scholarly journals Granulocyte-macrophage colony-stimulating factor induces a staurosporine inhibitable tyrosine phosphorylation of unique neutrophil proteins

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2446-2454
Author(s):  
RL Berkow
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Triton X ◽  
Stimulating Factor

Human neutrophils treated with chemotactic peptides or phorbol esters demonstrate tyrosine phosphorylation of a subset of proteins. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced a time- and concentration-dependent increase in the tyrosine phosphorylation of at least seven proteins. Three of these proteins with approximate molecular weights of 150, 95, and 70 Kd were unique to neutrophils treated with GM-CSF, and were not seen to be phosphorylated on tyrosine in neutrophils treated with the agonists FMLP or PMA, or the cytokines G-CSF and tumor necrosis factor. We found the 150-Kd protein to be localized within the cell particulate fraction and the 95- Kd protein within the cell cytosol. The 70-Kd phosphotyrosine protein was found in both fractions. When the neutrophils were treated with Triton X-100 (Sigma Chemical Co, St Louis, MO) to evaluate cytoskeletal associations of proteins, the 150 phosphotyrosine protein partitioned with the Triton X-100 insoluble cytoskeleton (TICS), and the 70-Kd protein partitioned with both the TICS and Triton X-100 soluble proteins. The GM-CSF-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor ST638. This was not seen with the putative C-kinase inhibitor, H-7. However, staurosporine was seen to inhibit tyrosine phosphorylation of neutrophil proteins by GM-CSF and in vitro tyrosine kinase activity of isolated neutrophil cytosol and particulate fractions. These data indicate that the three unique GM-CSF- induced phosphotyrosine-containing proteins may be responsible for the unique actions of GM-CSF and that staurosporine inhibits a tyrosine kinase responsible for the phosphorylation of these proteins.

scholarly journals Granulocyte-macrophage colony-stimulating factor induces a staurosporine inhibitable tyrosine phosphorylation of unique neutrophil proteins

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2446-2454 ◽  
Author(s):  
RL Berkow
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Triton X ◽  
Stimulating Factor

Abstract Human neutrophils treated with chemotactic peptides or phorbol esters demonstrate tyrosine phosphorylation of a subset of proteins. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced a time- and concentration-dependent increase in the tyrosine phosphorylation of at least seven proteins. Three of these proteins with approximate molecular weights of 150, 95, and 70 Kd were unique to neutrophils treated with GM-CSF, and were not seen to be phosphorylated on tyrosine in neutrophils treated with the agonists FMLP or PMA, or the cytokines G-CSF and tumor necrosis factor. We found the 150-Kd protein to be localized within the cell particulate fraction and the 95- Kd protein within the cell cytosol. The 70-Kd phosphotyrosine protein was found in both fractions. When the neutrophils were treated with Triton X-100 (Sigma Chemical Co, St Louis, MO) to evaluate cytoskeletal associations of proteins, the 150 phosphotyrosine protein partitioned with the Triton X-100 insoluble cytoskeleton (TICS), and the 70-Kd protein partitioned with both the TICS and Triton X-100 soluble proteins. The GM-CSF-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor ST638. This was not seen with the putative C-kinase inhibitor, H-7. However, staurosporine was seen to inhibit tyrosine phosphorylation of neutrophil proteins by GM-CSF and in vitro tyrosine kinase activity of isolated neutrophil cytosol and particulate fractions. These data indicate that the three unique GM-CSF- induced phosphotyrosine-containing proteins may be responsible for the unique actions of GM-CSF and that staurosporine inhibits a tyrosine kinase responsible for the phosphorylation of these proteins.

scholarly journals Involvement of tyrosine kinases in the activation of human peripheral blood neutrophils by granulocyte-macrophage colony-stimulating factor

