Mechanism of ribosomal p70S6 kinase activation by granulocyte macrophage colony-stimulating factor in neutrophils -: Cooperation of a MEK-related, THR421/SER424 kinase and a rapamycin-sensitive, mTOR-related THR389 kinase

We report here for the first time the detection of the ribosomal p70S6 kinase (p70S6K) in a hematopoietic cell, the neutrophil, and the stimulation of its enzymatic activity by granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF modified the V-max of the enzyme (from 7.2 to 20.5 pmol/min/mg) and induced a time- and dose-dependent phosphorylation on p70S6K residues Thr(389) and Thr(421)/Ser(424). The immunosuppressant macrolide rapamycin caused either a decrease in intensity of phospho-Thr(389) bands in Western blots, or as a downshift in the relative mobility of phospho-Thr(421)/ Ser(424) bands ( consistent with the loss of phosphate), but not both simultaneously. The immunosuppressant FK506 failed to inhibit p70S6K activation, but was able to rescue the rapamycin-induced downshift, pointing to a role for the mammalian target of rapamycin ( mTOR) kinase. Rapamycin also caused an inhibition (IC50 0.2 nM) of the in vitro enzymatic activity of p70S6K. However, the inhibition of activity was not complete, but only a 40 - 50%, indicating that neutrophil p70S6K activity has a rapamycin-resistant component. This component was totally inhibited by pre-incubating the cells with the mitogen-activated protein kinase ( MAPK) kinase (MEK) inhibitor PD-98059 prior to treatment with rapamycin. This indicated that a kinase from the MEK/ MAPK pathway also plays a role in p70S6K activation. Thus, GM-CSF causes the dual activation of a rapamycin-resistant, MAPK-related kinase, that targets Thr(421)/ Ser(424) S6K phosphorylation, and a rapamycin-sensitive, mTOR-related kinase, that targets Thr(389), both of which are needed in cooperation to achieve full activation of neutrophil p70S6K.