The signal transduction through Grb2/Ash in hematopoietic cells.
Grb2/Ash is composed of one SH2 and two SH3 domains and functions as an adapter linking tyrosine-kinase receptors and Ras in fibroblasts. The SH2 domain binds to tyrosine-phosphorylated proteins and the SH3 domain binds to protein containing proline-rich regions. However, the mechanisms of signal transduction through Grb2/Ash in hematopoietic cells are still unclear. By means of the binding experiments using the GST fusion protein including the full length Grb2/Ash, we have found that Shc and unidentified 130-kDa and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine-phosphorylated by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) in a human leukemia cell line UT-7. We have purified the 130-kDa protein (pp 130) using GST-GRB2/Ash affinity column. The amino-acid sequence analysis showed that the pp130 was identical to the human c-cbl proto-oncogene product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash. Moreover, c-Cbl (pp 130) becomes tyrosine-phosphorylated rapidly and transiently depending on GM-CSF and EPO stimulation. However, we could not find the homologous regions with guanine nucleotide exchange factors or GTPase-activating proteins in the c-cbl gene. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF and EPO in hematopoietic cells, and c-Cbl and Grb2/Ash might also transduce a signal that is different from the signal leading to Ras regulation. Recently, we have shown that the proto-oncogene vav product (Vav) is also tyrosine-phosphorylated by treatment with GM-CSF and EPO and is constitutively associated with the SH3 domain of Grb2/Ash in UT-7. Another guanine nucleotide exchange factor Sos is also associated with Grb2/Ash in UT-7. It has been reported that Vav has guanine nucleotide exchange activity and activates Ras in vitro and in vivo. These data suggest that tyrosine kinases, the adapter Grb2/Ash, and the guanine nucleotide exchange factor Vav and Sos are members of a signaling pathway leading to Ras activation in hematopoietic cells.
Odai
H
H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Hanazono
Y
Y
Sasaki
K
K
Iwamatu
A
A
Yazaki
Y
Y
Hirai
H
H
D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D002846:Chromatography, Affinity; D066246:ErbB Receptors / Q000502:physiology; D004921:Erythropoietin / Q000494:pharmacology*; D051380:GRB2 Adaptor Protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006410:Hematopoiesis; D006801:Humans; D007938:Leukemia; D011506:Proteins / Q000502:physiology*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D050721:Proto-Oncogene Proteins c-cbl; D011519:Proto-Oncogenes; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C000715470:MAS1 protein, human; D011506:Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; C067419:CBL protein, human