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Distinct Ras effector pathways are involved in Fc epsilon R1 regulation of the transcriptional activity of Elk-1 and NFAT in mast cells.

Turner H, Cantrell DA - J. Exp. Med. (1997)

Bottom Line: We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells.The effector pathway for Ras activation of NFAT is not Raf-1/MEK.We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.

ABSTRACT
Activation of Ras GTPases is a conserved feature of antigen receptor signaling, including Fc epsilon R1 activation of mast cells. Antigenic cross-linking of the Fc epsilon R1 on mast cells results in secretion of allergic mediators and induction of immediate early and cytokine genes. Here we examine the role of Ras in coupling the Fc epsilon R1 to transcriptional regulation. The transcription factors Elk-1, an immediate early gene regulator and the nuclear factor of activated T cells (NFAT), in the context of the IL-4 gene, are identified as Ras targets in mast cells. Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1. We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells. Activation of the "classical" Ras/Raf-1/MEK/ ERK cascade is necessary and sufficient for Fc epsilon R1 induction of Elk-1. Ras function is required, but not sufficient for Fc epsilon R1 induction of NFAT. However, activation or inhibition of Ras markedly shifts the antigen dose-response for Fc epsilon R1 induction of NFAT. The effector pathway for Ras activation of NFAT is not Raf-1/MEK. We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras. These data place Ras in a crucial position in mast cells, regulating disparate nuclear targets. Moreover, we identify that two GTPases, Ras and Rac-1, are important regulators of NFAT, and therefore of cytokine expression in mast cells.

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(a) Activated Ras or Raf are sufficient for Elk-1 activation in  mast cells. 1 × 107 cells per point were transfected with either the Elk-1  reporter system alone or in combination with 10 μg pEF-V12Ras or 10 μg  pEF Raf-CAAX. Cells transfected with activated mutants were left unstimulated, while control cells were exposed to either 50 ng/ml PdBu or  1 μg/ml IgE/500ng/ml KLH-DNP in the concentrations indicated above.  (b and c) FcεR1 activation of Elk-1 is dependent on Ras. 1 × 107 cells per  point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of either RSV N17Ras or pEF dn  Raf-1. Cells were recovered and left unstimulated (NS) or exposed to  IgE/KLH-DNP as described.
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Figure 2: (a) Activated Ras or Raf are sufficient for Elk-1 activation in mast cells. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone or in combination with 10 μg pEF-V12Ras or 10 μg pEF Raf-CAAX. Cells transfected with activated mutants were left unstimulated, while control cells were exposed to either 50 ng/ml PdBu or 1 μg/ml IgE/500ng/ml KLH-DNP in the concentrations indicated above. (b and c) FcεR1 activation of Elk-1 is dependent on Ras. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of either RSV N17Ras or pEF dn Raf-1. Cells were recovered and left unstimulated (NS) or exposed to IgE/KLH-DNP as described.

Mentions: Elk-1 could be a target for a kinase cascade initiated by Ras. The prototypical kinase cascade transducing Ras signals is the Raf-1/MEK/ Erk pathway, and there is a precedent for Elk-1 activation via this mechanism in the fibroblast (31). To explore the role of Ras and Raf-1 signals in Elk-1 activation in mast cells, we examined the effects of activated Ras and Raf-1 mutants on Elk-1 activity in mast cells. The Raf-1 construct used here is a fusion protein of Raf-1 with a CAAX box motif that targets Raf-1 to the plasma membrane, rendering the exogenously expressed Raf-1 constitutively active (32). In these experiments, plasmids bearing the indicated mutants were cotransfected with the LexA Elk-1C fusion protein expression plasmid and the LexA OP.tkCAT reporter gene. The data in Fig. 2 a show that activated forms of both Ras and a Ras effector, Raf-1, are capable of inducing Elk-1 transactivation in RBL2H3 cells in the absence of other stimuli.


