Identification of Grb2 As a Novel Binding Partner of  Tumor Necrosis Factor (TNF) Receptor I

Abstract

Tumor necrosis factor α (TNF-α) 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-α–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-α to TNFR-I. However, the mechanism engaged in TNF-α–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-α–dependent activation of c-Raf-1 kinase; activation of c-Raf-1 kinase by TNF-α 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-α–dependent activation of c-Raf-1 kinase. Inhibition of the TNFR-I/FAN (factor associated with neutral sphingomyelinase) interaction, which is essential for TNF-α–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.

Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens

“…The PreS2-dependent activation of AP-1 or NF-B requires the cytoplasmic localization of the PreS2-domain. 13,14 Moreover, the specific inhibition of the TNF␣-dependent activation of c-Raf-1 kinase by a PreS2-TLM-PLAP fusion peptide competing the Grb2/TNF-RI interaction, which occurs in the cytosolic compartment, 17 confirms that the PreS2-TLM enables the translocation across the membrane. The specificity and effectiveness of these effects is demonstrated by the fact that low concentrations of 2 m protein or peptide added to the culture medium are sufficient to exhibit these intracellular effects, whereas the respective mutants fail to induce these effects.…”

“…In a previous investigation, 17 we observed that this PLAP motif mediates the interaction of TNF-RI with the C-terminal SH3 domain of the adapter molecule Grb2. This Grb2/TNF-RI-interaction is a prerequisite for the TNF␣-dependent activation of c-Raf-1 kinase.…”

TNF Activates Syk Protein Tyrosine Kinase Leading to TNF-Induced MAPK Activation, NF-κB Activation, and Apoptosis

“…It has been shown that Src and adapter proteins Grb2, crk, and p130 Cas are downstream mediators of Pyk2 leading to the activation of p44/p42 MAPK and JNK (48). The association of Grb2 directly with TNFR1 has been reported (49). Thus, it is possible that TNF-induced Syk activation recruits Pyk2, which in turn recruits Grb2, leading to MAPK activation.…”

Tumor necrosis factor signaling

“…185 Further, the adapter protein Grb2 binds to a PLAP motif of TNF-R1, thereby potentially linking this receptor via SOS to Ras, c-Raf and the ERKs. 186 This signal, however, appears not sufficient to efficiently activate the MAP kinases, as FAN/nSMasederived ceramide acting on the ceramide-activated protein kinase (CAP-K; 187 ) is necessary to fully activate c-Raf. 186 Consistent with these data, survival of osteoclasts by TNF is mediated by Akt and ERKs and can be blocked by inhibitors of the ERK-activating kinase MEK-1, but also by a peptide interfering with FAN/PLAP domain interaction.…”