scholarly journals Peptide antisera to human colony-stimulating factor 1 receptor detect ligand-induced conformational changes and a binding site for phosphatidylinositol 3-kinase.

1991 ◽  
Vol 11 (5) ◽  
pp. 2489-2495 ◽  
Author(s):  
J R Downing ◽  
S A Shurtleff ◽  
C J Sherr
Keyword(s):  
Binding Site ◽  
Tyrosine Phosphorylation ◽  
Stable Complex ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Vitro Assay ◽  
Juxtamembrane Region ◽  
Stimulating Factor ◽  
Phosphatidylinositol 3

A peptide antiserum (anti-A) directed to the intracellular, juxtamembrane region (residues 552 to 574) of the human colony-stimulating factor 1 receptor (CSF-1R) precipitated only ligand-activated, native receptors from solution but bound to unstimulated forms after their denaturation. Two peptide antisera (anti-KI1 and -KI2), directed to residues 679 to 700 and 701 to 721, respectively, in the CSF-1R kinase insert (KI) domain and including mapped sites of ligand-induced phosphorylation at Tyr-699 and Tyr-708, bound at least 80% of the receptor molecules expressed in either CSF-1-stimulated or unstimulated cells. Immune complexes formed with anti-KI1, anti-A, or a peptide antiserum to the CSF-1R carboxyl terminus (anti-C-ter) coprecipitated CSF-1R complexed to a phosphatidylinositol 3-kinase (PtdIns 3-K) from CSF-1-stimulated cells, whereas anti-KI2 serum did not. In an in vitro assay, binding of CSF-1R to PtdIns 3-K required receptor tyrosine phosphorylation but not CSF-1R-mediated phosphorylation of the lipid kinase, and the association was specifically blocked by anti-KI2 or antibodies to phosphotyrosine. Neither anti-KI1, anti-A, nor anti-C-ter serum inhibited binding. We conclude that (i) only a minority of ligand-activated receptors form a stable complex with PtdIns 3-K in vivo, (ii) efficient binding of the lipid kinase requires receptor tyrosine phosphorylation within the CSF-1R KI domain, and (iii) a region within the KI domain defined by residues 701 to 721 at least partially overlaps the PtdIns 3-K binding site.

Peptide antisera to human colony-stimulating factor 1 receptor detect ligand-induced conformational changes and a binding site for phosphatidylinositol 3-kinase

1991 ◽  
Vol 11 (5) ◽  
pp. 2489-2495
Author(s):  
J R Downing ◽  
S A Shurtleff ◽  
C J Sherr
Keyword(s):  
Binding Site ◽  
Tyrosine Phosphorylation ◽  
Stable Complex ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Vitro Assay ◽  
Juxtamembrane Region ◽  
Stimulating Factor ◽  
Phosphatidylinositol 3

A peptide antiserum (anti-A) directed to the intracellular, juxtamembrane region (residues 552 to 574) of the human colony-stimulating factor 1 receptor (CSF-1R) precipitated only ligand-activated, native receptors from solution but bound to unstimulated forms after their denaturation. Two peptide antisera (anti-KI1 and -KI2), directed to residues 679 to 700 and 701 to 721, respectively, in the CSF-1R kinase insert (KI) domain and including mapped sites of ligand-induced phosphorylation at Tyr-699 and Tyr-708, bound at least 80% of the receptor molecules expressed in either CSF-1-stimulated or unstimulated cells. Immune complexes formed with anti-KI1, anti-A, or a peptide antiserum to the CSF-1R carboxyl terminus (anti-C-ter) coprecipitated CSF-1R complexed to a phosphatidylinositol 3-kinase (PtdIns 3-K) from CSF-1-stimulated cells, whereas anti-KI2 serum did not. In an in vitro assay, binding of CSF-1R to PtdIns 3-K required receptor tyrosine phosphorylation but not CSF-1R-mediated phosphorylation of the lipid kinase, and the association was specifically blocked by anti-KI2 or antibodies to phosphotyrosine. Neither anti-KI1, anti-A, nor anti-C-ter serum inhibited binding. We conclude that (i) only a minority of ligand-activated receptors form a stable complex with PtdIns 3-K in vivo, (ii) efficient binding of the lipid kinase requires receptor tyrosine phosphorylation within the CSF-1R KI domain, and (iii) a region within the KI domain defined by residues 701 to 721 at least partially overlaps the PtdIns 3-K binding site.