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1842-1852 ◽  
Author(s):  
SR McColl ◽  
JF DiPersio ◽  
AC Caon ◽  
P Ho ◽  
PH Naccache
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Human Peripheral Blood ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The aim of the present study is to evaluate the involvement of human neutrophil tyrosine kinase(s) in the signal transduction mechanism of granulocyte-macrophage colony-stimulating factor (GM-CSF). Stimulation of neutrophils with GM-CSF resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 40, 55, 74, 97, 118, and 155 Kd, detected by immunoblot using a monoclonal antibody directed against phosphotyrosine. GM-CSF-induced tyrosine phosphorylation was inhibited in a dose- and time-dependent manner by the tyrosine kinase inhibitor erbstatin. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of GM-CSF on human neutrophils. Pretreatment of neutrophils with erbstatin before incubation with GM-CSF completely inhibited the GM-CSF-induced intracellular alkalinization, downregulation of the leukotriene B4 receptor, enhancement of fMet-Leu-Phe-induced intracellular calcium mobilization, as well as the accumulation of mRNA for the proto- oncogene c-fos. Taken together, these data suggest that tyrosine kinase activation in human neutrophils plays a critical regulatory role in both the stimulation and priming of neutrophil function by GM-CSF.

scholarly journals Involvement of tyrosine kinases in the activation of human peripheral blood neutrophils by granulocyte-macrophage colony-stimulating factor

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1842-1852
Author(s):  
SR McColl ◽  
JF DiPersio ◽  
AC Caon ◽  
P Ho ◽  
PH Naccache
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Human Peripheral Blood ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The aim of the present study is to evaluate the involvement of human neutrophil tyrosine kinase(s) in the signal transduction mechanism of granulocyte-macrophage colony-stimulating factor (GM-CSF). Stimulation of neutrophils with GM-CSF resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 40, 55, 74, 97, 118, and 155 Kd, detected by immunoblot using a monoclonal antibody directed against phosphotyrosine. GM-CSF-induced tyrosine phosphorylation was inhibited in a dose- and time-dependent manner by the tyrosine kinase inhibitor erbstatin. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of GM-CSF on human neutrophils. Pretreatment of neutrophils with erbstatin before incubation with GM-CSF completely inhibited the GM-CSF-induced intracellular alkalinization, downregulation of the leukotriene B4 receptor, enhancement of fMet-Leu-Phe-induced intracellular calcium mobilization, as well as the accumulation of mRNA for the proto- oncogene c-fos. Taken together, these data suggest that tyrosine kinase activation in human neutrophils plays a critical regulatory role in both the stimulation and priming of neutrophil function by GM-CSF.

scholarly journals Hck expression correlates with granulocyte-macrophage colony- stimulating factor-induced proliferation in HL-60 cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 94-103
Author(s):  
D Linnekin ◽  
OM Howard ◽  
L Park ◽  
W Farrar ◽  
D Ferris ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Protein Tyrosine ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The human myeloid cell line HL-60 expresses approximately 300 high- affinity granulocyte-macrophage colony-stimulating factor receptors (GM- CSFRs), yet treatment of these cells with GM-CSF does not result in enhanced cellular proliferation or increases in protein tyrosine phosphorylation. In contrast, GM-CSF induces rapid increases in protein tyrosine phosphorylation and proliferative responses in HL-60 cells pretreated for 3 days in dimethyl sulfoxide (DMSO). Similarly, HL-60 cells pretreated with retinoic acid or 1,25 dihydroxyvitamin D3 were also capable of responding to GM-CSF. Interestingly, each of these treatments resulted in increased expression of the src-like tyrosine kinase hck. Stimulation with GM-CSF increased hck autophosphorylation in DMSO-treated HL-60 cells, suggesting that hck is a component of the GM-CSF signal transduction pathway. To determine if hck has a role in the DMSO-induced recoupling of the GM-CSFR, we overexpressed hck in HL- 60 cells. The resulting cell line (HL-60/hck) expresses hck mRNA and protein at levels comparable with DMSO-treated HL-60 cells. Stimulation of HL-60/hck cells with GM-CSF results in activation of hck, increases in protein tyrosine phosphorylation, and increased proliferation. These results show that cytokine receptors can exist in an uncoupled form and suggest that in HL-60 cells, appropriate levels of the src-like tyrosine kinase hck are critical for functional coupling of the GM-CSFR to biologic responses.

