CD16-MEDIATED P21(RAS) ACTIVATION IS ASSOCIATED WITH SHC AND P36 TYROSINE PHOSPHORYLATION AND THEIR BINDING WITH GRB2 IN HUMAN NATURAL-KILLER-CELLS

The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the onc oprotein She have been implicated in a highly conserved mechanism that regulates p21(ras) activation. We investigated the involvement of the se adaptor proteins in the signaling pathway induced by CD16 or interl eukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphoryla ted upon CD 16 or IL-2 stimulation with different kinetics. Shc immuno precipitates from lysates of CD16- or IL-2-stimulated NK cells contain ed Grb2 and an unidentifed 145-kD tyrosine phosphoprotein. Grb2 immuno precipitates from anti-CD16-stimulated NK cells contained not only Shc , but also a 36-kD tyrosine phosphoprotein (p36). The interaction betw een Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated b y in vitro binding assays using a bacteriologically synthesized glutat hione S-transferase-Grb2SH2 fusion protein. We also present evidence t hat p21(ras) is activated by CD16 and IL-2R cross-linking. Accumulatio n of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphor ylation and Grb2 association of Shc and p36 adaptor proteins.