Tyrosine Phosphorylation of the c-cbl Proto-oncogene Protein Product and Association with Epidermal Growth Factor (EGF) Receptor upon EGF Stimulation

Abstract

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

D-Cbl, the Drosophila homologue of the c-Cbl proto-oncogene, interacts with the Drosophila EGF receptor in vivo, despite lacking C-terminal adaptor binding sites

“…Association of c-Cbl with EGFR is dependent on receptor phosphorylation and is thought to be mediated by both direct binding of the c-Cbl PTB domain and via Grb2 as an adaptor molecule (Bowtell and Langdon, 1995;Galisteo et al, 1995). Tyrosine phosphorylation of c-Cbl is also seen within minutes of EGF stimulation of serum starved cells.…”

“…Homologues of c-cbl were subsequently identi®ed in humans (Blake et al, 1991) and Caenorhabditis elegans (Yoon et al, 1995) and both humans and mice have been recently shown to have a second functional cbl family member, cbl-B (Keane et al, 1995). c-Cbl has been shown to directly associate with the EGF receptor, and is phosphorylated in an EGF receptor dependent manner (Bowtell and Langdon, 1995;Fukazawa et al, 1995;Galisteo et al, 1995;Meisner et al, 1995;Odai et al, 1995;Solto and Cantley, 1996;Tanaka et al, 1995). c-Cbl has recently been linked to signalling by several di erent cytokine receptors including the Fcg receptor (Matsuo et al, 1996), B-cell receptor (Cory et al, 1995;Panchamoorthy et al, 1996), T-cell receptor (Donovan et al, 1994(Donovan et al, , 1996Lupher et al, 1996;Reedquist et al, 1996), and c-Kit receptor (Wisniewski et al, 1996).…”

Specific association of phosphatidylinositol 3‐kinase with the protooncogene product Cbl in Fcγ receptor signaling

“…The phosphorylation of most of these proteins was observed within 15 s after stimulation and peaked at 1 min and then gradually decreased up to 16 min. Several lines of evidence have raised the possibility that tyrosine phosphorylation of Cbl might be involved in immune recognition systems in hematopoietic cells [11][12][13][14][15][16][17]. To test the tyrosine phosphorylation of Cbl upon FcvR stimulation, whole cell lysates were immunoprecipitated with anti-phosphotyrosine antibodies (PY20) and analyzed by blotting with anti-Cbl antibodies.…”

“…Although relatively few tyrosine phosphorylated peptides have as yet been identified, a protooncogene product, Cbl, has recently attracted attention among them [5]. Cbl is tyrosine phosphorylated upon stimulation of T-cell receptor, B-cell receptor and some cytokine receptors as well [11][12][13][14][15][16][17]. Some Src family tyrosine kinases are reported to be associated with Cbl in hematopoietic cells [5,12,15].…”

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

“…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. However, its implication is not restricted to pathways controlled by non-receptor tyrosine kinases, since c-Cbl was recently shown to be also phosphorylated by Epidermal Growth Factor Receptor (EGFR) (Birge et al, 1992) and platelet derived growth-factor receptor (PDGF-R) (Galisteo et al, 1995). Consistent with the structure of c-Cbl, it has been shown that c-Cbl could bind to transducing molecules such as Lck, Fyn (Reedquist et al, 1994), Syk, the p85 subunit of the PI 3-kinase, Shc , Nck (Rivero-Lezcano et al, 1994), Zap-70 (Fournel et al, 1996), Grb2 (Motto et al, 1994), Crk-II and the guanine nucleotide exchange factor C3G .…”