scholarly journals Granulocyte-macrophage colony-stimulating factor activates microtubule- associated protein 2 kinase in neutrophils via a tyrosine kinase- dependent pathway

Blood ◽  
1992 ◽  
Vol 79 (12) ◽  
pp. 3350-3354 ◽  
Author(s):  
MA Raines ◽  
DW Golde ◽  
M Daeipour ◽  
AE Nel
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Colony Stimulating Factor ◽  
Kinase Activation ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Receptors of the hematopoietin superfamily, including the granulocyte- macrophage colony-stimulating factor (GM-CSF) receptor, lack a tyrosine kinase domain as well as other sequences indicative of a known signaling mechanism. In this report, we identify the serine/threonine kinase, microtubule-associated protein 2 (MAP2) kinase, as an intermediate in the GM-CSF signal transduction pathway. Treatment of peripheral blood neutrophils or terminally differentiated HL-60 cells with GM-CSF induced a rapid and dose-dependent increase in MAP2 kinase activity. Maximal activity occurred within 5 minutes and the kinetics of the response varied depending on the target cell (prolonged in neutrophils and transient in neutrophilic HL-60 cells). MAP2 kinase activity in these cells correlates with the induction of a 42-Kd tyrosine phosphoprotein. Furthermore, tyrosine phosphorylation is necessary for MAP2 kinase activation since its activity is inhibited by treatment with the tyrosine kinase inhibitor, erbstatin analog. These data suggest that tyrosine phosphorylation is important in GM-CSF- mediated signal transduction and that MAP2 kinase activation may be a central biochemical event involved in its signaling.

scholarly journals Hck expression correlates with granulocyte-macrophage colony- stimulating factor-induced proliferation in HL-60 cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 94-103 ◽  
Author(s):  
D Linnekin ◽  
OM Howard ◽  
L Park ◽  
W Farrar ◽  
D Ferris ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Protein Tyrosine ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The human myeloid cell line HL-60 expresses approximately 300 high- affinity granulocyte-macrophage colony-stimulating factor receptors (GM- CSFRs), yet treatment of these cells with GM-CSF does not result in enhanced cellular proliferation or increases in protein tyrosine phosphorylation. In contrast, GM-CSF induces rapid increases in protein tyrosine phosphorylation and proliferative responses in HL-60 cells pretreated for 3 days in dimethyl sulfoxide (DMSO). Similarly, HL-60 cells pretreated with retinoic acid or 1,25 dihydroxyvitamin D3 were also capable of responding to GM-CSF. Interestingly, each of these treatments resulted in increased expression of the src-like tyrosine kinase hck. Stimulation with GM-CSF increased hck autophosphorylation in DMSO-treated HL-60 cells, suggesting that hck is a component of the GM-CSF signal transduction pathway. To determine if hck has a role in the DMSO-induced recoupling of the GM-CSFR, we overexpressed hck in HL- 60 cells. The resulting cell line (HL-60/hck) expresses hck mRNA and protein at levels comparable with DMSO-treated HL-60 cells. Stimulation of HL-60/hck cells with GM-CSF results in activation of hck, increases in protein tyrosine phosphorylation, and increased proliferation. These results show that cytokine receptors can exist in an uncoupled form and suggest that in HL-60 cells, appropriate levels of the src-like tyrosine kinase hck are critical for functional coupling of the GM-CSFR to biologic responses.

scholarly journals Up-regulation of the amount of Giα2 associated with the plasma membrane in human neutrophils stimulated by granulocyte-macrophage colony-stimulating factor