Distinct Ras effector pathways are involved in Fc epsilon R1 regulation of the transcriptional activity of Elk-1 and NFAT in mast cells.

Turner H, Cantrell DA - J. Exp. Med. (1997)

(a) Activated Ras or Raf are sufficient for Elk-1 activation in  mast cells. 1 × 107 cells per point were transfected with either the Elk-1  reporter system alone or in combination with 10 μg pEF-V12Ras or 10 μg  pEF Raf-CAAX. Cells transfected with activated mutants were left unstimulated, while control cells were exposed to either 50 ng/ml PdBu or  1 μg/ml IgE/500ng/ml KLH-DNP in the concentrations indicated above.  (b and c) FcεR1 activation of Elk-1 is dependent on Ras. 1 × 107 cells per  point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of either RSV N17Ras or pEF dn  Raf-1. Cells were recovered and left unstimulated (NS) or exposed to  IgE/KLH-DNP as described.
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Related In: Results  -  Collection

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Figure 2: (a) Activated Ras or Raf are sufficient for Elk-1 activation in mast cells. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone or in combination with 10 μg pEF-V12Ras or 10 μg pEF Raf-CAAX. Cells transfected with activated mutants were left unstimulated, while control cells were exposed to either 50 ng/ml PdBu or 1 μg/ml IgE/500ng/ml KLH-DNP in the concentrations indicated above. (b and c) FcεR1 activation of Elk-1 is dependent on Ras. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of either RSV N17Ras or pEF dn Raf-1. Cells were recovered and left unstimulated (NS) or exposed to IgE/KLH-DNP as described.
Mentions: Elk-1 could be a target for a kinase cascade initiated by Ras. The prototypical kinase cascade transducing Ras signals is the Raf-1/MEK/ Erk pathway, and there is a precedent for Elk-1 activation via this mechanism in the fibroblast (31). To explore the role of Ras and Raf-1 signals in Elk-1 activation in mast cells, we examined the effects of activated Ras and Raf-1 mutants on Elk-1 activity in mast cells. The Raf-1 construct used here is a fusion protein of Raf-1 with a CAAX box motif that targets Raf-1 to the plasma membrane, rendering the exogenously expressed Raf-1 constitutively active (32). In these experiments, plasmids bearing the indicated mutants were cotransfected with the LexA Elk-1C fusion protein expression plasmid and the LexA OP.tkCAT reporter gene. The data in Fig. 2 a show that activated forms of both Ras and a Ras effector, Raf-1, are capable of inducing Elk-1 transactivation in RBL2H3 cells in the absence of other stimuli.

Bottom Line: We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells.The effector pathway for Ras activation of NFAT is not Raf-1/MEK.We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.

ABSTRACT
Activation of Ras GTPases is a conserved feature of antigen receptor signaling, including Fc epsilon R1 activation of mast cells. Antigenic cross-linking of the Fc epsilon R1 on mast cells results in secretion of allergic mediators and induction of immediate early and cytokine genes. Here we examine the role of Ras in coupling the Fc epsilon R1 to transcriptional regulation. The transcription factors Elk-1, an immediate early gene regulator and the nuclear factor of activated T cells (NFAT), in the context of the IL-4 gene, are identified as Ras targets in mast cells. Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1. We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells. Activation of the "classical" Ras/Raf-1/MEK/ ERK cascade is necessary and sufficient for Fc epsilon R1 induction of Elk-1. Ras function is required, but not sufficient for Fc epsilon R1 induction of NFAT. However, activation or inhibition of Ras markedly shifts the antigen dose-response for Fc epsilon R1 induction of NFAT. The effector pathway for Ras activation of NFAT is not Raf-1/MEK. We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras. These data place Ras in a crucial position in mast cells, regulating disparate nuclear targets. Moreover, we identify that two GTPases, Ras and Rac-1, are important regulators of NFAT, and therefore of cytokine expression in mast cells.

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