Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I

Abstract

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine. Its pleiotropic biological properties are signaled through two distinct cell surface receptors: the TNF receptor type I (TNFR-I) and the TNF receptor type II. Neither of the two receptors possesses tyrosine kinase activity. A large majority of TNF-alpha-dependent activities can be mediated by TNFR-I. Recently, c-Raf-1 kinase was identified as an intracellular target of a signal transduction cascade initiated by binding of TNF-alpha to TNFR-I. However, the mechanism engaged in TNF-alpha-dependent activation of c-Raf-1 kinase is still enigmatic. Here we report that the cytosolic adapter protein Grb2 is a novel binding partner of TNFR-I. Grb2 binds with its COOH-terminal SH3 domain to a PLAP motif within TNFR-I and with its NH2-terminal SH3 domain to SOS (son of sevenless). A PLAP deletion mutant of TNFR-I fails to bind Grb2. The TNFR-I/Grb2 interaction is essential for the TNF-alpha-dependent activation of c-Raf-1 kinase; activation of c-Raf-1 kinase by TNF-alpha can be blocked by coexpression of Grb2 mutants harboring inactivating point mutations in the NH2- or COOH-terminal SH3 domain, cell-permeable peptides that disrupt the Grb2/TNFR-I interaction or transdominant negative Ras. Functionality of the TNFR-I/Grb2/SOS/Ras interaction is a prerequisite but not sufficient for TNF-alpha-dependent activation of c-Raf-1 kinase. Inhibition of the TNFR-I/FAN (factor associated with neutral sphingomyelinase) interaction, which is essential for TNF-alpha-dependent activation of the neutral sphingomyelinase, either by cell-permeable peptides or by deletion of the FAN binding domain, prevents activation of c-Raf-1 kinase. In conclusion, binding of the Grb2 adapter protein via its COOH-terminal SH3 domain to the nontyrosine kinase receptor TNFR-I results in activation of a signaling cascade known so far to be initiated, in the case of the tyrosine kinase receptors, by binding of the SH2 domain of Grb2 to phosphotyrosine.

Figure 1

Grb2 and TNFR-I are direct binding partners. (A) Purified hexa-His–tagged cytoplasmic domain of TNFR-I (cTNFR-I; aa 205–426) (lanes 1–3 and 5, 1 μg; lane 4, 0.1 μg) was added to purified Grb2 (lanes 1–4, 6, and 7, 0.8 μg) in a volume of 300 μl. The mixtures were precipitated by addition of Ni-NTA–agarose (lanes 2, 4, 6, and 7) (specific for hexa-His–tagged c-TNFR-I), by a polyclonal sera specific for Grb2. Analysis of the precipitates for the presence of Grb2 was performed by Western blot using a monoclonal Grb2-specific antiserum. In lane 7, one-fifth of the precipitate was loaded to avoid overloading. Lanes 5 and 6 served as negative controls. (B) Coimmunoprecipitation of Grb2 and TNFR-I from the lysate of 10⁶ CCL13 cells. The lysates were precipitated by polyclonal rabbit-derived sera against Grb2 or goat-derived serum against TNFR-I and analyzed for the presence of Grb2 (left) or TNFR-I (right) by Western blot, using a mouse mAb specific for Grb2 or a polyclonal rabbit-derived serum specific for TNFR-I. Precipitations with an unrelated rabbit serum (rabbit anti-PreS2; left, lane 4; right, lane 2) or an unrelated goat serum (goat anti-PreS2; left, lane 5; right, lane 4) served as negative control. Total cellular lysates (left and right, lane 1) were used as positive controls.

