DISCRETE PROTEIN INTERACTIONS WITH THE GRB2 C-CBL COMPLEX IN SCF-MEDIATED AND TPO-MEDIATED MYELOID CELL-PROLIFERATION/

Hemopoietic cell proliferation is mediated by nontyrosine and tyrosine kinases that signal via uncommon and common sets of downstream effect or molecules including the Grb2/c-Chl. In the present study we evaluat ed tyrosine phosphorylation of c-Cbl and the interaction of the Grb2/c -Cbl complex with signaling proteins upon activation of non-tyrosine ( c-Mpl) and tyrosine kinase (c-Kit) receptors leading to myeloid cell p roliferation. By using the growth factor dependent M-07e cell line, we found that both c-Mpl and c-Kit ligands, namely: SCF and TPO, induce c-Cbl tyrosine phosphorylation. In these cells the adaptor protein Grb 2 constitutively binds a substantial fraction of c-Cbl through the N-t erminal SH3 domain. In vitro experiments showed that the stable Grb2/c -Cbl complex interacts, through the Grb2 SH2 domain, with the SCF-acti vated c-Kit. By contrast stimulation with TPO leads to the formation o f a Grb2 complex containing JAK2. In vitro and in vivo experiments sup port the hypothesis that Grb2 mediates the association of c-Kit with c -Cbl. Moreover we found that, upon SCF stimulation, the Grb2/c-Cbl com plex recruits She, probably via Grb2. By contrast the Ras exchanger fa ctor (Sos1) was not detected in anti-c-Cbl immunoprecipitates suggesti ng that Grb2/Sos1 and Grb2/c-Cbl are present in different complexes. T aken together our results demonstrate that c-Cbl plays an important ro le in coupling both tyrosine and non-tyrosine kinase receptors to down stream effector molecules and that different signaling molecules inter act with Grb2/c-Cbl complex when non-tyrosine or tyrosine kinase recep tors are activated.