scholarly journals Association between phosphatidylinositol-3 kinase, Cbl and other tyrosine phosphorylated proteins in colony-stimulating factor-1-stimulated macrophages

1996 ◽  
Vol 320 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Varuni KANAGASUNDARAM ◽  
Anthony JAWOROWSKI ◽  
John A HAMILTON
Keyword(s):  
Cell Line ◽  
Exchange Chromatography ◽  
Stable Complex ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphorylated Proteins ◽  
Cell Extracts ◽  
Stimulating Factor ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Colony stimulating factor-1 (CSF-1) stimulation of the macrophage cell line BAC1.2F5 and murine bone marrow-derived macrophages resulted in tyrosine phosphorylation of phosphatidylinositol-3 kinase (PI-3 kinase) p85α and its stable association with several tyrosine phosphorylated proteins, including CSF-1 receptor (p165), p120, p95 and p55–p60. p120 co-migrated with the product of the protooncogene c-cbl in anti-p85α immunoprecipitates, and associated with p85α in a rapid and transient manner. Reciprocal experiments confirmed the presence of p85α in anti-Cbl immunoprecipitates on CSF-1 stimulation of macrophages. PI-3 kinase immunoprecipitates from the myeloid FDC-P1 cell line expressing mutant CSF-1 receptor (Y721F), which does not associate with PI-3 kinase, still contained Cbl. The identity of the tyrosine phosphorylated protein p95 remains unknown. The interaction between p85α and the tyrosine phosphorylated proteins survived anion-exchange chromatography, suggesting perhaps the presence of a stable complex; furthermore, in CSF-1-treated BAC1.2F5 cell extracts, only one of the two pools of PI-3 kinase separated by chromatography was present in this putative complex. The association did not appear to correlate with proliferation, since a similar interaction between p85α and tyrosine phosphorylated proteins was also observed in poorly proliferating resident peritoneal macrophages stimulated with CSF-1. The possible significance of these findings for CSF-1-regulated macrophage functions is discussed.

scholarly journals Both Src-Dependent and -Independent Mechanisms Mediate Phosphatidylinositol 3-Kinase Regulation of Colony-Stimulating Factor 1-Activated Mitogen-Activated Protein Kinases in Myeloid Progenitors

2000 ◽  
Vol 20 (18) ◽  
pp. 6779-6798 ◽  
Author(s):  
Angel W.-M. Lee ◽  
David J. States
Keyword(s):  
Kinase Inhibitors ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulating Factor ◽  
In Cells ◽  
Phosphatidylinositol 3 ◽  
Myeloid Progenitors

ABSTRACT Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (ΔKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or ΔKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing ΔKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or ΔKI receptors. However, only in ΔKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.

The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase

Nature ◽  
1989 ◽  
Vol 342 (6250) ◽  
pp. 699-702 ◽  
Author(s):  
Lyuba Varticovski ◽  
Brian Druker ◽  
Deborah Morrison ◽  
Lewis Cantley ◽  
Thomas Roberts
Keyword(s):  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulating Factor ◽  
Phosphatidylinositol 3

Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells

1993 ◽  
Vol 13 (10) ◽  
pp. 6572-6585
Author(s):  
M Dosil ◽  
S Wang ◽  
I R Lemischka
Keyword(s):  
Growth Factors ◽  
Tyrosine Phosphorylation ◽  
Receptor Tyrosine Kinase ◽  
Chimeric Receptor ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Gtpase Activating Protein ◽  
Ras Gtpase ◽  
Mitogenic Signalling ◽  
Stimulating Factor

