Analysis of cell proliferation by GM-CSF

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GM-CSF regulates proliferation and differentiation of various hematopoietic cells. We have been analyzing signal transduction of human GM-CSF rcceptor and found that there are multiple distinct signaling pathways through human GM-CSF receptor. By using mutant receptors, we found that we can activate or inactivate certain signaling pathway. In this study, we tried to analyze signals for cell proliferation using the mutant hGM-CSF receptors which can activate proliferation without activation of MAPK cascade. We clones several genes by subtraction but we found difficulty to reveal function of these new genes since the cloning methods was non-functional. To clone and analyze genes by biochemical methods, we tried to establish in vitro reconstitution system of signaling pathway by fractionated cell lysates. We succeeded to make an in vitro system of STAT5 activation from hematopoietic cell lysate, but failed to apply this system to c-myc transcriptional induction. We cloned hGM-CSSF receptor binding proteins by pull-down analysis, and analyzed function of these cloned genes by STAT5 in vitro system.