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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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Effect of TPO stimulation on Shc, Vav, the receptor  itself, Raf-1, and MAPK.  Growth factor-deprived BAF-mplwt and BAF-mplΔ7 cells  were either left untreated or  stimulated with TPO for the indicated times and cell extracts  were prepared. Immunoprecipitations were performed with antibodies to Shc (a), Vav (b), and  myc (c) and the immunoprecipitates were blotted with antiphosphotyrosine antibodies (a–c). To  confirm equal loading of protein,  membranes were stripped and  reprobed with the antibodies  used for immunoprecipitations  (lower panel of a–c). In (c) a  higher amount of c-mplΔ7 protein was immunoprecipitated. (d)  Antiphosphotyrosine immunoblot of total cell lysates. (e) Cell lysates were immunoblotted with  an antibody to Raf-1. The lower  mobility of Raf-1 seen after  stimulation with TPO in BAF-mplwt and BAF-mplΔ7 reflects  the increased phosphorylation of  Raf-1 on serine. (f) Cell lysates  were immunoblotted with anti-active MAPK antibodies which  recognize the active forms of  Erk-1 and Erk-2 (different exposures of the same membrane are  shown in the upper and middle  panel). Membranes were stripped  and reprobed with anti-Erk2 antibodies to confirm equal protein  loading.
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Figure 4: Effect of TPO stimulation on Shc, Vav, the receptor itself, Raf-1, and MAPK. Growth factor-deprived BAF-mplwt and BAF-mplΔ7 cells were either left untreated or stimulated with TPO for the indicated times and cell extracts were prepared. Immunoprecipitations were performed with antibodies to Shc (a), Vav (b), and myc (c) and the immunoprecipitates were blotted with antiphosphotyrosine antibodies (a–c). To confirm equal loading of protein, membranes were stripped and reprobed with the antibodies used for immunoprecipitations (lower panel of a–c). In (c) a higher amount of c-mplΔ7 protein was immunoprecipitated. (d) Antiphosphotyrosine immunoblot of total cell lysates. (e) Cell lysates were immunoblotted with an antibody to Raf-1. The lower mobility of Raf-1 seen after stimulation with TPO in BAF-mplwt and BAF-mplΔ7 reflects the increased phosphorylation of Raf-1 on serine. (f) Cell lysates were immunoblotted with anti-active MAPK antibodies which recognize the active forms of Erk-1 and Erk-2 (different exposures of the same membrane are shown in the upper and middle panel). Membranes were stripped and reprobed with anti-Erk2 antibodies to confirm equal protein loading.

Mentions: Our results demonstrate that c-mplΔ7 is able to mediate TPO-stimulated proliferation without activation of the Jak-STAT pathway. We therefore asked if other signaling pathways previously described for c-mpl (8–11) were activated in TPO-stimulated BAF-mplΔ7 or BAF-mplΔ8 cells. As shown in Fig. 4, stimulation of both c-mplwt and c-mplΔ7 induced tyrosine phosphorylation of Shc (a), Vav (b) and c-mpl (c). In constrast, c-mplΔ8 was completely inactive (data not shown). Phosphorylation of Shc and Vav was slightly reduced and phosphorylation of the receptor itself was markedly reduced in BAF-mplΔ7 cells as compared to BAF-mplwt cells. A phosphotyrosine blot of total cell lysates after TPO stimulation (Fig. 4 d) was in agreement with the above observations: protein tyrosine phosphorylation was still detectable in BAF-mplΔ7 cells but the number of proteins phosphorylated and the degree of phosphorylation was reduced compared to BAF-mplwt cells. No tyrosine phosphorylated proteins were detected in lysates from TPO-stimulated BAF-mplΔ8 cells (data not shown). These results suggest that c-mplΔ7 mediates activation of tyrosine kinase(s) other than Jaks. The mutation in box1 in c-mplΔ8 appears to disrupt activation of not only the Jaks but also the additional or alternative tyrosine kinase(s) active in BAF-mplΔ7 cells.


