Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

Abstract

In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less a ected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.

Tyrosine-317 of p52Shc mediates androgen-stimulated proliferation signals in human prostate cancer cells

“…Bars denote the range of duplicates p52 Shc in androgen-stimulated proliferation of prostate cancer cells M-S Lee et al the observations that Shc proteins mediate signals from NDF-activated ErbB-2 in human prostate cancer cells (Grasso et al, 1997). Alternatively, DHT-AR complexes activate c-Src activity by which p52 Shc is phosphorylated (Lennartsson et al, 1999;Migliaccio et al, 2000). However, this may not be the case since the activation of c-Src by DHT-AR complexes after 10 nM DHT treatment is a rapid and transient event within 6 h (Migliaccio et al, 2000; MS Lee, TC Yuan and MF Lin, unpublished observations), while 10 nM DHT effect on Y317 phosphorylation of p52 Shc is observed at 48 h after treatment (Figure 4b).…”

Effects of an EGFR-binding affibody molecule on intracellular signaling pathways

“…Antibodies recognizing phosphorylated Erk (Thr 202/Tyr 204) or Akt (Ser 473) were from Cell Signaling Technology (Beverly, MA, USA). The reagents applied have previously been used and described (20).…”

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

“…Cells were stimulated with 100ng/mL FL (Prospec Tany, Rehovot, Israel) for the indicated periods of time at 37°C, washed once with cold PBS and lysed. Lysates were subjected to immunoprecipitation and Western blotting as described elsewhere [23,25]. Immunodetection was performed by enhanced chemoluminescence using Immobilon Western chemoluminescent HRP substrate (Millipore Corporation, USA) and a CCD camera (LAS-3000, Fujifilm, Tokyo, Japan).…”