THE PROTOONCOGENE PRODUCT C-CBL BECOMES TYROSINE-PHOSPHORYLATED BY STIMULATION WITH GM-CSF OR EPO AND CONSTITUTIVELY BINDS TO THE SH3 DOMAIN OF GRB2 ASH IN HUMAN HEMATOPOIETIC-CELLS/

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythrop oietin (Epo) are hematopoietic growth factors that regulate proliferat ion and differentiation of hematopoietic cells, They elicit and contro l a cascade of biochemical events, the earliest of which is tyrosine p hosphorylation of several cellular proteins, Grb2/Ash is composed of S H2 and SH3 domains. The SH2 domain binds to tyrosine-phosphorylated pr oteins, and the SH3 domains bind to proteins containing proline-rich r egions, It is considered that Grb2/Ash functions as an adapter protein linking tyrosine kinases and Ras in downstream of receptors for growt h factors in fibroblasts, However, the mechanisms of signal transducti on through Grb2/Ash and the roles of proteins associated with Grb2/Ash remain to be determined in hematopoietic cells, By means of the bindi ng experiments using the glutathione S-transferase fusion protein incl uding the full-length Grb2/Ash, we have found that She and unidentifie d 130- and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine phosphorylated by treatment with GM-CSF or Epo in a huma n leukemia cell line, UT-7. We have purified the 130-kDa protein (pp13 0) using the glutathione S-transferase-Grb2/Ash affinity column, The a mino acid sequence analysis of the three peptides derived from the in situ protease digestion of the purified pp130 showed that the pp130 wa s identical to the human c-cbl proto-oncogene product (c-Cbl), c-Cbl c onstitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash, and the binding of Grb2/A sh to c-Cbl or Sos was not altered by GM CSF stimulation, Moreover, c- Cbl(pp130) becomes tyrosine phosphorylated rapidly and transiently dep ending on GM-CSF or Epo stimulation, These findings strongly suggest t hat c-Cbl is implicated in the signal transduction of GM-CSF or Epo in hematopoietic cells and that c-Cbl is involved in another signaling p athway different from the Ras signaling pathway.