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.
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 affected. 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.
Lennartsson
J
J
The Ludwig Institute for Cancer Research, Biomedical Centre, S-751 24 Uppsala, Sweden.
Blume-Jensen
P
P
Hermanson
M
M
Pontén
E
E
Carlberg
M
M
Rönnstrand
L
L
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001011:Aorta / Q000166:cytology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D016762:Genes, fos; D006801:Humans; D008024:Ligands; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000502:physiology*; D019009:Proto-Oncogene Proteins c-kit / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019089:Stem Cell Factor / Q000494:pharmacology / Q000502:physiology; D013552:Swine; D019061:src-Family Kinases / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D008024:Ligands; D011506:Proteins; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019089:Stem Cell Factor; D019000:Phosphotyrosine; D019009:Proto-Oncogene Proteins c-kit; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)