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

Hildt E, Oess S - J. Exp. Med. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Klinikum der Universität Ulm, Ulm, 89081 Germany.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193078&req=5

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.

Mentions: In the case of tyrosine kinase receptor–dependent activation of c-Raf-1 kinase, the SH3 domains of Grb2 are known to interact with the SOS proteins, which interfere with Ras (22, 26, 27). Based on the observation that integrity of the NH2-terminal SH3 domain of Grb2 is essential to trigger TNF-α–dependent activation of c-Raf-1 kinase, it was investigated whether Grb2, while bound to TNFR-I with its COOH-terminal SH3 domain, still interacts with SOS1/2 proteins with its NH2-terminal SH3 domain. Hexa-His–tagged cytoplasmic domain of TNFR-I was added to cellular lysates derived from 293 cells. The mixture was precipitated using either anti-Grb2– or anti-SOS1/2–specific antibodies, or Ni-NTA–agarose, which specifically precipitates 6H-cytTNFR-I. Western blot analysis of the precipitates demonstrated that cytTNFR-I indeed coprecipitates with both Grb2 and SOS (Fig. 4 A).


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

Hildt E, Oess S - J. Exp. Med. (1999)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193078&req=5

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.
Mentions: In the case of tyrosine kinase receptor–dependent activation of c-Raf-1 kinase, the SH3 domains of Grb2 are known to interact with the SOS proteins, which interfere with Ras (22, 26, 27). Based on the observation that integrity of the NH2-terminal SH3 domain of Grb2 is essential to trigger TNF-α–dependent activation of c-Raf-1 kinase, it was investigated whether Grb2, while bound to TNFR-I with its COOH-terminal SH3 domain, still interacts with SOS1/2 proteins with its NH2-terminal SH3 domain. Hexa-His–tagged cytoplasmic domain of TNFR-I was added to cellular lysates derived from 293 cells. The mixture was precipitated using either anti-Grb2– or anti-SOS1/2–specific antibodies, or Ni-NTA–agarose, which specifically precipitates 6H-cytTNFR-I. Western blot analysis of the precipitates demonstrated that cytTNFR-I indeed coprecipitates with both Grb2 and SOS (Fig. 4 A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Klinikum der Universität Ulm, Ulm, 89081 Germany.

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.

Show MeSH
Related in: MedlinePlus