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The thrombopoietin receptor can mediate proliferation without activation of the Jak-STAT pathway.

Dorsch M, Fan PD, Danial NN, Rothman PB, Goff SP - J. Exp. Med. (1997)

Bottom Line: Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation.This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels.These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Cellular, and Biophysical Studies, Columbia University, College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

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Activation of PI 3-kinase in TPO-stimulated BAF-mplwt  and BAF-mplΔ7 cells. Growth factor–deprived cells were left untreated  or were stimulated with TPO for 5 min and cell extracts were prepared.  Immunoprecipitations were performed with anti-phosphotyrosine antibodies and the immunoprecipitates were analyzed for PI 3-kinase activity.  Formation of PI 3-P was inhibited by inclusion of 100 nM wortmannin  (+ Wort.) in the kinase reaction. The ratios of labeled PI 3-P in stimulated samples/unstimulated samples are shown as fold activation. The results shown represent one out of three experiments with similar outcomes.
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Figure 6: Activation of PI 3-kinase in TPO-stimulated BAF-mplwt and BAF-mplΔ7 cells. Growth factor–deprived cells were left untreated or were stimulated with TPO for 5 min and cell extracts were prepared. Immunoprecipitations were performed with anti-phosphotyrosine antibodies and the immunoprecipitates were analyzed for PI 3-kinase activity. Formation of PI 3-P was inhibited by inclusion of 100 nM wortmannin (+ Wort.) in the kinase reaction. The ratios of labeled PI 3-P in stimulated samples/unstimulated samples are shown as fold activation. The results shown represent one out of three experiments with similar outcomes.

Mentions: Previous studies have implicated PI 3-kinase in the mitogenic response induced by a number of cytokines (6, 30, 31). To study this pathway we analyzed PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates from BAF-mplwt, BAF-mplΔ7 cells and BAF-mplΔ8 cells before and after TPO stimulation. c-mplΔ7 mediated an increase in PI 3-kinase activity comparable to the wild-type receptor (Fig. 6), indicating that Jak activation is not a prerequisite for PI 3-kinase activation. Mutant c-mplΔ8 showed no increase in PI 3-kinase activity. Incubation of BAF-mplwt and BAF-mplD7 cells with increasing concentrations of the PI 3-kinase inhibitor wortmannin (1, 10, 100, and 1,000 nM) resulted in a concentration-dependent decrease in TPO-dependent proliferation as monitored by [3H]thymidine incorporation after 48 h. Approximatively 50% inhibition of maximal proliferation was observed at a concentration of 100 nM wortmannin, similar to results obtained by others for erythropoietin- or IL-7–induced proliferation (30, 31); proliferation was completely abolished at 1 μM (data not shown). These results suggest that PI 3-kinase may be an essential player in the generation of the mitogenic response by TPO. In this context it is of interest that the proliferation-defective mutant c-mplΔ8 does not activate PI 3-kinase (Fig. 6) but that the proliferation-competent C-terminal truncation mutant c-mplΔ7ΔC still mediates PI 3-kinase activation (M. Dorsch, and S.P. Goff, unpublished observation).


The thrombopoietin receptor can mediate proliferation without activation of the Jak-STAT pathway.

Dorsch M, Fan PD, Danial NN, Rothman PB, Goff SP - J. Exp. Med. (1997)

Activation of PI 3-kinase in TPO-stimulated BAF-mplwt  and BAF-mplΔ7 cells. Growth factor–deprived cells were left untreated  or were stimulated with TPO for 5 min and cell extracts were prepared.  Immunoprecipitations were performed with anti-phosphotyrosine antibodies and the immunoprecipitates were analyzed for PI 3-kinase activity.  Formation of PI 3-P was inhibited by inclusion of 100 nM wortmannin  (+ Wort.) in the kinase reaction. The ratios of labeled PI 3-P in stimulated samples/unstimulated samples are shown as fold activation. The results shown represent one out of three experiments with similar outcomes.
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Related In: Results  -  Collection

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Figure 6: Activation of PI 3-kinase in TPO-stimulated BAF-mplwt and BAF-mplΔ7 cells. Growth factor–deprived cells were left untreated or were stimulated with TPO for 5 min and cell extracts were prepared. Immunoprecipitations were performed with anti-phosphotyrosine antibodies and the immunoprecipitates were analyzed for PI 3-kinase activity. Formation of PI 3-P was inhibited by inclusion of 100 nM wortmannin (+ Wort.) in the kinase reaction. The ratios of labeled PI 3-P in stimulated samples/unstimulated samples are shown as fold activation. The results shown represent one out of three experiments with similar outcomes.
Mentions: Previous studies have implicated PI 3-kinase in the mitogenic response induced by a number of cytokines (6, 30, 31). To study this pathway we analyzed PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates from BAF-mplwt, BAF-mplΔ7 cells and BAF-mplΔ8 cells before and after TPO stimulation. c-mplΔ7 mediated an increase in PI 3-kinase activity comparable to the wild-type receptor (Fig. 6), indicating that Jak activation is not a prerequisite for PI 3-kinase activation. Mutant c-mplΔ8 showed no increase in PI 3-kinase activity. Incubation of BAF-mplwt and BAF-mplD7 cells with increasing concentrations of the PI 3-kinase inhibitor wortmannin (1, 10, 100, and 1,000 nM) resulted in a concentration-dependent decrease in TPO-dependent proliferation as monitored by [3H]thymidine incorporation after 48 h. Approximatively 50% inhibition of maximal proliferation was observed at a concentration of 100 nM wortmannin, similar to results obtained by others for erythropoietin- or IL-7–induced proliferation (30, 31); proliferation was completely abolished at 1 μM (data not shown). These results suggest that PI 3-kinase may be an essential player in the generation of the mitogenic response by TPO. In this context it is of interest that the proliferation-defective mutant c-mplΔ8 does not activate PI 3-kinase (Fig. 6) but that the proliferation-competent C-terminal truncation mutant c-mplΔ7ΔC still mediates PI 3-kinase activation (M. Dorsch, and S.P. Goff, unpublished observation).

Bottom Line: Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation.This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels.These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Cellular, and Biophysical Studies, Columbia University, College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

Show MeSH
Related in: MedlinePlus