Figure 1

Grb2 and TNFR-I are direct binding partners. (A) Purified hexa-His–tagged cytoplasmic domain of TNFR-I (cTNFR-I; aa 205–426) (lanes 1–3 and 5, 1 μg; lane 4, 0.1 μg) was added to purified Grb2 (lanes 1–4, 6, and 7, 0.8 μg) in a volume of 300 μl. The mixtures were precipitated by addition of Ni-NTA–agarose (lanes 2, 4, 6, and 7) (specific for hexa-His–tagged c-TNFR-I), by a polyclonal sera specific for Grb2. Analysis of the precipitates for the presence of Grb2 was performed by Western blot using a monoclonal Grb2-specific antiserum. In lane 7, one-fifth of the precipitate was loaded to avoid overloading. Lanes 5 and 6 served as negative controls. (B) Coimmunoprecipitation of Grb2 and TNFR-I from the lysate of 10⁶ CCL13 cells. The lysates were precipitated by polyclonal rabbit-derived sera against Grb2 or goat-derived serum against TNFR-I and analyzed for the presence of Grb2 (left) or TNFR-I (right) by Western blot, using a mouse mAb specific for Grb2 or a polyclonal rabbit-derived serum specific for TNFR-I. Precipitations with an unrelated rabbit serum (rabbit anti-PreS2; left, lane 4; right, lane 2) or an unrelated goat serum (goat anti-PreS2; left, lane 5; right, lane 4) served as negative control. Total cellular lysates (left and right, lane 1) were used as positive controls.

Figure 1

Grb2 and TNFR-I are direct binding partners. (A) Purified hexa-His–tagged cytoplasmic domain of TNFR-I (cTNFR-I; aa 205–426) (lanes 1–3 and 5, 1 μg; lane 4, 0.1 μg) was added to purified Grb2 (lanes 1–4, 6, and 7, 0.8 μg) in a volume of 300 μl. The mixtures were precipitated by addition of Ni-NTA–agarose (lanes 2, 4, 6, and 7) (specific for hexa-His–tagged c-TNFR-I), by a polyclonal sera specific for Grb2. Analysis of the precipitates for the presence of Grb2 was performed by Western blot using a monoclonal Grb2-specific antiserum. In lane 7, one-fifth of the precipitate was loaded to avoid overloading. Lanes 5 and 6 served as negative controls. (B) Coimmunoprecipitation of Grb2 and TNFR-I from the lysate of 10⁶ CCL13 cells. The lysates were precipitated by polyclonal rabbit-derived sera against Grb2 or goat-derived serum against TNFR-I and analyzed for the presence of Grb2 (left) or TNFR-I (right) by Western blot, using a mouse mAb specific for Grb2 or a polyclonal rabbit-derived serum specific for TNFR-I. Precipitations with an unrelated rabbit serum (rabbit anti-PreS2; left, lane 4; right, lane 2) or an unrelated goat serum (goat anti-PreS2; left, lane 5; right, lane 4) served as negative control. Total cellular lysates (left and right, lane 1) were used as positive controls.

Figure 2

A PLAP motif in the cytoplasmic domain of TNFR-I mediates the interaction with Grb2. (A) Purified hexa-His–tagged cytTNFR-I (1 μg) was added to cellular lysate (500 μg) derived from TNF-α–stimulated 293 cells. Coprecipitation of Grb2 by addition of Ni-NTA–agarose, which precipitates the 6H-cytTNFR-I, was inhibited by increasing concentrations of a peptide (functional peptide [FP], lanes 4–6) harboring the recognition sequence TTKPLAP, whereas a mutated peptide (MP) (TTKKLAP, lanes 7–9) did not significantly affect the interaction of Grb2 and TNFR-I. An unrelated peptide served as additional control (CP) (DRQIKIWFQNRRMKWKK; lanes 1–3). The peptides were used in concentrations of  0.1 (lanes 2, 5, and 8) and 4 μg/ml (lanes 3, 6, and 9). The precipitates were analyzed by Western blot using a Grb2-specific antiserum. (B) Left: after transient transfection of 293 cells with p6H-TNFR-I (encoding for a fusion protein of an NH₂-terminal hexa-His tag and full length TNFR-I) (lanes 5 and 6), with p6H-TNFR-IΔ-PLAP (encoding for the hexa-His–tagged PLAP deletion mutant (lanes 1–3), or with the vector pCDNA.3 (lane 4), cellular lysates were precipitated by addition of Ni-NTA–agarose (lanes 2, 4, and 5) by a Grb2-specific polyclonal serum (lanes 3 and 6) or by an unrelated rabbit-derived serum (anti-PreS2; lane 1). The subsequent Western blot analysis was performed using a Grb2-specific, mouse-derived mAb. Right: lysates derived from p6H-TNFR-I– (lanes 1, 2, 4, and 5), p6H-TNFR-IΔPLAP– (lanes 3 and 6), or pCDNA.3- (lane 7) transfected cells were precipitated with Ni-NTA–agarose (lanes 4–6), a Grb2-specific polyclonal rabbit derived serum (lanes 2, 3, and 7), or an unrelated rabbit serum (anti-PreS2; lane 1). The precipitates were analyzed using a hexa-His tag–specific antibody.

Figure 2

A PLAP motif in the cytoplasmic domain of TNFR-I mediates the interaction with Grb2. (A) Purified hexa-His–tagged cytTNFR-I (1 μg) was added to cellular lysate (500 μg) derived from TNF-α–stimulated 293 cells. Coprecipitation of Grb2 by addition of Ni-NTA–agarose, which precipitates the 6H-cytTNFR-I, was inhibited by increasing concentrations of a peptide (functional peptide [FP], lanes 4–6) harboring the recognition sequence TTKPLAP, whereas a mutated peptide (MP) (TTKKLAP, lanes 7–9) did not significantly affect the interaction of Grb2 and TNFR-I. An unrelated peptide served as additional control (CP) (DRQIKIWFQNRRMKWKK; lanes 1–3). The peptides were used in concentrations of  0.1 (lanes 2, 5, and 8) and 4 μg/ml (lanes 3, 6, and 9). The precipitates were analyzed by Western blot using a Grb2-specific antiserum. (B) Left: after transient transfection of 293 cells with p6H-TNFR-I (encoding for a fusion protein of an NH₂-terminal hexa-His tag and full length TNFR-I) (lanes 5 and 6), with p6H-TNFR-IΔ-PLAP (encoding for the hexa-His–tagged PLAP deletion mutant (lanes 1–3), or with the vector pCDNA.3 (lane 4), cellular lysates were precipitated by addition of Ni-NTA–agarose (lanes 2, 4, and 5) by a Grb2-specific polyclonal serum (lanes 3 and 6) or by an unrelated rabbit-derived serum (anti-PreS2; lane 1). The subsequent Western blot analysis was performed using a Grb2-specific, mouse-derived mAb. Right: lysates derived from p6H-TNFR-I– (lanes 1, 2, 4, and 5), p6H-TNFR-IΔPLAP– (lanes 3 and 6), or pCDNA.3- (lane 7) transfected cells were precipitated with Ni-NTA–agarose (lanes 4–6), a Grb2-specific polyclonal rabbit derived serum (lanes 2, 3, and 7), or an unrelated rabbit serum (anti-PreS2; lane 1). The precipitates were analyzed using a hexa-His tag–specific antibody.

Figure 3

The TNFR-I/Grb2 interaction is essential for TNF-dependent activation of c-Raf-1 kinase and subsequent activation of AP-1. (A) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (15 μg/ml) covering the PLAP sequence (functional peptide [FP]: DRQIKIWFQNRRMKWKKTTKPLAP; left, lane 4; right, lane 5) or a mutated peptide ([MP]: DRQIKIWFQNRRMKWKKTTKKLAP; left, lane 2; right, lane 4). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml) (left, lanes 1, 2, and 4) or to EGF (5 ng/ml) (right, lanes 3–5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate and compared with unstimulated cells (left, lane 3; right, lanes 1 and 2). (B) 70Z3 cells were transfected with pCDNA.3 (lane 1), p6H-TNFR-I (lane 2), and p6H-TNFR-IΔPLAP (lane 3). After stimulation with TNF-α (200 U/ml), the activity of c-Raf-1 kinase was determined by immunocomplex assay. Activity of c-Raf-1 kinase in pCDNA.3-transfected cells was set arbitrarily as 1. (C) Immunocomplex assay of c-Raf-1 kinase activity in 293 cells transiently transfected with pCDNA.3 (lanes 1, 2, and 6), pΔNSH3-Grb2 (lane 3), pΔSH2Grb2 (lane 4), and pΔCSH3Grb2 (lane 5) after TNF-α (lanes 3–6) or EGF (lane 2) stimulation. Unstimulated cells (lane 1) served as negative control. (D) Reporter gene assay in 293 cells transiently cotransfected with pCDNA.3, pΔNSH3-Grb2, pΔSH2Grb2, pΔCSH3Grb2, and pHCR13.1 encoding a transdominant negative mutant of c-Raf-1 kinase 20 or pRasN17 and the reporter plasmid p3xAP-1–CAT after TNF-α stimulation (100 U/ml) for 24 h. The determination of the induced CAT amount was performed using a commercially available ELISA system. Activities given as fold induction are mean values of two independent experiments.

