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, m-TOR-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 Vmax of the enzyme (from 7.2 to 20.5 pmol/min/mg) and induced a time- and dose-dependent phosphorylation on p70S6K residues Thr389 and Thr421/Ser424. The immunosuppressant macrolide rapamycin caused either a decrease in intensity of phospho-Thr389 bands in Western blots, or as a downshift in the relative mobility of phospho-Thr421/Ser424 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 Thr421/Ser424 S6K phosphorylation, and a rapamycin-sensitive, mTOR-related kinase, that targets Thr389, both of which are needed in cooperation to achieve full activation of neutrophil p70S6K.
Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Jason A
JA
Lehman
Victor
V
Calvo
Julian
J
Gomez-Cambronero
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007700:Kinetics; D020935:MAP Kinase Signaling System; D051379:Mice; D008954:Models, Biological; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D004791:Enzyme Inhibitors; D005419:Flavonoids; D007166:Immunosuppressive Agents; D013912:Threonine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011494:Protein Kinases; C546843:mTOR protein, mouse; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D000469:Alkaline Phosphatase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus