The signal transduction pathway of erythropoietin involves three forms of mitogen-activated protein (MAP) kinase in UT7 erythroleukemia cells.
The survival and proliferation of the UT-7 human leukemic cell line is strictly dependent on the presence of either interleukin 3, granulocyte-macrophage colony-stimulating factor or erythropoietin. In these cells, erythropoietin stimulation led to the rapid phosphorylation of several proteins including the erythropoietin receptor and proteins with molecular masses around 45 kDa which could be mitogen-activated protein (MAP) kinases. Separation of cytosol from resting or erythropoietin-stimulated UT-7 cells by anion-exchange chromatography revealed two peaks of myelin basic protein kinase activity. The kinase activity of the first peak was independent of erythropoietin treatment of the cells and corresponded to an unidentified 50-kDa kinase, whereas the second peak was only present in erythropoietin-stimulated cells and corresponded to three forms of MAP kinases with molecular masses of 45, 44 and 42 kDa. The three forms were separated by hydrophobic chromatography and were shown to be activated in erythropoietin-stimulated cells. The 44-kDa and 42-kDa forms corresponded to extracellular signal-regulated kinase (ERK)-1 and ERK-2, respectively. Evidence was obtained showing that the 45-kDa form is not a shifted form of ERK-1 but corresponded to a less well defined form of MAP kinase which may be the previously described ERK-4. MAP kinase activation was detected after 1 min erythropoietin stimulation and remained detectable after more than 1 hour. A role for MAP kinase activation in erythropoietin-stimulated cell proliferation was suggested by the simultaneous inhibition of erythropoietin-induced MAP kinase stimulation and cell proliferation. The potential activator of MAP kinase, RAF-1, was hyperphosphorylated in erythropoietin-stimulated cells and its autophosphorylation activity was strongly increased. The protein adaptor Shc was heavily phosphorylated in UT-7 erythropoietin-stimulated cells and associated strongly with a unidentified 145-kDa protein. However, Shc bound poorly to the activated erythropoietin receptor and most Shc proteins were cytosolic in both unstimulated and erythropoietin-stimulated cells. In contrast, Grb2 associated efficiently with the activated erythropoietin receptor and a significant part of Grb2 was associated to a particulate subcellular fraction upon erythropoietin stimulation.
Institut Cochin de Génétique Moléculaire (ICGM), Institut de la Santé et de la Recherche Médicale (INSERM U363), Université René Descartes, Paris, France.
S
S
Gobert
V
V
Duprez
C
C
Lacombe
S
S
Gisselbrecht
P
P
Mayeux
D048868:Adaptor Proteins, Signal Transducing; D001692:Biological Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D004921:Erythropoietin / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000473:pathology; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007527:Isoenzymes; D011506:Proteins; D004921:Erythropoietin; D014443:Tyrosine; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases