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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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Induction of GAS-binding activity in BAF-mplwt but not  BAF-mplΔ7 cells. (a) Growth factor–deprived cells were left untreated or  were stimulated with TPO. The time points and the TPO concentrations  analyzed are indicated. Cell extracts were prepared and analysed by  EMSA using the IRF-1 GAS probe. GAS-binding activity was detected  in BAF-mplwt but not BAF-mplΔ7 cells. (b) The identity of the GAS-binding complexes in BAF-mplwt cells (5′ stimulation) was examined in  supershift assays with antibodies specific for STAT1, 3, and 5 ( STAT5a  and STAT5b antibodies were pooled). (c) Antiphosphotyrosine blot of  STAT3 immunoprecipitates shows that STAT3 is tyrosine phosphorylated after TPO-stimulation of the wild-type but not the mutant receptor.  Membrane was stripped and reprobed with anti-STAT3 antibodies to  confirm equal protein loading.
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Figure 3: Induction of GAS-binding activity in BAF-mplwt but not BAF-mplΔ7 cells. (a) Growth factor–deprived cells were left untreated or were stimulated with TPO. The time points and the TPO concentrations analyzed are indicated. Cell extracts were prepared and analysed by EMSA using the IRF-1 GAS probe. GAS-binding activity was detected in BAF-mplwt but not BAF-mplΔ7 cells. (b) The identity of the GAS-binding complexes in BAF-mplwt cells (5′ stimulation) was examined in supershift assays with antibodies specific for STAT1, 3, and 5 ( STAT5a and STAT5b antibodies were pooled). (c) Antiphosphotyrosine blot of STAT3 immunoprecipitates shows that STAT3 is tyrosine phosphorylated after TPO-stimulation of the wild-type but not the mutant receptor. Membrane was stripped and reprobed with anti-STAT3 antibodies to confirm equal protein loading.

Mentions: We next analyzed whether the failure of c-mplΔ7 to activate Jaks was also reflected in a lack of activation of their major targets, the STAT proteins. Tyrosine phosphorylation of STATs by Jaks leads to activation of their DNA-binding activity (6, 7). Stimulation of the TPO receptor has been described to activate STAT1, 3, and 5 (10–12). Using a DNA probe (GAS-element) (7) which can detect several activated STATs (including STAT1, 3, and 5), we measured STAT DNA-binding activity in lysates prepared from TPO-stimulated BAF-mplwt and BAF-mplΔ7 cells (Fig. 3 a), and also 32D-mplwt and 32D-mplΔ7 cells (data not shown) in an electrophoretic mobility-shift assay (EMSA). Complex formation was detected in cells expressing the wild-type receptor but not in cells expressing the mutant receptor. The GAS-binding activity was seen as early as 5 min after TPO stimulation and was still present after 1 h of stimulation (Fig. 3 a) whereas no GAS-binding activity was detected in BAF-mplΔ7 cells at any of the time points analyzed. Increasing the concentration of TPO up to 500 or 1,000 ng/ml did not enhance the GAS-binding activity in BAF-mplwt cells and did not result in any detectable activity in BAF-mplΔ7 cells (Fig. 3 a). The identity of the STATs present in the different complexes detected in TPO-stimulated BAF-mplwt cells was analyzed by supershift assays with antibodies to STAT1, 3, and 5 (Fig. 3 b). The complex with the lowest mobility was supershifted with anti-STAT5 antibodies. Antibodies to STAT1 supershifted the complex with the highest mobility. The complex with intermediate mobility was diminished by anti-STAT1 and anti-STAT3 antibodies indicating that the complex probably consists of STAT1/STAT3 heterodimers. To confirm the activation of STAT3, an anti-phosphotyrosine immunoblot of STAT3 immunoprecipitates was performed showing phosphorylation of STAT3 by the wild-type receptor but not by the mutant receptor (Fig. 3 c). The inability of c-mplΔ7 to induce a STAT-DNA complex is consistent with the observed lack of Jak activation in TPO-stimulated BAF-mplΔ7 cells and 32D-mplΔ7 cells. Moreover, this result excludes the possibility that another, as yet unidentified Jak kinase is activated by the mutant receptor to induce STAT DNA-binding activity.


