Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase.
Mast cell growth factor (MGF, the ligand for c-kit receptor) can stimulate proliferation of factor dependent myeloid cell line, M07e, and MGF synergizes with granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3 in this effect. The effect of MGF on protein tyrosine kinase activity in M07e cells was investigated by immunoblotting with anti-phosphotyrosine mAb and this was compared with effects of GM-CSF. MGF stimulation rapidly induced or enhanced at least 12 tyrosine phosphorylated bands. Major bands had molecular weights of 145, 120, 110, 98, 62, 55 and 42 kD. P145, the most prominent phosphorylated protein, was identified as c-kit product using anti-c-kit-mAb (YB5.B8), suggesting ligand-dependent receptor autophosphorylation. Five of six tyrosine phosphorylated bands induced or enhanced by GM-CSF stimulation comigrated with those tyrosine phosphorylated by MGF (138, 120, 76, 55 and 42 kD). P42 was identified, at least in part, as mitogen-activated protein (MAP) kinase. MGF induced tyrosine phosphorylation of a complex of GTPase-activating protein (GAP, 120 kD) and GAP associated proteins (p62/p190) as detected by anti-GAP Ab immunoprecipitation followed by immunoblotting with anti-phosphotyrosine mAb. GM-CSF also stimulated slightly but consistently tyrosine phosphorylation of GAP and p190 but not p62. Both MGF and GM-CSF enhanced Raf-1 phosphorylation and increased Raf-1 associated kinase activity in vitro. Phosphoamino acid analysis revealed Raf-1 phosphorylation by these two growth factors occurred almost exclusively on serine residues. No tyrosine phosphorylation of Raf-1 protein was detected. These data suggest shared and unshared components of signaling pathways of both factors, which may be involved in cell proliferation.
Miyazawa
K
K
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis.
Hendrie
P C
PC
Mantel
C
C
Wood
K
K
Ashman
L K
LK
Broxmeyer
H E
HE
D001853:Bone Marrow / Q000502:physiology; D001854:Bone Marrow Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D020690:GTPase-Activating Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D007377:Interleukin-3 / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology*; D011506:Proteins / Q000378:metabolism / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D019009:Proto-Oncogene Proteins c-kit; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
D020690:GTPase-Activating Proteins; D007377:Interleukin-3; D011506:Proteins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011494:Protein Kinases; D019009:Proto-Oncogene Proteins c-kit; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases