Authors
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 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. 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 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. p70S6K 1The abbreviations used are: p70S6K, ribosomal p70-S6 kinase; GM-CSF, granulocyte macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; EPO, erythropoietin; IL-8, interleukin-8; PI3K, phosphatidylinositol 3-kinase; PDK1, 3-phosphoinositide-dependent protein kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MEKi, MEK inhibitor PD-98059; mTOR, mammalian target of rapamycin; FKBP, FK506-binding protein; FRAP, FKBP12-rapamycin-associated protein; PP2B, protein phosphatase 2B. catalyzes the phosphorylation of the S6 protein (1Nemenoff R.A. Price D.J. Mendelsohn M.J. Carter E.A. Avruch J. J. Biol. 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A. 1998; 95: 1432-1437Crossref PubMed Scopus (947) Google Scholar, 13Saitoh M. Pullen N. Brennan P. Cantrell D. Dennis P.B. Thomas G. J. Biol. Chem. 2002; 227: 20104-20112Abstract Full Text Full Text PDF Scopus (144) Google Scholar). In addition, a “module IV” containing Ser411, Ser418, Thr421, and Ser424 are phosphorylated in tandem prior to full activation of the enzyme (3Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (487) Google Scholar). Phosphorylation of Thr412 and Ser371 plays a central role in the regulation of p70S6K activity in vivo (13Saitoh M. Pullen N. Brennan P. Cantrell D. Dennis P.B. Thomas G. J. Biol. Chem. 2002; 227: 20104-20112Abstract Full Text Full Text PDF Scopus (144) Google Scholar, 14Weng Q.P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). In the living cell, ribosomal p70S6K is activated through a complex network of signaling molecules (15Avruch J. Belham C. Weng Q. Hara K. Yonezawa K. Prog Mol. Cell. Biol. 2001; 26: 115-154Google Scholar, 16Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (608) Google Scholar). The generation of 3-phosphoinositide lipid products by PI3K is required for the phosphorylation of two activating sites in p70S6K: Thr229 and Thr389 (17Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 18Balendran A. Currie R. Armstrong C.G. Avruch J. Alessi D.R. J. Biol. Chem. 1999; 274: 37400-37406Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Thr229 is phosphorylated by PDK1 (19Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, N. Dennis P.B. M. A. Kozma S.C. 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GM-CSF has a of on and that the activation of two signaling the and in J. C. Sci. 1996; PubMed Scopus Google Scholar). of to the cell and phosphorylation of the ribosomal kinase is central in has that G-CSF, a hematopoietic factor to GM-CSF, and cell 95: PubMed Google Scholar), activation of of cell signaling p70S6K or GM-CSF its cell through We that MAPK activation in to GM-CSF is in cells as the neutrophil and plays a role in J. J. Cell 2001; PubMed Google Scholar), and that a the MAPK and the p70S6K J. Biochem. Res. 2002; PubMed Scopus Google Scholar). report that p70S6K is in that GM-CSF causes an in phosphorylation of Thr389 and Thr421/Ser424 to an in its enzymatic We also show for the first time that the by which GM-CSF ribosomal S6K is through a of activation of two signaling and and was from was from was from the kinase inhibitor kinase used for and from from was from from was from PD-98059 was from FK506 and phosphatase from from was from kinase used for was from kinase and kinase from Cell p70S6 kinase and cell from on a by and D. J. PubMed Scopus Google Scholar). of from the of an as was with of to and the and and for The was in of and for in a and by and the neutrophil was in has indicated that neutrophil the for neutrophil not is as the of in or in the time of the and used and Western was on report J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) with as in and with or and with GM-CSF and in 0.2 of in in a for with to to an and with and of 0.2 of and and The of cell to and used for the was a of with to in for The and with cell as indicated a a by with with and with complex in a of with and with for protein and used for In with the used for to that protein was by protein in by and that the in protein not for in phosphorylation with the p70S6 p70S6K enzymatic activity was by an kinase as J. J. Cell 2001; PubMed Google Scholar, J. Biochem. Res. 2002; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) to kinase activity in in and with or and with GM-CSF and in of for and on for with in with the to as indicated complex in a of 40 with of and used in an in vitro kinase the for was of the S6 kinase in kinase kinase inhibitor and of kinase inhibitor of to the of of from the of kinase containing the with the cell or The was for in a and by 40 of the and for in with S6 kinase from In used as the p70S6K the in vitro kinase S6 is of a complex that is on residues in vitro in to 40 S ribosomal from the of the in 11Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (590) Google as indicated in the and Western with of and The phosphatase enzyme was in a of of phosphatase and the of to to achieve a of and for with The was by the on an and to a of in to the to used for as indicated by with as G. A. A. 2001; PubMed Scopus Google Scholar) with was in vivo by the of GM-CSF to a neutrophil cell for and with of of of and of in for that and for in a in of and in the for as and in of by on a was Cell and as p70S6K and GM-CSF and Phosphorylation here that protein in to by the ribosomal p70S6K the for the mammalian S6K1 We also able to its enzymatic activity in vitro in cell and in The detection of p70S6K activity is in with GM-CSF a and of S6K of ribosomal p70S6K activity is by the that the and ribosomal 40 S the protein was GM-CSF activity by in The of p70S6K in is in activation to GM-CSF is activity is as a of protein and is a in V max (from 7.2 to 20.5 and also in the (from to by GM-CSF stimulation that GM-CSF a p70S6K in of neutrophil p70S6K. with GM-CSF and as indicated The kinase was the was as a of time of the in vitro enzyme protein and protein a was from which the V max and activity and GM-CSF was able to the phosphorylation of p70S6K in in GM-CSF a phosphorylation of p70S6K on Thr389, which is of the residues critical for S6K activation. The of the phosphorylated in with that of the GM-CSF phosphorylation of p70S6K on two Thr421/Ser424 that are also for enzyme The is with GM-CSF as as that the in both phosphorylation and enzyme activity by GM-CSF are phosphorylation is for phospho-Thr421/Ser424 as as for phospho-Thr389 Phosphorylation of Thr389 is as an in of the The Thr421/Ser424 dual phosphorylation is GM-CSF, and is by both a in of the and by an mobility The also of with and with the to In vitro kinase activity also a to GM-CSF activity is and and as the of phosphorylation in and with enzymatic Thus, the in that p70S6K is in that GM-CSF its enzymatic activity in a time and dose-dependent the V max for its and that phosphorylation in Thr389, Thr421, and in p70S6K in of the that for the in both phosphorylation and first was the of the immunosuppressant a inhibitor of mTOR, of the of p70S6K (17Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In rapamycin inhibited p70S6K Thr389 phosphorylation in a The intensity of phospho-Thr389 but The of rapamycin on the dual phosphorylation Thr421/Ser424 was also but and that rapamycin causes a mobility in the phospho-Thr421/Ser424 in the of In for the of the in GM-CSF rapamycin as a and the in GM-CSF as from the to a with rapamycin is with p70S6K as a phosphorylated in in the is a Western of to relative mobility The in has a to that of also in to was with phospho-Thr389 the intensity with rapamycin GM-CSF a is to indicating that phosphorylation of Thr421/Ser424 was not by rapamycin. the to Thr421/Ser424 was not rapamycin to inhibit the of dual phosphorylation in to GM-CSF with a of rapamycin that of rapamycin as as the mobility of p70S6K, and to the was protein in the as in but the was not a of Thus, rapamycin causes a in mobility of p70S6K but was a rapamycin-resistant component of inhibition of p70S6K by rapamycin is not was as a of p70S6K activity in cell D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (590) Google Scholar, C.J. J. Blenis J. 1992; PubMed Scopus Google Scholar, J. Kuo C.J. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar), rapamycin on kinase activity and rapamycin has D. Chen J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, E. Chen J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). a of rapamycin inhibition and p70S6K activity in S. G. Fumagalli S. Pende M. Kozma S.C. Thomas G. C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). an inhibitor of a S6K to the of rapamycin on We needed to that the mobility downshift from to a is to the of phosphorylated the control neutrophil with of phosphatase in used the to p70S6K and in a to that in the that as the phosphatase for the phosphorylated p70S6K from the phosphatase to the a This is to the cells and We that a of the in with rapamycin in to the loss of the of phosphatase used in in to the downshift, a to the most to the that phosphatase the residues of a target of the The that rapamycin has a on the phosphorylation of p70S6K induced by GM-CSF to a of in the of signaling of macrolide immunosuppressant to rapamycin. FK506 is a of the FK506-binding but its by different from rapamycin J. Kuo C.J. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). The is mTOR-related FK506 not the of phosphorylation of p70S6K downshift and the inhibition of p70S6K phosphorylation caused by rapamycin was by of cells with FK506 The phosphorylated a to the mobility of the This is to that used by in of (17Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, J. Kuo C.J. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). FK506 which is also the for a the two It has that FK506 the induced by rapamycin (17Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, J. Kuo C.J. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, B.E. G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R.B. Dennis P.B. J.W. N.A. Kozma S.C. Thomas G. EMBO J. 1995; PubMed Scopus Google Scholar, M.J. J. Google Scholar). MAPK the in p70S6K in rapamycin not p70S6K, in the in vitro enzymatic activity of p70S6K by rapamycin. in of cells with rapamycin prior to with GM-CSF caused an inhibition of enzymatic activity with an of 0.2 However, inhibition was only of the of as as not that neutrophil p70S6K to phosphorylation, has a rapamycin-resistant component. the regulation of component on the from that is a the MAPK and the p70S6K pathway J. Biochem. Res. 2002; PubMed Scopus Google Scholar), as as on Price D.J. S. J. Avruch J. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar) indicated that a of sites in the p70S6K phosphorylated in vitro by as or We the MEK/MAPK pathway for the rapamycin-resistant component of S6K activation. in the MEK inhibitor PD-98059 also caused a inhibition of p70S6K However, cells with a of PD-98059 and rapamycin prior to stimulation with GM-CSF, the loss of activity was complete, with to that on the and the are an of the of the rapamycin MEKi, by in show that the of rapamycin and on the of to of in to p70S6K in a in to show that not of rapamycin with the inhibition of p70S6K, a of rapamycin and in only not the of rapamycin vitro kinase activity of is not inhibited with not that the of phosphorylation, a of rapamycin and a downshift in the and to the with and with phospho-Thr389 to that with the in vitro treatment with phosphatase of that a of It that the is the used Thr389, which to the of the full of the rapamycin We report here for the first time the of p70S6K in activity and and its by the hematopoietic growth factor The in in of the that cells are is not the kinase that is to in is and by of a In also the of p70S6K activation as two cell signaling Phosphorylation of either Thr389 or Thr421/Ser424 on p70S6K is but not to achieve full activation. to during cell as is the first full report p70S6K in for that in a hematopoietic cell, its and its of regulation with The enzyme for to 20.5 of is in with in the for of and cell cells with and and D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (590) Google Scholar, D.J. R.A. Avruch J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google Scholar, Thomas G. 1991; PubMed Scopus Google Scholar, M. Thomas G. PubMed Scopus Google Scholar). the enzyme to activity in the D.J. R.A. Avruch J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Thomas G. 1991; PubMed Scopus Google Scholar, M. Thomas G. PubMed Scopus Google Scholar). are two that the of p70S6K in either cells or In a of the role of in of of T. T. E. T. T. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) that p70S6K p70S6K is also activated on the of phosphorylation in S6K J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, since p70S6K is not in rapamycin the the PI3K, as a pathway of and regulation in p70S6K in in a of enzyme activity and of the phosphorylation of residues and with with and with an also p70S6K that the kinase. Phosphorylation of 40 S ribosomal the S6 as in also to the p70S6K activity in the of p70S6K regulation by GM-CSF in the first being the of rapamycin. We that inhibit Thr389 phosphorylation as by R.B. Dennis P.B. J.W. N.A. Kozma S.C. Thomas G. EMBO J. 1995; PubMed Scopus Google Scholar). that a of Thr389 to p70S6K and the enzyme to activated by or growth However, an kinase the here in activated by GM-CSF in the of since residues for phosphorylation in the the Thr389 to an and the in cells a activity and was able to activated by but only to a of the R.B. Dennis P.B. J.W. N.A. Kozma S.C. Thomas G. 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