Abstract
Acute myeloid leukemia (AML) is characterised by defects in the regulation of hematopoietic cell survival, differentiation and proliferation. IL-3 and GM-CSF exert potent survival and proliferative effects on a range of myeloid progenitor cells. Previously we have identified a novel cytoplasmic motif of the human GM-CSF/IL-3 receptor beta chain responsible for coordinating survival and proliferative responses in primary hematopoietic cells. The motif contains a serine residue (585) that is phosphorylated in response to cytokine and couples to the PI3K/AKT pathway via the 14-3-3 adaptor protein and is required for cell survival (Guthridge et al,
Molecular Cell 6:99, 2000
;Blood 103:820, 2004
). An adjacent tyrosine residue (577) within the motif is phosphorylated via Jak2 and associates with proliferative function. We investigated the phosphorylation status of the beta chain motif in response to GM-CSF stimulation in blasts cells from 12 patients with AML, and in blood monocytes from 6 healthy volunteers. Leukemic samples consisted of >99% blasts after buffy coat separation according to cytospin morphology. All leukemic blasts and blood monocyte samples expressed alpha and beta chain by flow cytometry. The phosphorylation status of residues 577 and 585 were examined utilising beta chain phospho-specific antibodies, after stimulation of AML blasts or blood monocytes with GM-CSF and beta chain immunoprecipitation. Human monocyte preparations all showed inducible serine 585 phosphorylation and tyrosine 577 phosphorylation. In contrast AML blasts displayed strong constitutive serine 585 phosphorylation in 10 out of 12 patients. No discernible tyrosine 577 phosphorylation in the absence of cytokine was observed for the AML samples. Beta chain phosphorylation pattern was independent of FAB classification. Inhibition of Jak2 did not prevent serine 585 phosphorylation of the receptor. Constitutive Akt activation was observed in AML blasts. The data provides evidence for dysregulation of a novel GM-CSF/IL-3 receptor mediated survival pathway involving serine/threonine kinases, 14-3-3 and Akt that is active in primary AML blasts in the majority of patients. This data would suggest that dysregulated activation of serine/threonine kinases and/or phosphatases at the receptor level may contribute to leukemogenesis.Author notes
Corresponding author
2005, The American Society of Hematology
2005