Figure 3

The TNFR-I/Grb2 interaction is essential for TNF-dependent activation of c-Raf-1 kinase and subsequent activation of AP-1. (A) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (15 μg/ml) covering the PLAP sequence (functional peptide [FP]: DRQIKIWFQNRRMKWKKTTKPLAP; left, lane 4; right, lane 5) or a mutated peptide ([MP]: DRQIKIWFQNRRMKWKKTTKKLAP; left, lane 2; right, lane 4). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml) (left, lanes 1, 2, and 4) or to EGF (5 ng/ml) (right, lanes 3–5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate and compared with unstimulated cells (left, lane 3; right, lanes 1 and 2). (B) 70Z3 cells were transfected with pCDNA.3 (lane 1), p6H-TNFR-I (lane 2), and p6H-TNFR-IΔPLAP (lane 3). After stimulation with TNF-α (200 U/ml), the activity of c-Raf-1 kinase was determined by immunocomplex assay. Activity of c-Raf-1 kinase in pCDNA.3-transfected cells was set arbitrarily as 1. (C) Immunocomplex assay of c-Raf-1 kinase activity in 293 cells transiently transfected with pCDNA.3 (lanes 1, 2, and 6), pΔNSH3-Grb2 (lane 3), pΔSH2Grb2 (lane 4), and pΔCSH3Grb2 (lane 5) after TNF-α (lanes 3–6) or EGF (lane 2) stimulation. Unstimulated cells (lane 1) served as negative control. (D) Reporter gene assay in 293 cells transiently cotransfected with pCDNA.3, pΔNSH3-Grb2, pΔSH2Grb2, pΔCSH3Grb2, and pHCR13.1 encoding a transdominant negative mutant of c-Raf-1 kinase 20 or pRasN17 and the reporter plasmid p3xAP-1–CAT after TNF-α stimulation (100 U/ml) for 24 h. The determination of the induced CAT amount was performed using a commercially available ELISA system. Activities given as fold induction are mean values of two independent experiments.

Figure 3

The TNFR-I/Grb2 interaction is essential for TNF-dependent activation of c-Raf-1 kinase and subsequent activation of AP-1. (A) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (15 μg/ml) covering the PLAP sequence (functional peptide [FP]: DRQIKIWFQNRRMKWKKTTKPLAP; left, lane 4; right, lane 5) or a mutated peptide ([MP]: DRQIKIWFQNRRMKWKKTTKKLAP; left, lane 2; right, lane 4). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml) (left, lanes 1, 2, and 4) or to EGF (5 ng/ml) (right, lanes 3–5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate and compared with unstimulated cells (left, lane 3; right, lanes 1 and 2). (B) 70Z3 cells were transfected with pCDNA.3 (lane 1), p6H-TNFR-I (lane 2), and p6H-TNFR-IΔPLAP (lane 3). After stimulation with TNF-α (200 U/ml), the activity of c-Raf-1 kinase was determined by immunocomplex assay. Activity of c-Raf-1 kinase in pCDNA.3-transfected cells was set arbitrarily as 1. (C) Immunocomplex assay of c-Raf-1 kinase activity in 293 cells transiently transfected with pCDNA.3 (lanes 1, 2, and 6), pΔNSH3-Grb2 (lane 3), pΔSH2Grb2 (lane 4), and pΔCSH3Grb2 (lane 5) after TNF-α (lanes 3–6) or EGF (lane 2) stimulation. Unstimulated cells (lane 1) served as negative control. (D) Reporter gene assay in 293 cells transiently cotransfected with pCDNA.3, pΔNSH3-Grb2, pΔSH2Grb2, pΔCSH3Grb2, and pHCR13.1 encoding a transdominant negative mutant of c-Raf-1 kinase 20 or pRasN17 and the reporter plasmid p3xAP-1–CAT after TNF-α stimulation (100 U/ml) for 24 h. The determination of the induced CAT amount was performed using a commercially available ELISA system. Activities given as fold induction are mean values of two independent experiments.

Figure 3

The TNFR-I/Grb2 interaction is essential for TNF-dependent activation of c-Raf-1 kinase and subsequent activation of AP-1. (A) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (15 μg/ml) covering the PLAP sequence (functional peptide [FP]: DRQIKIWFQNRRMKWKKTTKPLAP; left, lane 4; right, lane 5) or a mutated peptide ([MP]: DRQIKIWFQNRRMKWKKTTKKLAP; left, lane 2; right, lane 4). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml) (left, lanes 1, 2, and 4) or to EGF (5 ng/ml) (right, lanes 3–5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate and compared with unstimulated cells (left, lane 3; right, lanes 1 and 2). (B) 70Z3 cells were transfected with pCDNA.3 (lane 1), p6H-TNFR-I (lane 2), and p6H-TNFR-IΔPLAP (lane 3). After stimulation with TNF-α (200 U/ml), the activity of c-Raf-1 kinase was determined by immunocomplex assay. Activity of c-Raf-1 kinase in pCDNA.3-transfected cells was set arbitrarily as 1. (C) Immunocomplex assay of c-Raf-1 kinase activity in 293 cells transiently transfected with pCDNA.3 (lanes 1, 2, and 6), pΔNSH3-Grb2 (lane 3), pΔSH2Grb2 (lane 4), and pΔCSH3Grb2 (lane 5) after TNF-α (lanes 3–6) or EGF (lane 2) stimulation. Unstimulated cells (lane 1) served as negative control. (D) Reporter gene assay in 293 cells transiently cotransfected with pCDNA.3, pΔNSH3-Grb2, pΔSH2Grb2, pΔCSH3Grb2, and pHCR13.1 encoding a transdominant negative mutant of c-Raf-1 kinase 20 or pRasN17 and the reporter plasmid p3xAP-1–CAT after TNF-α stimulation (100 U/ml) for 24 h. The determination of the induced CAT amount was performed using a commercially available ELISA system. Activities given as fold induction are mean values of two independent experiments.

Figure 3

The TNFR-I/Grb2 interaction is essential for TNF-dependent activation of c-Raf-1 kinase and subsequent activation of AP-1. (A) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (15 μg/ml) covering the PLAP sequence (functional peptide [FP]: DRQIKIWFQNRRMKWKKTTKPLAP; left, lane 4; right, lane 5) or a mutated peptide ([MP]: DRQIKIWFQNRRMKWKKTTKKLAP; left, lane 2; right, lane 4). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml) (left, lanes 1, 2, and 4) or to EGF (5 ng/ml) (right, lanes 3–5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate and compared with unstimulated cells (left, lane 3; right, lanes 1 and 2). (B) 70Z3 cells were transfected with pCDNA.3 (lane 1), p6H-TNFR-I (lane 2), and p6H-TNFR-IΔPLAP (lane 3). After stimulation with TNF-α (200 U/ml), the activity of c-Raf-1 kinase was determined by immunocomplex assay. Activity of c-Raf-1 kinase in pCDNA.3-transfected cells was set arbitrarily as 1. (C) Immunocomplex assay of c-Raf-1 kinase activity in 293 cells transiently transfected with pCDNA.3 (lanes 1, 2, and 6), pΔNSH3-Grb2 (lane 3), pΔSH2Grb2 (lane 4), and pΔCSH3Grb2 (lane 5) after TNF-α (lanes 3–6) or EGF (lane 2) stimulation. Unstimulated cells (lane 1) served as negative control. (D) Reporter gene assay in 293 cells transiently cotransfected with pCDNA.3, pΔNSH3-Grb2, pΔSH2Grb2, pΔCSH3Grb2, and pHCR13.1 encoding a transdominant negative mutant of c-Raf-1 kinase 20 or pRasN17 and the reporter plasmid p3xAP-1–CAT after TNF-α stimulation (100 U/ml) for 24 h. The determination of the induced CAT amount was performed using a commercially available ELISA system. Activities given as fold induction are mean values of two independent experiments.

