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/
H. Odai et al., 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/, The Journal of biological chemistry, 270(18), 1995, pp. 10800-10805
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.