THE SIGNAL-TRANSDUCTION PATHWAY OF ERYTHROPOIETIN INVOLVES 3 FORMS OFMITOGEN-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, granulocy te-macrophage colony-stimulating factor or erythropoietin. In these ce lls, erythropoietin stimulation led to the rapid phosphorylation of se veral proteins including the erythropoietin receptor and proteins with molecular masses around 45 kDa which could be mitogen-activated prote in (MAP) kinases. Separation of cytosol from resting or erythropoietin -stimulated UT-7 cells by anion-exchange chromatography revealed two p eaks of myelin basic protein kinase activity. The kinase activity of t he 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 correspo nded 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-regulate d kinase (ERK)-1 and ERK-2, respectively. Evidence was obtained showin g 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 eryth ropoietin stimulation and remained detectable after more than 1 hour. A role for MAP kinase activation in erythropoietin stimulated cell pro liferation was suggested by the simultaneous inhibition of erythropoie tin-induced MAP kinase stimulation and cell proliferation. The potenti al activator of MAP kinase, RAF-1, was hyperphosphorylated in erythrop oietin-stimulated cells and its autophosphorylation activity was stron gly increased. The protein adaptor She was heavily phosphorylated in U T-7 erythropoietin-stimulated cells and associated strongly with a uni dentified 145-kDa protein. However, Shc bound poorly to the activated erythropoietin receptor and most Shc proteins were cytosolic in both u nstimulated and erythropoietin-stimulated cells. In contrast, Grb2 ass ociated efficiently with the activated erythropoietin receptor and a s ignificant part of Grb2 was associated to a particulate subcellular fr action upon erythropoietin stimulation.