scholarly journals Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3698-3704 ◽  
Author(s):  
SE Nicholson ◽  
U Novak ◽  
SF Ziegler ◽  
JE Layton
Keyword(s):  
Amino Acids ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Signaling Molecules ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Kinase Activation ◽  
Stimulating Factor

The protein tyrosine kinases JAK1 and JAK2 are phosphorylated tyrosine after the interaction of granulocyte colony-stimulating factor (G-CSF) with its transmembrane receptor. So too is Stat3, a member of the STAT family of transcriptional activators thought to be activated by the JAK kinases. Truncated G-CSF receptor (G-CSF-R) mutants were used to determine the different regions of the cytoplasmic domain necessary for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK. We have shown that G-CSF-induced tyrosine phosphorylation and kinase activation of JAK2 requires the membrane proximal 57 amino acids of the cytoplasmic domain. In contrast, maximal Stat3 tyrosine phosphorylation required amino acids 96 to 183 of the G-CSF-R cytoplasmic domain, Stat3 DNA binding could occur with a receptor truncated 96 amino acids from the transmembrane domain and containing a single tyrosine residue, but was reduced in comparison with the full- length receptor. Together with the tyrosine phosphorylation of Stat3, this finding suggests that additional Stat3 does not appear to be required for proliferation. MAP kinase tyrosine phosphorylation correlated with both the proliferative response and JAK2 activation.

scholarly journals Point mutations in the conserved box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3117-3126 ◽  
Author(s):  
BR Avalos ◽  
MG Hunter ◽  
JM Parker ◽  
SK Ceselski ◽  
BJ Druker ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Amino Acid ◽  
Tyrosine Phosphorylation ◽  
Cytoplasmic Domain ◽  
Early Event ◽  
Sequence Motif ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL-3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy-terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF-mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG-CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR.

scholarly journals Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1760-1764 ◽  
Author(s):  
SS Tian ◽  
P Lamb ◽  
HM Seidel ◽  
RB Stein ◽  
J Rosen
Keyword(s):  
Dna Binding ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Dna Binding Activity ◽  
Dna Binding Complexes ◽  
Stimulating Factor

Abstract Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates proliferation and differentiation of progenitor cells of neutrophils by signaling through its receptor (G-CSFR). Although the G- CSFR belongs to the cytokine receptor superfamily, which lacks an intracellular kinase domain, G-CSF-induced tyrosine phosphorylation of cellular proteins is critical for its biologic activities. We report here that JAK1 and JAK2 tyrosine kinases are tyrosine phosphorylated in response to G-CSF induction. We also demonstrate that the DNA-binding protein STAT3 (also called the acute-phase response factor [APRF], activated by interleukin-6) is an early target of G-CSF-induced tyrosine phosphorylation. G-CSF induces two DNA-binding complexes; the major complex contains tyrosine phosphorylated STAT3 protein and the minor complex appears to be a heterodimer of the STAT1 (previously p91, a component of DNA-binding complexes activated by interferons) and STAT3 proteins. Antiphosphotyrosine antibody interferes with the DNA binding activity of activated STAT3, indicating that tyrosine phosphorylation of STAT3 is important for the DNA binding activity. These results identify a signal transduction pathway activated in response to G-CSF and provide a mechanism for the rapid modulation of gene expression by G-CSF.

scholarly journals Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation

1993 ◽  
Vol 13 (12) ◽  
pp. 7774-7781
Author(s):  
F Dong ◽  
C van Buitenen ◽  
K Pouwels ◽  
L H Hoefsloot ◽  
B Löwenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Splice Variant ◽  
Transmembrane Domain ◽  
Myeloid Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Myeloid Progenitor Cells ◽  
Carboxy Terminal ◽  
Stimulating Factor

The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

scholarly journals Distinct regions of the human granulocyte-colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction.

1993 ◽  
Vol 13 (4) ◽  
pp. 2384-2390 ◽  
Author(s):  
S F Ziegler ◽  
T A Bird ◽  
K K Morella ◽  
B Mosley ◽  
D P Gearing ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatoma Cell ◽  
Signal Transduction Pathway ◽  
Cytoplasmic Domain ◽  
Sequence Motifs ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Human Hepatoma Cell ◽  
Functional Region ◽  
Stimulating Factor

Using two different cell systems, we show that the cytoplasmic domain of the granulocyte-colony-stimulating factor receptor (G-CSFR) may be composed of at least two functional regions. The first, within the membrane-proximal 57 amino acids, is absolutely required to deliver a proliferative signal. This region contains two sequence motifs conserved between members of the hematopoietin receptor family. The second functional region resides between amino acids 57 and 96. This region is required for the induction of acute-phase plasma protein gene expression when the G-CSFR is transfected into human hepatoma cell lines. The G-CSFR-transfected hepatoma cells respond to G-CSF by increasing the production of the same set of plasma proteins as stimulated by interleukin-6, suggesting that the two cytokines share a common signal transduction pathway.

scholarly journals Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation.

1993 ◽  
Vol 13 (12) ◽  
pp. 7774-7781 ◽  
Author(s):  
F Dong ◽  
C van Buitenen ◽  
K Pouwels ◽  
L H Hoefsloot ◽  
B Löwenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Splice Variant ◽  
Transmembrane Domain ◽  
Myeloid Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Myeloid Progenitor Cells ◽  
Carboxy Terminal ◽  
Stimulating Factor

The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

Activation of Akt kinase by granulocyte colony-stimulating factor (G-CSF): evidence for the role of a tyrosine kinase activity distinct from the janus kinases

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1656-1662 ◽  
Author(s):  
Fan Dong ◽  
Andrew C. Larner
Keyword(s):  
Amino Acids ◽  
Tyrosine Kinases ◽  
Cell Surface Receptor ◽  
Signaling Cascades ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cytokine Activation ◽  
Stimulating Factor ◽  
Stimulation Of ◽  
Factor G

Activation of the serine/threonine kinase Akt has been shown to be a critical component for growth factor and cytokine stimulation of cell survival. Although some of the immediate upstream activators of Akt have been defined, the roles of tyrosine kinases in the activation of Akt are not well delineated. Granulocyte colony-stimulating factor (G-CSF) regulates the proliferation, differentiation, and survival of neutrophilic granulocytes. G-CSF exerts its actions by stimulating several signaling cascades after binding its cell surface receptor. Both Jak (Janus) and Src families of tyrosine kinases are stimulated by incubation of cells with G-CSF. In this report, we show that G-CSF stimulation of cells leads to activation of Akt. The membrane-proximal 55 amino acids of the G-CSF receptor cytoplasmic domain are sufficient for mediating Akt activation. However, activation of Akt appears to be downregulated by the receptor's carboxy-terminal region of 98 amino acids, a region that has been shown to be truncated in some patients with acute myeloid leukemia associated with severe congenital neutropenia. Furthermore, we demonstrate that G-CSF–induced activation of Akt requires the activities of Src family kinases but can be clearly dissociated from G-CSF–stimulated activation of Stats (signal transducers and activators of transcripton) by the Jak kinases. Thus, cytokine activation of the Jak/Stat and other signaling cascades can be functionally separated.

scholarly journals Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1760-1764 ◽  
Author(s):  
SS Tian ◽  
P Lamb ◽  
HM Seidel ◽  
RB Stein ◽  
J Rosen
Keyword(s):  
Dna Binding ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Signal Transduction Pathway ◽  
Binding Activity ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Dna Binding Activity ◽  
Dna Binding Complexes ◽  
Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates proliferation and differentiation of progenitor cells of neutrophils by signaling through its receptor (G-CSFR). Although the G- CSFR belongs to the cytokine receptor superfamily, which lacks an intracellular kinase domain, G-CSF-induced tyrosine phosphorylation of cellular proteins is critical for its biologic activities. We report here that JAK1 and JAK2 tyrosine kinases are tyrosine phosphorylated in response to G-CSF induction. We also demonstrate that the DNA-binding protein STAT3 (also called the acute-phase response factor [APRF], activated by interleukin-6) is an early target of G-CSF-induced tyrosine phosphorylation. G-CSF induces two DNA-binding complexes; the major complex contains tyrosine phosphorylated STAT3 protein and the minor complex appears to be a heterodimer of the STAT1 (previously p91, a component of DNA-binding complexes activated by interferons) and STAT3 proteins. Antiphosphotyrosine antibody interferes with the DNA binding activity of activated STAT3, indicating that tyrosine phosphorylation of STAT3 is important for the DNA binding activity. These results identify a signal transduction pathway activated in response to G-CSF and provide a mechanism for the rapid modulation of gene expression by G-CSF.

Distinct regions of the human granulocyte-colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction

1993 ◽  
Vol 13 (4) ◽  
pp. 2384-2390
Author(s):  
S F Ziegler ◽  
T A Bird ◽  
K K Morella ◽  
B Mosley ◽  
D P Gearing ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatoma Cell ◽  
Signal Transduction Pathway ◽  
Cytoplasmic Domain ◽  
Sequence Motifs ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Human Hepatoma Cell ◽  
Functional Region ◽  
Stimulating Factor

Using two different cell systems, we show that the cytoplasmic domain of the granulocyte-colony-stimulating factor receptor (G-CSFR) may be composed of at least two functional regions. The first, within the membrane-proximal 57 amino acids, is absolutely required to deliver a proliferative signal. This region contains two sequence motifs conserved between members of the hematopoietin receptor family. The second functional region resides between amino acids 57 and 96. This region is required for the induction of acute-phase plasma protein gene expression when the G-CSFR is transfected into human hepatoma cell lines. The G-CSFR-transfected hepatoma cells respond to G-CSF by increasing the production of the same set of plasma proteins as stimulated by interleukin-6, suggesting that the two cytokines share a common signal transduction pathway.

scholarly journals Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4435-4444 ◽  
Author(s):  
SS Tian ◽  
P Tapley ◽  
C Sincich ◽  
RB Stein ◽  
J Rosen ◽  
...  
Keyword(s):  
Immediate Early Genes ◽  
Signaling Molecules ◽  
Immediate Early ◽  
Terminal Deletion ◽  
Deletion Mutants ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Multiple Signaling ◽  
Stimulating Factor ◽  
Dependent Pathway

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of proliferation and differentiation of neutrophilic granulocyte precursor cells. G-CSF activates multiple signaling molecules, including the JAK1 and JAK2 kinases and the STAT transcription factors. To investigate G-CSF signaling events regulated by the JAK-STAT pathway, we have generated UT7-epo cells stably expressing either wild-type (wt) G-CSF receptor or a series of C-terminal deletion mutants. Gel mobility shift and immunoprecipitation/Western analysis showed that STAT5 is rapidly activated by G-CSF in cells expressing the wt G-CSF receptor, in addition to the previously reported STAT3 and STAT1. Mutants lacking any tyrosine residues in the cytoplasmic domain maintain their ability to activate STAT5 and STAT1 but cannot activate STAT3, implying that STAT5 and STAT1 activation does not require receptor tyrosine phosphorylation. We also observed significant changes in the ratio of STAT1:STAT3:STAT5 activated by various G-CSF receptor C-terminal deletion mutants. These mutant receptors were further used to investigate the role of JAKs and STATs in G-CSF-mediated responses in these cells. We found that JAK activation correlates with G-CSF-induced cell proliferation, whereas STAT activation is not required. We have also identified three classes of G-CSF immediate early genes, whose activation correlates with the activation of distinct JAK-STAT pathways. Our data show that, whereas c-fos is regulated through a pathway independent of STAT activation, oncostatin M, IRF-1, and egr-1 are regulated by an STAT5-dependent pathway and fibrinogen is regulated by an STAT3-dependent pathway. In conclusion, our results suggest that G-CSF regulates its complex biologic activities by selectively activating distinct early response genes through different JAK-STAT signaling molecules.

scholarly journals G-CSF receptor activation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2 phosphatase

Blood ◽  
2011 ◽  
Vol 118 (4) ◽  
pp. 1077-1086 ◽  
Author(s):  
Muneyoshi Futami ◽  
Quan-sheng Zhu ◽  
Zakary L. Whichard ◽  
Ling Xia ◽  
Yuehai Ke ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Src Kinase ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
In Cells

Abstract Src activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF–stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.

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