The signal transduction through Grb2/Ash in hematopoietic cells

Leukemia. 1997 Apr:11 Suppl 3:405-7.

Abstract

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.

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Cell Line
  • Chromatography, Affinity
  • ErbB Receptors / physiology
  • Erythropoietin / pharmacology*
  • GRB2 Adaptor Protein
  • Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology*
  • Hematopoiesis
  • Humans
  • Leukemia
  • Proteins / physiology*
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins / biosynthesis*
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / isolation & purification
  • Proto-Oncogene Proteins c-cbl
  • Proto-Oncogenes
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction*
  • Ubiquitin-Protein Ligases*
  • src Homology Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • MAS1 protein, human
  • Proteins
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Erythropoietin
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Proto-Oncogene Proteins c-cbl
  • Ubiquitin-Protein Ligases
  • ErbB Receptors
  • CBL protein, human