Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2578-2585 ◽  
Author(s):  
Carinne Lecoq-Lafon ◽  
Frédérique Verdier ◽  
Serge Fichelson ◽  
Stany Chrétien ◽  
Sylvie Gisselbrecht ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Direct Consequence ◽  
Receptor Activation ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitors ◽  
Epo Receptor ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Phosphatidylinositol 3

Abstract Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2578-2585 ◽  
Author(s):  
Carinne Lecoq-Lafon ◽  
Frédérique Verdier ◽  
Serge Fichelson ◽  
Stany Chrétien ◽  
Sylvie Gisselbrecht ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Direct Consequence ◽  
Receptor Activation ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitors ◽  
Epo Receptor ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Phosphatidylinositol 3

Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

scholarly journals Granulocyte-Macrophage Colony-Stimulating Factor–Activated Signaling Pathways in Human Neutrophils. I. Tyrosine Phosphorylation-Dependent Stimulation of Phosphatidylinositol 3-Kinase and Inhibition by Phorbol Esters

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1035-1044 ◽  
Author(s):  
Amin Al-Shami ◽  
Sylvain G. Bourgoin ◽  
Paul H. Naccache
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phorbol Esters ◽  
Pi3 Kinase ◽  
Human Neutrophils ◽  
Phosphatidylinositol 3 Kinase ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Stimulation Of

Abstract Phosphatidylinositol 3-kinase (PI3-kinase) is a cytosolic enzyme that plays key roles in mediating signaling through many receptors. The heterodimeric form of PI3-kinase is made up of a regulatory subunit, p85, and a catalytic subunit, p110. Although granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate PI3-kinase, the mechanisms by which this activation is mediated and regulated are incompletely understood. Here we show that treatment of human neutrophils with GM-CSF induced both time- and concentration-dependent increases in the level of tyrosine phosphorylation of p85. The ability of GM-CSF to activate PI3-kinase was abolished by pretreating the cells with erbstatin, a tyrosine kinase inhibitor. The simultaneous treatment of the cells with GM-CSF and phorbol esters such as phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) significantly inhibited both the tyrosine phosphorylation of p85 and the activation of PI3-kinase. The inhibitory effects of phorbol esters were not induced by their inactive analogues and they were selective to the stimulation of tyrosine phosphorylation of p85 since phorbol esters did not alter the enhancement of the pattern of tyrosine phosphorylation of other cellular proteins, including that of Jak2 induced by GM-CSF. However, PMA significantly inhibited the in situ tyrosine phosphorylation and the activation of lyn observed in response to GM-CSF. The results suggest that the activation of PI3-kinase by GM-CSF is mediated by the tyrosine phosphorylation of p85 and that this activation is downregulated by PKC possibly via the inhibition of lyn.

scholarly journals Tyrosine phosphorylation of the erythropoietin receptor: role for differentiation and mitogenic signal transduction

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 598-606 ◽  
Author(s):  
S Gobert ◽  
F Porteu ◽  
S Pallu ◽  
O Muller ◽  
M Sabbah ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Expression Vectors ◽  
Tyrosine Residue ◽  
Phosphatidylinositol 3 Kinase ◽  
Intracellular Domain ◽  
Tyrosine Residues ◽  
Epo Receptor ◽  
Phosphatidylinositol 3

The erythropoietin (Epo) receptor belongs to the cytokine receptor superfamily. Although the cytokine receptors do not possess a tyrosine kinase consensus sequence in the intracellular domain, rapid stimulation of a tyrosine kinase activity occurs after activation by the ligand. We and others have shown that Epo induces the tyrosine phosphorylation of its cognate receptor as well as phosphorylation of other proteins. In this report, we examined the role of the receptor tyrosine residues in signal transduction. Eight tyrosine residues are located within the intracellular domain of the murine Epo receptor. A single tyrosine residue is present in the region previously shown to be sufficient for proliferative signal transduction. This tyrosine (Tyr 343) was mutated to phenylalanine. Moreover, mutant receptors were also generated with either a tyrosine residue or a phenylalanine residue at position 343 and with a COOH terminal truncation that removed the 7 other tyrosine residues. Expression vectors carrying these mutated receptors were transfected into the interleukin-3-dependent murine cell line Ba/F3. Epo-induced growth was sustained efficiently by all these receptors, although receptors without any tyrosine residues conferred a significantly reduced mitogenic activity. Moreover, all receptors were able to mediate Epo-dependant accumulation of beta-globin mRNA. The mutated receptors all induced the tyrosine phosphorylation of several cellular proteins after Epo stimulation. However, the truncated receptors induced the phosphorylation of a reduced number of proteins, suggesting that phosphorylated tyrosines of the receptor could have a role in the recruitment either of a tyrosine kinase or of tyrosine kinase substrate proteins. The receptors were all able to mediate Epo- induced activation of phosphatidylinositol 3-kinase, although truncated receptors no longer bound phosphatidylinositol 3-kinase.

scholarly journals rab5 GTPase Regulates Adenovirus Endocytosis

1999 ◽  
Vol 73 (11) ◽  
pp. 9664-9668 ◽  
Author(s):  
Tanja Rauma ◽  
Juha Tuukkanen ◽  
Jeffrey M. Bergelson ◽  
Gerene Denning ◽  
Timo Hautala
Keyword(s):  
Tyrosine Phosphorylation ◽  
Hela Cells ◽  
Intracellular Signaling ◽  
Virus Entry ◽  
Dominant Negative ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT Adenovirus interaction with αv integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca2+]c-mediated signaling or for tyrosine phosphorylation of pp125FAK, p130CAS, and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.

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 of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

2000 ◽  
Vol 279 (2) ◽  
pp. E266-E274 ◽  
Author(s):  
Paul G. Drake ◽  
Alejandro Balbis ◽  
Jiong Wu ◽  
John J. M. Bergeron ◽  
Barry I. Posner
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Metabolic Effects ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

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