The Proto-oncogene 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

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) are hematopoietic growth factors that regulate proliferation and differentiation of hematopoietic cells. They elicit and control a cascade of biochemical events, the earliest of which is tyrosine phosphorylation of several cellular proteins. Grb2/Ash is composed of SH2 and SH3 domains. The SH2 domain binds to tyrosine-phosphorylated proteins, and the SH3 domains bind to proteins containing proline-rich regions. It is considered that Grb2/Ash functions as an adapter protein linking tyrosine kinases and Ras in downstream of receptors for growth factors in fibroblasts. However, the mechanisms of signal transduction through Grb2/Ash and the roles of proteins associated with Grb2/Ash remain to be determined in hematopoietic cells. By means of the binding experiments using the glutathione S-transferase fusion protein including the full-length Grb2/Ash, we have found that Shc and unidentified 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 human leukemia cell line, UT-7. We have purified the 130-kDa protein (pp130) using the glutathione S-transferase-Grb2/Ash affinity column. The amino acid sequence analysis of the three peptides derived from the in situ protease digestion of the purified pp130 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, and the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation. Moreover, c-Cbl (pp130) becomes tyrosine phosphorylated rapidly and transiently depending on GM-CSF or Epo stimulation. These findings strongly suggest that 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 pathway different from the Ras signaling pathway.

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2

“…In this regard, Grb2 appeared as a good candidate able to connect the multimolecular complex nucleated by c-Cbl, since (1) Grb2 and c-Cbl have been shown to form stable complexes in HL-60 and Jurkat cells, via the Nterminal SH3 domain of Grb2 and the proline-rich motif of the C-terminus of c-Cbl (Donovan et al, 1994;Rivero-Lezcano et al, 1994) and (2) human CSF-1R harbors a docking motif for Grb2 SH2 domain on Tyr 699 (Tyr 697 for murine) (Van Der Geer and Hunter, 1993). In fact, when endogenous Grb2 was immunoprecipitated from c-FmsCHO cells, a signi®cant amount of c-Cbl was recruited in the pellet, even in the absence of CSF-1, indicating that Grb2 was constitutively associated with c-Cbl, in agreement with a previous report (Odai et al, 1995). CSF-1R was also found to interact with Grb2 only in the presence of CSF-1, thus supporting the idea that Grb2 could connect c-Cbl to the autophosphorylated CSF-1R.…”

“…In as much as (1) the human CSF-1R harbors the consensus sequence responsible for the docking of the Grb2-SH2 domain on Tyr 699 (Van Der Greer and Hunter, 1993), and (2) it has been shown that the SH3 domain of Grb2 associated constitutively to the prolinerich region of c-Cbl (Odai et al, 1995), we considered the possibility that Grb2 might bridge the CSF-1R and the cCbl/Crk-II complex. Lysates from unstimulated or CSF-1-stimulated c-FmsCHO cells, untreated or boiled in 1% SDS were diluted tenfold in LB then precipitated with GST-Crk-SH2 and ®nally reprobed with anti-Grb2 antibodies.…”

“…Recently, c-Cbl has been reported to be highly phosphorylated upon stimulation through T cell receptor (TCR) (Donovan et al, 1994), B cell receptor (BCR) , Fcg receptor , granulocyte macrophage-colony stimulating factor receptor (GM-CSFR) and erythroprotein receptor (EPOR) (Odai et al, 1995). These data strongly suggest that c-Cbl is likely to play a major role in the events occurring downstream of hematopoietic receptors coupled to c-Src-like kinases.…”

The phosphatidylinositol 3′ kinase pathway is required for the survival signal of leukocyte tyrosine kinase

“…Previously we have shown that Shc is a substrate of LTK, which binds to the NPXY sequence at the carboxyl terminal domain (amino acids 859 ± 862) and connects Grb2 and LTK (Ueno et al, 1995). Because c-Cbl is known to associate with Shc and Grb2 (Odai et al, 1995;Fukazawa et al, 1996;Panchamoorthy et al, 1996), we hypothesized that tyrosine 862 is critical for c-Cbl phosphorylation. To test this, we examined if several mutant EL receptors can phosphorylate c-Cbl.…”

Tyrosine kinase activity of the EGF receptor is enhanced by the expression of oncogenic 70Z-Cbl

“…Cbl lacks a de®ned catalytic domain but is rapidly phosphorylated on tyrosine residues following the stimulation of a wide range of cell surface receptors which include growth factor receptors, immunoglobulin receptors, antigen receptors and integrin receptors (Donovan et al, 1994;Tanaka et al, 1995;Galisteo et al, 1995;Bowtell and Langdon, 1995;Odai et al, 1995;Marcilla et al, 1995;Wang et al, 1996;Cory et al, 1995;Kontani et al, 1996;Ojaniemi et al, 1997). Indeed in many cell types Cbl appears to be one of the most prominent and rapidly phosphorylated substrates of protein tyrosine kinases (Andoniou et al, 1994;Donovan et al, 1994;.…”