Figure 4

SOS coprecipitates with Grb2 and TNFR-I. (A) In the presence (+; lanes 1–4) or absence (−; lanes 5–7) of purified hexa-His–tagged cyt(c)TNFR-I (1 μg), cellular lysates derived from 293 cells were precipitated with Ni-NTA–agarose (lanes 2 and 5), SOS1/2-specific antiserum (lanes 3 and 6), and Grb2-specific antiserum (lanes 4 and 7). The precipitates were analyzed for the presence of cytTNFR-I by Western blot using a hexa-His tag–specific antibody. In lane 1, the mixture of cytTNFR-I combined with cellular lysate was loaded directly as the positive control. (B) 70Z3 cells stably producing wild-type TNFR-I or a deletion mutant (TNFRID308–340) lacking the nSMase-activating domain were stimulated with TNF-α (200 U/ml) for 15 min, and c-Raf-1 kinase activity was determined by immunocomplex assay. (C) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (lanes 2 and 4) covering the FAN binding sequence (functional peptide [FP], 5 μg/ml). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml; lanes 2 and 3) or EGF (5 ng/ml; lanes 1 and 5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate. (D) Schematic representation of the proposed model of TNF-α–dependent activation of c-Raf-1 kinase. A PLAP motif recruits the Grb2 adapter protein through its COOH-terminal SH3 domain to TNFR-I. The NH₂-terminal SH3 domain of Grb2 interacts with SOS, which recruits via Ras c-Raf-1 kinase to the membrane. TNF-α–dependent activation of nSMase is mediated by binding of FAN to a distinct domain of TNFR-I (12, 13). Activation of nSMase results in the production of ceramide and, consequently, in an activation of CAP kinase. Both TNF-α–responsive pathways, the activation of Ras via Grb2 and the activation of nSMase, cooperatively stimulate c-Raf-1 kinase.

Figure 4

SOS coprecipitates with Grb2 and TNFR-I. (A) In the presence (+; lanes 1–4) or absence (−; lanes 5–7) of purified hexa-His–tagged cyt(c)TNFR-I (1 μg), cellular lysates derived from 293 cells were precipitated with Ni-NTA–agarose (lanes 2 and 5), SOS1/2-specific antiserum (lanes 3 and 6), and Grb2-specific antiserum (lanes 4 and 7). The precipitates were analyzed for the presence of cytTNFR-I by Western blot using a hexa-His tag–specific antibody. In lane 1, the mixture of cytTNFR-I combined with cellular lysate was loaded directly as the positive control. (B) 70Z3 cells stably producing wild-type TNFR-I or a deletion mutant (TNFRID308–340) lacking the nSMase-activating domain were stimulated with TNF-α (200 U/ml) for 15 min, and c-Raf-1 kinase activity was determined by immunocomplex assay. (C) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (lanes 2 and 4) covering the FAN binding sequence (functional peptide [FP], 5 μg/ml). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml; lanes 2 and 3) or EGF (5 ng/ml; lanes 1 and 5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate. (D) Schematic representation of the proposed model of TNF-α–dependent activation of c-Raf-1 kinase. A PLAP motif recruits the Grb2 adapter protein through its COOH-terminal SH3 domain to TNFR-I. The NH₂-terminal SH3 domain of Grb2 interacts with SOS, which recruits via Ras c-Raf-1 kinase to the membrane. TNF-α–dependent activation of nSMase is mediated by binding of FAN to a distinct domain of TNFR-I (12, 13). Activation of nSMase results in the production of ceramide and, consequently, in an activation of CAP kinase. Both TNF-α–responsive pathways, the activation of Ras via Grb2 and the activation of nSMase, cooperatively stimulate c-Raf-1 kinase.

