AbstractBACKGROUND AND OBJECTIVES: B1647 is a cell line derived from bone marrow cells of a patient with acute myeloid leukemia (M2) with a complete erythro-megakaryocytic phenotype and bears both k and p isoforms of c-mpl. Interestingly, spontaneous B1647 cell proliferation is significantly potentiated by thrombopoietin (TPO). DESIGN AND METHODS: We aimed to evaluate the proliferative signal transduction events following the activation of c-mpl and we stimulated B1647 cells with TPO 40 ng/mL for 3, 7, 15 and 30 minutes; cells were then lysed and whole lysates were immunoprecipitated with anti-phosphotyrosine antibodies. RESULTS: In our hands, TPO stimulation induced phosphorylation of several substrate proteins in B1647 cells. The increase in tyrosine phosphorylation from background spontaneous activation was transient, maximal after 10 minutes and declined to reach constitutive levels after 30 minutes. In particular, protein substrates between 50 and 140 kDa appeared to be selectively phosphorylated by TPO. We demonstrated that Jak2, Stat3 and Shc were activated in B1647 cells after TPO, as already shown for different cell lines by other authors. Moreover, Lyn kinase activation was detected. Grb2 co-immunoprecipitated with phosphorylated proteins. The phosphorylation of Syk kinase was not demonstrated, whereas Vav was activated by TPO. INTERPRETATION AND CONCLUSIONS: The pattern of protein phosphorylation determined in B1647 cells by TPO testifies the role of this cytokine in sustaining cell growth and indicates Lyn tyrosine kinase as a possible target protein in transduction of the TPO proliferative signal.
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Vol. 87 No. 12 (2002): December, 2002 : Articles
Ferrata Storti Foundation, Pavia, Italy
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