Growth Factor-Induced Phosphorylation of c-rasp21 in Normal Hemopoietic Progenitor Cells

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Abstract
Normal murine hemopoietic progenitor cells (colony-forming cells, CFC), representing 0.2% of the bone marrow cell population, were purified to homogeneity by fluorescence-activated cell sorting. CFC require the presence of the murine hemopoietic regulator, granulocyte-macrophage-colony stimulating factor (GM-CSF) for survival, proliferation, and differentiation along the myeloid pathway. An analysis of protein phosphorylation in GM-CSF-stimulated CFC over a 20-hr period demonstrated three phosphoproteins of approximate MW 21 kd and pI 6.2, 5.7 and 5.2 p21-6.2 persisted for 14 hr, while p21-5.7 and p21-5.2 were only detected during the first 5 hr of the analysis. The phosphate turnover time in all three p21 proteins was less than 3 hr and p21-5.2 contains an alkaline-resistant phosphorylation site. Low levels of p21-6.2 were also detected in unstimulated CFC. The observation of these phosphoproteins led us to investigate c-ras p21 in CFC. Immune precipitation with the anti-Ha/Ki-ras p21 monoclonal antibody (Y13-259) showed that expression of c-ras p21 in CFC was independent of GM-CSF stimulation, but that phosphorylated c-ras p21 was present only after GM-CSF stimulation. CFC contained one-tenth of the amount of phosphorylated c-ras p21 per cell compared with v-Ha-ras-transformed fibroblasts. It is possible that the phosphorylation of c-ras p21 in CFC has a significant role in the growth factor-directed molecular cascade responsible for normal hemopoietic development.