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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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TPO stimulates c-fos and c-myc synthesis in BAF-mplwt and  BAF-mplΔ7 cells. Growth factor–deprived cells were washed twice and  incubated for 30 min at a density of 107 per ml in RPMI 1640 deficient in  methionine and cysteine (ICN). Cells were metabolically labeled as described (36) by adding 0.5 mCi of [35S]methionine (Translabel; ICN) per  ml to the cell suspension. TPO (200 ng/ml) was added simultaneously  and cells were incubated for the indicated times. Unstimulated (U) cells  were incubated with [35S]methionine in the absence of TPO for 1 h. Cell  extracts were prepared and c-fos and c-myc were immunoprecipitated  with antibodies to c-fos (top) or c-myc (bottom). Immunoprecipitates were  resolved by SDS-PAGE (7.5% gel) and analyzed by fluorography. Signals  were quantified with a PhosphorImager.
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Figure 5: TPO stimulates c-fos and c-myc synthesis in BAF-mplwt and BAF-mplΔ7 cells. Growth factor–deprived cells were washed twice and incubated for 30 min at a density of 107 per ml in RPMI 1640 deficient in methionine and cysteine (ICN). Cells were metabolically labeled as described (36) by adding 0.5 mCi of [35S]methionine (Translabel; ICN) per ml to the cell suspension. TPO (200 ng/ml) was added simultaneously and cells were incubated for the indicated times. Unstimulated (U) cells were incubated with [35S]methionine in the absence of TPO for 1 h. Cell extracts were prepared and c-fos and c-myc were immunoprecipitated with antibodies to c-fos (top) or c-myc (bottom). Immunoprecipitates were resolved by SDS-PAGE (7.5% gel) and analyzed by fluorography. Signals were quantified with a PhosphorImager.

Mentions: c-mplΔ7 also retained the ability of the wild-type receptor (28, 29) to induce phosphorylation of the serine-threonine kinases Raf-1 (Fig. 4 e) and MAPK (Fig. 4 f), and upregulation of c-fos and c-myc expression (Fig. 5). While the c-mplΔ7-mediated effect on Raf-1 was comparable to the wild-type receptor, the phosphorylation of MAPK induced by the mutant receptor was reduced in its intensity and duration (Fig. 4 f). Induction of c-fos and c-myc protein synthesis was reduced approximatively threefold in BAF-mplΔ7 cells as compared to BAF-mplwt cells. In an effort to further investigate the importance of these signals for Jak-independent proliferation, we generated the mutant c-mplΔ7ΔC by introducing an additional COOH-terminal truncation (aa 601-625) in the c-mplΔ7 mutant; it has been previously shown that this region is required for both Shc activation and receptor phosphorylation (13, 21). This double mutant failed to induce tyrosine phosphorylation of Jak and Shc and phosphorylation of Raf-1 but nevertheless was sufficient to mediate proliferation in BAF/3 cells although maximal proliferation was reduced about twofold when compared with the c-mplΔ7 mutant (data not shown). These data suggest that the mitogenic signal required neither Jak activation nor Shc or Raf-1 phosphorylation.


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)

TPO stimulates c-fos and c-myc synthesis in BAF-mplwt and  BAF-mplΔ7 cells. Growth factor–deprived cells were washed twice and  incubated for 30 min at a density of 107 per ml in RPMI 1640 deficient in  methionine and cysteine (ICN). Cells were metabolically labeled as described (36) by adding 0.5 mCi of [35S]methionine (Translabel; ICN) per  ml to the cell suspension. TPO (200 ng/ml) was added simultaneously  and cells were incubated for the indicated times. Unstimulated (U) cells  were incubated with [35S]methionine in the absence of TPO for 1 h. Cell  extracts were prepared and c-fos and c-myc were immunoprecipitated  with antibodies to c-fos (top) or c-myc (bottom). Immunoprecipitates were  resolved by SDS-PAGE (7.5% gel) and analyzed by fluorography. Signals  were quantified with a PhosphorImager.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199166&req=5

Figure 5: TPO stimulates c-fos and c-myc synthesis in BAF-mplwt and BAF-mplΔ7 cells. Growth factor–deprived cells were washed twice and incubated for 30 min at a density of 107 per ml in RPMI 1640 deficient in methionine and cysteine (ICN). Cells were metabolically labeled as described (36) by adding 0.5 mCi of [35S]methionine (Translabel; ICN) per ml to the cell suspension. TPO (200 ng/ml) was added simultaneously and cells were incubated for the indicated times. Unstimulated (U) cells were incubated with [35S]methionine in the absence of TPO for 1 h. Cell extracts were prepared and c-fos and c-myc were immunoprecipitated with antibodies to c-fos (top) or c-myc (bottom). Immunoprecipitates were resolved by SDS-PAGE (7.5% gel) and analyzed by fluorography. Signals were quantified with a PhosphorImager.
Mentions: c-mplΔ7 also retained the ability of the wild-type receptor (28, 29) to induce phosphorylation of the serine-threonine kinases Raf-1 (Fig. 4 e) and MAPK (Fig. 4 f), and upregulation of c-fos and c-myc expression (Fig. 5). While the c-mplΔ7-mediated effect on Raf-1 was comparable to the wild-type receptor, the phosphorylation of MAPK induced by the mutant receptor was reduced in its intensity and duration (Fig. 4 f). Induction of c-fos and c-myc protein synthesis was reduced approximatively threefold in BAF-mplΔ7 cells as compared to BAF-mplwt cells. In an effort to further investigate the importance of these signals for Jak-independent proliferation, we generated the mutant c-mplΔ7ΔC by introducing an additional COOH-terminal truncation (aa 601-625) in the c-mplΔ7 mutant; it has been previously shown that this region is required for both Shc activation and receptor phosphorylation (13, 21). This double mutant failed to induce tyrosine phosphorylation of Jak and Shc and phosphorylation of Raf-1 but nevertheless was sufficient to mediate proliferation in BAF/3 cells although maximal proliferation was reduced about twofold when compared with the c-mplΔ7 mutant (data not shown). These data suggest that the mitogenic signal required neither Jak activation nor Shc or Raf-1 phosphorylation.

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