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)

Induction of GAS-binding activity in BAF-mplwt but not  BAF-mplΔ7 cells. (a) Growth factor–deprived cells were left untreated or  were stimulated with TPO. The time points and the TPO concentrations  analyzed are indicated. Cell extracts were prepared and analysed by  EMSA using the IRF-1 GAS probe. GAS-binding activity was detected  in BAF-mplwt but not BAF-mplΔ7 cells. (b) The identity of the GAS-binding complexes in BAF-mplwt cells (5′ stimulation) was examined in  supershift assays with antibodies specific for STAT1, 3, and 5 ( STAT5a  and STAT5b antibodies were pooled). (c) Antiphosphotyrosine blot of  STAT3 immunoprecipitates shows that STAT3 is tyrosine phosphorylated after TPO-stimulation of the wild-type but not the mutant receptor.  Membrane was stripped and reprobed with anti-STAT3 antibodies to  confirm equal protein loading.
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Related In: Results  -  Collection

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Figure 3: Induction of GAS-binding activity in BAF-mplwt but not BAF-mplΔ7 cells. (a) Growth factor–deprived cells were left untreated or were stimulated with TPO. The time points and the TPO concentrations analyzed are indicated. Cell extracts were prepared and analysed by EMSA using the IRF-1 GAS probe. GAS-binding activity was detected in BAF-mplwt but not BAF-mplΔ7 cells. (b) The identity of the GAS-binding complexes in BAF-mplwt cells (5′ stimulation) was examined in supershift assays with antibodies specific for STAT1, 3, and 5 ( STAT5a and STAT5b antibodies were pooled). (c) Antiphosphotyrosine blot of STAT3 immunoprecipitates shows that STAT3 is tyrosine phosphorylated after TPO-stimulation of the wild-type but not the mutant receptor. Membrane was stripped and reprobed with anti-STAT3 antibodies to confirm equal protein loading.
Mentions: We next analyzed whether the failure of c-mplΔ7 to activate Jaks was also reflected in a lack of activation of their major targets, the STAT proteins. Tyrosine phosphorylation of STATs by Jaks leads to activation of their DNA-binding activity (6, 7). Stimulation of the TPO receptor has been described to activate STAT1, 3, and 5 (10–12). Using a DNA probe (GAS-element) (7) which can detect several activated STATs (including STAT1, 3, and 5), we measured STAT DNA-binding activity in lysates prepared from TPO-stimulated BAF-mplwt and BAF-mplΔ7 cells (Fig. 3 a), and also 32D-mplwt and 32D-mplΔ7 cells (data not shown) in an electrophoretic mobility-shift assay (EMSA). Complex formation was detected in cells expressing the wild-type receptor but not in cells expressing the mutant receptor. The GAS-binding activity was seen as early as 5 min after TPO stimulation and was still present after 1 h of stimulation (Fig. 3 a) whereas no GAS-binding activity was detected in BAF-mplΔ7 cells at any of the time points analyzed. Increasing the concentration of TPO up to 500 or 1,000 ng/ml did not enhance the GAS-binding activity in BAF-mplwt cells and did not result in any detectable activity in BAF-mplΔ7 cells (Fig. 3 a). The identity of the STATs present in the different complexes detected in TPO-stimulated BAF-mplwt cells was analyzed by supershift assays with antibodies to STAT1, 3, and 5 (Fig. 3 b). The complex with the lowest mobility was supershifted with anti-STAT5 antibodies. Antibodies to STAT1 supershifted the complex with the highest mobility. The complex with intermediate mobility was diminished by anti-STAT1 and anti-STAT3 antibodies indicating that the complex probably consists of STAT1/STAT3 heterodimers. To confirm the activation of STAT3, an anti-phosphotyrosine immunoblot of STAT3 immunoprecipitates was performed showing phosphorylation of STAT3 by the wild-type receptor but not by the mutant receptor (Fig. 3 c). The inability of c-mplΔ7 to induce a STAT-DNA complex is consistent with the observed lack of Jak activation in TPO-stimulated BAF-mplΔ7 cells and 32D-mplΔ7 cells. Moreover, this result excludes the possibility that another, as yet unidentified Jak kinase is activated by the mutant receptor to induce STAT DNA-binding activity.

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