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)

Effect of TPO stimulation on Shc, Vav, the receptor  itself, Raf-1, and MAPK.  Growth factor-deprived BAF-mplwt and BAF-mplΔ7 cells  were either left untreated or  stimulated with TPO for the indicated times and cell extracts  were prepared. Immunoprecipitations were performed with antibodies to Shc (a), Vav (b), and  myc (c) and the immunoprecipitates were blotted with antiphosphotyrosine antibodies (a–c). To  confirm equal loading of protein,  membranes were stripped and  reprobed with the antibodies  used for immunoprecipitations  (lower panel of a–c). In (c) a  higher amount of c-mplΔ7 protein was immunoprecipitated. (d)  Antiphosphotyrosine immunoblot of total cell lysates. (e) Cell lysates were immunoblotted with  an antibody to Raf-1. The lower  mobility of Raf-1 seen after  stimulation with TPO in BAF-mplwt and BAF-mplΔ7 reflects  the increased phosphorylation of  Raf-1 on serine. (f) Cell lysates  were immunoblotted with anti-active MAPK antibodies which  recognize the active forms of  Erk-1 and Erk-2 (different exposures of the same membrane are  shown in the upper and middle  panel). Membranes were stripped  and reprobed with anti-Erk2 antibodies to confirm equal protein  loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199166&req=5

Figure 4: Effect of TPO stimulation on Shc, Vav, the receptor itself, Raf-1, and MAPK. Growth factor-deprived BAF-mplwt and BAF-mplΔ7 cells were either left untreated or stimulated with TPO for the indicated times and cell extracts were prepared. Immunoprecipitations were performed with antibodies to Shc (a), Vav (b), and myc (c) and the immunoprecipitates were blotted with antiphosphotyrosine antibodies (a–c). To confirm equal loading of protein, membranes were stripped and reprobed with the antibodies used for immunoprecipitations (lower panel of a–c). In (c) a higher amount of c-mplΔ7 protein was immunoprecipitated. (d) Antiphosphotyrosine immunoblot of total cell lysates. (e) Cell lysates were immunoblotted with an antibody to Raf-1. The lower mobility of Raf-1 seen after stimulation with TPO in BAF-mplwt and BAF-mplΔ7 reflects the increased phosphorylation of Raf-1 on serine. (f) Cell lysates were immunoblotted with anti-active MAPK antibodies which recognize the active forms of Erk-1 and Erk-2 (different exposures of the same membrane are shown in the upper and middle panel). Membranes were stripped and reprobed with anti-Erk2 antibodies to confirm equal protein loading.
Mentions: Our results demonstrate that c-mplΔ7 is able to mediate TPO-stimulated proliferation without activation of the Jak-STAT pathway. We therefore asked if other signaling pathways previously described for c-mpl (8–11) were activated in TPO-stimulated BAF-mplΔ7 or BAF-mplΔ8 cells. As shown in Fig. 4, stimulation of both c-mplwt and c-mplΔ7 induced tyrosine phosphorylation of Shc (a), Vav (b) and c-mpl (c). In constrast, c-mplΔ8 was completely inactive (data not shown). Phosphorylation of Shc and Vav was slightly reduced and phosphorylation of the receptor itself was markedly reduced in BAF-mplΔ7 cells as compared to BAF-mplwt cells. A phosphotyrosine blot of total cell lysates after TPO stimulation (Fig. 4 d) was in agreement with the above observations: protein tyrosine phosphorylation was still detectable in BAF-mplΔ7 cells but the number of proteins phosphorylated and the degree of phosphorylation was reduced compared to BAF-mplwt cells. No tyrosine phosphorylated proteins were detected in lysates from TPO-stimulated BAF-mplΔ8 cells (data not shown). These results suggest that c-mplΔ7 mediates activation of tyrosine kinase(s) other than Jaks. The mutation in box1 in c-mplΔ8 appears to disrupt activation of not only the Jaks but also the additional or alternative tyrosine kinase(s) active in BAF-mplΔ7 cells.

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