Flk2/Flt3 is a recently identified receptor tyrosine kinase expressed in brain, placenta, testis, and primitive hematopoietic cells. The mitogenic signalling potential and biochemical properties of Flk2/Flt3 have been analyzed by using a chimeric receptor composed of the extracellular domain of the human colony-stimulating factor 1 receptor and the transmembrane and cytoplasmic domains of murine Flk2/Flt3. We demonstrate that colony-stimulating factor 1 stimulation of the Flk2/Flt3 kinase in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a full proliferative response in the absence of other growth factors. In transfected interleukin 3 (IL-3)-dependent Ba/F3 lymphoid cells, activation of the chimeric receptor can abrogate IL-3 requirement and sustain long-term proliferation. We show that phospholipase C-gamma 1, Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, Vav, Fyn, and Src are components of the Flk2/Flt3 signal transduction pathway. In addition, we demonstrate that phospholipase C-gamma 1, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, and Src family tyrosine kinases, but not Ras GTPase-activating protein, Vav, or Nck, physically associate with the Flk2/Flt3 cytoplasmic domain. Cell-type-specific differences in tyrosine phosphorylation of p85 and Shc are observed. A comparative analysis of the Flk2/Flt3 signal cascade with those of the endogenous platelet-derived growth factor and IL-3 receptors indicates that Flk2/Flt3 displays specific substrate preferences. Furthermore, tyrosine phosphorylation of p85 and Shc is similarly affected by totally different growth factors in the same cellular background.

scholarly journals Phosphatidylinositol 3 Kinase/Akt Signal Relay Cooperates with Smad in Bone Morphogenetic Protein-2-Induced Colony Stimulating Factor-1 (CSF-1) Expression and Osteoclast Differentiation

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 4989-4998 ◽  
Author(s):  
Chandi C. Mandal ◽  
Goutam Ghosh Choudhury ◽  
Nandini Ghosh-Choudhury
Keyword(s):  
Binding Protein ◽  
Osteoclast Differentiation ◽  
Osteoclast Formation ◽  
Bone Morphogenetic Protein 2 ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Osteoblast Activity ◽  
Stimulating Factor ◽  
Phosphatidylinositol 3

Murine spleen cells produce mature osteoclasts when cocultured with osteoblastic cells. Colony-stimulating factor (CSF)-1 is the growth factor required for differentiating the monocyte-macrophage precursor cells into preosteoclasts. Bone morphogenic protein (BMP) signaling in osteoblasts regulates bone mass in mice, suggesting a role of BMP in osteoclastogenesis along with osteoblast activity. The intracellular signal transduction cross talk regulating the osteoblastic production of CSF-1 as a mechanism of BMP-induced osteoclastogenesis is described in this report. We have recently described the involvement of Smad 1/5 in BMP-2-induced CSF-1 expression and osteoclast formation. In this study, using the pharmacological inhibitors and the adenovirus (Ad) vectors expressing dominant-negative (DN) phosphatidylinositol 3 kinase (PI3K), the PI3K-signaling inhibitor, phosphatase and tensin homolog deleted in chromosome 10 (PTEN) or DN Akt kinase in the in vitro coculture assay, we show an essential role of the lipid kinase cascade in BMP-2-mediated multinucleated osteoclast formation and CSF-1 mRNA expression, transcription, and secretion. Inhibition of PI3K/Akt signaling blocked the binding of Smads 1/5 to the CSF-1 BMP-responsive element present in the CSF-1 promoter, resulting in attenuation of Smad-dependent CSF-1 transcription. Furthermore, PI3K inhibition and DN Akt prevented association of the transcriptional coactivator, CREB (cAMP response element binding protein) binding protein (CBP), with Smads 1/5. Together, these data for the first time demonstrate that PI3K-dependent Akt activation regulates BMP-2-induced CSF-1 expression and provides a mechanism for osteoblastic cell-assisted osteoclast differentiation.

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