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

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine. Its pleiotropic biological properties are signaled through two distinct cell su rface receptors: the TNF receptor type I (TNFR-I) and the TNF receptor type II. Neither of the two receptors possesses tyrosine kinase activity. A lar ge majority of TNF-alpha-dependent activities can be mediated by TNFR-I. Re cently, c-Raf-1 kinase was identified as an intracellular target of a signa l 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 p artner of TNFR-I. Grb2 binds with its COOH-terminal SH3 domain to a FLAP mo tif within TNFR-I and with its NH2-terminal SH3 domain to SOS (son of seven less). A FLAP 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 coexpr ession of Grb2 mutants harboring inactivating point mutations in the NH2- o r COOH-terminal SH3 domain, cell-permeable peptides that disrupt the Grb2/T NFR-I interaction or transdominant negative Ras. Functionality of the TNFR- I/Grb2/SOS/Ras interaction is a prerequisite but not sufficient for TNF-alp ha-dependent activation of c-Raf-1 kinase. Inhibition of the TNFR-I/FAN (fa ctor associated with neutral. sphingomyelinase) interaction, which is essen tial for TNF-alpha-dependent activation of the neutral sphingomyelinase, ei ther by cell-permeable peptides or by deletion of the the FAN binding domai n, prevents activation of c-Raf-1 kinase. In conclusion, binding of the Grb 2 adapter protein via its COOH-terminal SH3 domain to the nontyrosine kinas e 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.