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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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Mitogenic response of BAF/3 cells expressing c-mpl mutants.  (a) Schematic representation of c-mplwt and deletion mutants c-mplΔ7 and  c-mplΔ8. (b) Stable expression of c-mplwt, c-mplΔ7 or c-mplΔ8 in BAF/ 3 transfectants. Cell lysates prepared from 2 × 106 cells were resolved on a  7.5% SDS-PAGE gel, transferred to a nitrocellulose membrane and immunoblotted with a mAb against the myc-epitope. (c) TPO-induced proliferation of BAF-mplwt, BAF-mplΔ7, and BAF-mplΔ8 cells. [3H]thymidine incorporation as an indicator of cellular proliferation was measured at  different TPO concentrations. The mean of triplicate counts for each data  point is shown.
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Figure 1: Mitogenic response of BAF/3 cells expressing c-mpl mutants. (a) Schematic representation of c-mplwt and deletion mutants c-mplΔ7 and c-mplΔ8. (b) Stable expression of c-mplwt, c-mplΔ7 or c-mplΔ8 in BAF/ 3 transfectants. Cell lysates prepared from 2 × 106 cells were resolved on a 7.5% SDS-PAGE gel, transferred to a nitrocellulose membrane and immunoblotted with a mAb against the myc-epitope. (c) TPO-induced proliferation of BAF-mplwt, BAF-mplΔ7, and BAF-mplΔ8 cells. [3H]thymidine incorporation as an indicator of cellular proliferation was measured at different TPO concentrations. The mean of triplicate counts for each data point is shown.

Mentions: A series of deletion mutants of c-mpl was constructed; two selected mutants are depicted in Fig. 1 a. Mutant c-mplΔ7 lacks the first 10 amino acids (aa) (KWQFPAHYRR, aa 505-514; reference 2) of the cytoplasmic domain but retains an intact box1, whereas mutant c-mplΔ8 retains the juxtamembrane region but lacks the NH2-terminal half of box1 (LRHALWPS, aa 515-522). Cell lines stably expressing the wild-type and mutant receptors were established by transfection of the IL-3–dependent cell line BAF/3. Comparable levels of receptor expression were detected in cells expressing c-mplwt (BAF-mplwt), c-mplΔ7 (BAF-mplΔ7), and c-mplΔ8 (BAF-mplΔ8) (Fig. 1 b). The transfected cells were then analyzed for their mitogenic response to TPO (Fig. 1 c). Expression of c-mplwt conferred responsiveness to TPO as shown previously (4, 5) (Fig. 1 c). BAF-mplΔ7 cells also showed a strong proliferative response to TPO, though higher levels of TPO were required when compared to BAF-mplwt. BAF-mplΔ8 cells were completely unresponsive to TPO (Fig. 1 C) and parental BAF/3 cells (not shown), demonstrating that TPO-responsiveness required expression of a functional receptor in these cells. These results indicate that the first 10 aa of the c-mpl cytoplasmic domain are dispensable for a mitogenic response, whereas an intact NH2-terminal half of box1 is absolutely required. BAF-mplΔ7 cells retained their proliferative capacity in TPO for a prolonged period of time (>3 mo, data not shown), suggesting that the mutant receptor provides the signals necessary for long-term survival.


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)

Mitogenic response of BAF/3 cells expressing c-mpl mutants.  (a) Schematic representation of c-mplwt and deletion mutants c-mplΔ7 and  c-mplΔ8. (b) Stable expression of c-mplwt, c-mplΔ7 or c-mplΔ8 in BAF/ 3 transfectants. Cell lysates prepared from 2 × 106 cells were resolved on a  7.5% SDS-PAGE gel, transferred to a nitrocellulose membrane and immunoblotted with a mAb against the myc-epitope. (c) TPO-induced proliferation of BAF-mplwt, BAF-mplΔ7, and BAF-mplΔ8 cells. [3H]thymidine incorporation as an indicator of cellular proliferation was measured at  different TPO concentrations. The mean of triplicate counts for each data  point is shown.
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Related In: Results  -  Collection

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Figure 1: Mitogenic response of BAF/3 cells expressing c-mpl mutants. (a) Schematic representation of c-mplwt and deletion mutants c-mplΔ7 and c-mplΔ8. (b) Stable expression of c-mplwt, c-mplΔ7 or c-mplΔ8 in BAF/ 3 transfectants. Cell lysates prepared from 2 × 106 cells were resolved on a 7.5% SDS-PAGE gel, transferred to a nitrocellulose membrane and immunoblotted with a mAb against the myc-epitope. (c) TPO-induced proliferation of BAF-mplwt, BAF-mplΔ7, and BAF-mplΔ8 cells. [3H]thymidine incorporation as an indicator of cellular proliferation was measured at different TPO concentrations. The mean of triplicate counts for each data point is shown.
Mentions: A series of deletion mutants of c-mpl was constructed; two selected mutants are depicted in Fig. 1 a. Mutant c-mplΔ7 lacks the first 10 amino acids (aa) (KWQFPAHYRR, aa 505-514; reference 2) of the cytoplasmic domain but retains an intact box1, whereas mutant c-mplΔ8 retains the juxtamembrane region but lacks the NH2-terminal half of box1 (LRHALWPS, aa 515-522). Cell lines stably expressing the wild-type and mutant receptors were established by transfection of the IL-3–dependent cell line BAF/3. Comparable levels of receptor expression were detected in cells expressing c-mplwt (BAF-mplwt), c-mplΔ7 (BAF-mplΔ7), and c-mplΔ8 (BAF-mplΔ8) (Fig. 1 b). The transfected cells were then analyzed for their mitogenic response to TPO (Fig. 1 c). Expression of c-mplwt conferred responsiveness to TPO as shown previously (4, 5) (Fig. 1 c). BAF-mplΔ7 cells also showed a strong proliferative response to TPO, though higher levels of TPO were required when compared to BAF-mplwt. BAF-mplΔ8 cells were completely unresponsive to TPO (Fig. 1 C) and parental BAF/3 cells (not shown), demonstrating that TPO-responsiveness required expression of a functional receptor in these cells. These results indicate that the first 10 aa of the c-mpl cytoplasmic domain are dispensable for a mitogenic response, whereas an intact NH2-terminal half of box1 is absolutely required. BAF-mplΔ7 cells retained their proliferative capacity in TPO for a prolonged period of time (>3 mo, data not shown), suggesting that the mutant receptor provides the signals necessary for long-term survival.

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