activity, another downstream target of PI3K; however, wortmannin could only partially inhibit this activation (Fig. 4 E). These data suggest that PI3K is solely responsible for the redox signal between Ras and Akt kinase and is partially responsible for the signal between Ras and ERK1/2. It is likely that other effector pathways, such as Raf kinase, also contribute to the signaling between Ras and ERK1/2.
      To date, the precise biochemical events triggered by redox agents have been ill-defined. In this report, we identify a Ras effector, PI3K, which orchestrates redox-triggered downstream signaling events. The rapid progress being made in understanding PI3K signaling will likely also have a major impact on the elucidation of crucial events in redox signaling. It will be very interesting to identify other effectors that redox-activated Ras may selectively recruit. This will help in understanding various physiological processes such as long-lasting neuronal responses and the cellular response to redox stress during host defense. Clinically, these studies may have far-reaching implications in intervention directed at various pathophysiological disorders in which redox signaling is thought to play crucial roles. These may include hypertension, diabetes, renal failure, atherosclerosis, and Alzheimer's disease.

      ACKNOWLEDGEMENTS

      We thank Dr. M. D. Waterfield for continuous support and Dr. Michael A. White for generously providing the Ras effector mutants.

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