Figure 4

SOS coprecipitates with Grb2 and TNFR-I. (A) In the presence (+; lanes 1–4) or absence (−; lanes 5–7) of purified hexa-His–tagged cyt(c)TNFR-I (1 μg), cellular lysates derived from 293 cells were precipitated with Ni-NTA–agarose (lanes 2 and 5), SOS1/2-specific antiserum (lanes 3 and 6), and Grb2-specific antiserum (lanes 4 and 7). The precipitates were analyzed for the presence of cytTNFR-I by Western blot using a hexa-His tag–specific antibody. In lane 1, the mixture of cytTNFR-I combined with cellular lysate was loaded directly as the positive control. (B) 70Z3 cells stably producing wild-type TNFR-I or a deletion mutant (TNFRID308–340) lacking the nSMase-activating domain were stimulated with TNF-α (200 U/ml) for 15 min, and c-Raf-1 kinase activity was determined by immunocomplex assay. (C) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (lanes 2 and 4) covering the FAN binding sequence (functional peptide [FP], 5 μg/ml). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml; lanes 2 and 3) or EGF (5 ng/ml; lanes 1 and 5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate. (D) Schematic representation of the proposed model of TNF-α–dependent activation of c-Raf-1 kinase. A PLAP motif recruits the Grb2 adapter protein through its COOH-terminal SH3 domain to TNFR-I. The NH₂-terminal SH3 domain of Grb2 interacts with SOS, which recruits via Ras c-Raf-1 kinase to the membrane. TNF-α–dependent activation of nSMase is mediated by binding of FAN to a distinct domain of TNFR-I (12, 13). Activation of nSMase results in the production of ceramide and, consequently, in an activation of CAP kinase. Both TNF-α–responsive pathways, the activation of Ras via Grb2 and the activation of nSMase, cooperatively stimulate c-Raf-1 kinase.

Figure 4

SOS coprecipitates with Grb2 and TNFR-I. (A) In the presence (+; lanes 1–4) or absence (−; lanes 5–7) of purified hexa-His–tagged cyt(c)TNFR-I (1 μg), cellular lysates derived from 293 cells were precipitated with Ni-NTA–agarose (lanes 2 and 5), SOS1/2-specific antiserum (lanes 3 and 6), and Grb2-specific antiserum (lanes 4 and 7). The precipitates were analyzed for the presence of cytTNFR-I by Western blot using a hexa-His tag–specific antibody. In lane 1, the mixture of cytTNFR-I combined with cellular lysate was loaded directly as the positive control. (B) 70Z3 cells stably producing wild-type TNFR-I or a deletion mutant (TNFRID308–340) lacking the nSMase-activating domain were stimulated with TNF-α (200 U/ml) for 15 min, and c-Raf-1 kinase activity was determined by immunocomplex assay. (C) 12-h serum-deprived 293 cells were grown for 6 h in the presence of cell-permeable peptides (lanes 2 and 4) covering the FAN binding sequence (functional peptide [FP], 5 μg/ml). Thereafter, cells were exposed for 15 min to TNF-α (100 U/ml; lanes 2 and 3) or EGF (5 ng/ml; lanes 1 and 5) in addition to the cell-permeable peptides. Activity of c-Raf-1 kinase was determined by immunocomplex assay using 6H-MEK as substrate. (D) Schematic representation of the proposed model of TNF-α–dependent activation of c-Raf-1 kinase. A PLAP motif recruits the Grb2 adapter protein through its COOH-terminal SH3 domain to TNFR-I. The NH₂-terminal SH3 domain of Grb2 interacts with SOS, which recruits via Ras c-Raf-1 kinase to the membrane. TNF-α–dependent activation of nSMase is mediated by binding of FAN to a distinct domain of TNFR-I (12, 13). Activation of nSMase results in the production of ceramide and, consequently, in an activation of CAP kinase. Both TNF-α–responsive pathways, the activation of Ras via Grb2 and the activation of nSMase, cooperatively stimulate c-Raf-1 kinase.