Growth factor-induced phosphorylation of c-ras p21 in normal hemopoietic progenitor cells.
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.
Stanley
I J
IJ
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Victoria, Australia.
Nicola
N A
NA
Burgess
A W
AW
D000818:Animals; D003115:Colony-Stimulating Factors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology*; D006410:Hematopoiesis / Q000187:drug effects / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D011519:Proto-Oncogenes / Q000187:drug effects
D003115:Colony-Stimulating Factors; D006133:Growth Substances; D011518:Proto-Oncogene Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016283:Proto-Oncogene Proteins p21(ras)