Ribosomal p70S6K basal activity increases upon induction of differentiation of myelomonocytic leukemic cell lines HL60, AML14 and MPD

Abstract

The role of ribosomal p70S6K in the cell cycle has been studied extensively, and it is known that this enzyme is crucial for cell advancement through G 1. Conversely, the participation of p70S6K in cell differentiation is not well understood. We have studied the response of p70S6K to the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in three differentiation-capable leukemic cell lines (MPD, AML-14 and HL-60) and in normal mature neutrophils. Immature leukemic cells starved for 16 h showed a robust (∼3.5-fold over controls) p70S6K phosphorylation on T 421/S 424 residues in response to an acute (5 min) 10 nM GM-CSF stimulation. On the other hand, cells that had been induced to differentiate and express granulocytic phenotypes, showed an increased (∼6-fold) basal level of p70S6K T 421/S 424 phosphorylation over immature cells, as well as an increased baseline tyrosyl phosphorylation of the GM-CSF receptor β subunit (GM-CSF·Rβ). However, the differentiated cells displayed a weak (∼1.4-fold over controls) response to GM-CSF even at prolonged incubation times (20 min). In vitro p70S6K enzymatic activity paralleled p70S6K T 421/S 424 phosphorylation in both high basal, unstimulated, levels in immature cells and a low degree of response to GM-CSF. Lastly, peripheral blood mature neutrophils had low basal GM-CSF·Rβ and p70S6K activity, with both parameters being robustly stimulated following addition of GM-CSF, a situation in contrast with the cell lines, indicative perhaps of their incomplete terminal differentiation. In summary, these findings show the increase in basal phosphorylation of p70S6K upon granulocytic differentiation of myeloid leukemic cells and their responses to GM-CSF that are closely paralleled with tyrosyl phosphorylation of its receptor, and help in pointing to specific cell signaling molecules that are different in leukemic blasts from normal leukocytes.