1993 ◽  
Vol 292 (1) ◽  
pp. 183-187 ◽  
Author(s):  
M Durstin ◽  
S R McColl ◽  
J Gomez-Cambronero ◽  
P H Naccache ◽  
R I Sha'afi
Keyword(s):  
Plasma Membrane ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Protein Synthesis Inhibitor ◽  
Colony Stimulating Factor ◽  
Synthesis Inhibitor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Preincubation of human neutrophils with the human cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) results in an increase in the amount of alpha-subunit of Gi2 (Gi alpha 2) associated with the plasma membrane and a corresponding decrease in the amount associated with the granule fractions. Similar results are obtained with interleukin-8. GM-CSF has no effect on the distribution of Gi alpha 3. The effect of GM-CSF on Gi alpha 2 is time-dependent, and, although a significant effect can be observed after incubation for 5 min with GM-CSF, the enhancement increases with increasing time. Genistein, a protein tyrosine kinase inhibitor, and 1,2-bis-(O-aminophenoxyl)ethane-NNN'N'-tetra-acetic acid (BAPTA), an intracellular Ca2+ chelator, decrease the stimulatory effect of GM-CSF. On the other hand, the protein-synthesis inhibitor cycloheximide does not affect the action of GM-CSF. Also, although preincubation of human neutrophils with GM-CSF increases the levels of Gi alpha 2 in the plasma membrane it does not alter the total amount of cellular Gi alpha 2. In addition, the level of Gi alpha 2 mRNA, unlike that of the proto-oncogene c-fos, is not increased in cells treated with GM-CSF. This indicates that the observed increase in the amount of Gi alpha 2 associated with the plasma membrane is not due to the synthesis of new Gi alpha 2. These data provide insight into the mechanism by which GM-CSF may prime human neutrophils for increased responsiveness to subsequent stimulation by G-protein-dependent agonists.

scholarly journals Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol.

1990 ◽  
Vol 172 (3) ◽  
pp. 767-777 ◽  
Author(s):  
S Bourgoin ◽  
E Plante ◽  
M Gaudry ◽  
P H Naccache ◽  
P Borgeat ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Mechanism Of Action ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.

scholarly journals Granulocyte-macrophage colony-stimulating factor activates microtubule- associated protein 2 kinase in neutrophils via a tyrosine kinase- dependent pathway

Blood ◽  
1992 ◽  
Vol 79 (12) ◽  
pp. 3350-3354 ◽  
Author(s):  
MA Raines ◽  
DW Golde ◽  
M Daeipour ◽  
AE Nel
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Colony Stimulating Factor ◽  
Kinase Activation ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Receptors of the hematopoietin superfamily, including the granulocyte- macrophage colony-stimulating factor (GM-CSF) receptor, lack a tyrosine kinase domain as well as other sequences indicative of a known signaling mechanism. In this report, we identify the serine/threonine kinase, microtubule-associated protein 2 (MAP2) kinase, as an intermediate in the GM-CSF signal transduction pathway. Treatment of peripheral blood neutrophils or terminally differentiated HL-60 cells with GM-CSF induced a rapid and dose-dependent increase in MAP2 kinase activity. Maximal activity occurred within 5 minutes and the kinetics of the response varied depending on the target cell (prolonged in neutrophils and transient in neutrophilic HL-60 cells). MAP2 kinase activity in these cells correlates with the induction of a 42-Kd tyrosine phosphoprotein. Furthermore, tyrosine phosphorylation is necessary for MAP2 kinase activation since its activity is inhibited by treatment with the tyrosine kinase inhibitor, erbstatin analog. These data suggest that tyrosine phosphorylation is important in GM-CSF- mediated signal transduction and that MAP2 kinase activation may be a central biochemical event involved in its signaling.

scholarly journals Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 37-45 ◽  
Author(s):  
D Metcalf ◽  
CG Begley ◽  
GR Johnson ◽  
NA Nicola ◽  
MA Vadas ◽  
...  
Keyword(s):  
Colony Formation ◽  
Specific Activity ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Direct Stimulation ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Marrow Cultures

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.

Sign in / Sign up

Export Citation Format

Share Document