scholarly journals Modification of Phosphatidylinositol 3-Kinase SH2 Domain Binding Properties by Abl- or Lck-mediated Tyrosine Phosphorylation at Tyr-688

1998 ◽  
Vol 273 (7) ◽  
pp. 3994-4000 ◽  
Author(s):  
Maria von Willebrand ◽  
Scott Williams ◽  
Manju Saxena ◽  
Jennifer Gilman ◽  
Pankaj Tailor ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Sh2 Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Binding Properties ◽  
Phosphatidylinositol 3

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase

1993 ◽  
Vol 13 (3) ◽  
pp. 1737-1745
Author(s):  
J A Cooper ◽  
A Kashishian
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Sh2 Domain ◽  
Phosphorylation Sites ◽  
Phosphatidylinositol 3 Kinase ◽  
Binding Properties ◽  
Sh2 Domains ◽  
Phosphatidylinositol 3

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Determination of Gab1 (Grb2-Associated Binder-1) Interaction with Insulin Receptor-Signaling Molecules

1998 ◽  
Vol 12 (7) ◽  
pp. 914-923 ◽  
Author(s):  
Stéphane Rocchi ◽  
Sophie Tartare-Deckert ◽  
Joseph Murdaca ◽  
Marina Holgado-Madruga ◽  
Albert J. Wong ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Hybrid System ◽  
Sh2 Domain ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Two Hybrid ◽  
Phosphatidylinositol 3

Abstract The newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.

scholarly journals In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

1993 ◽  
Vol 13 (3) ◽  
pp. 1737-1745 ◽  
Author(s):  
J A Cooper ◽  
A Kashishian
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Sh2 Domain ◽  
Phosphorylation Sites ◽  
Phosphatidylinositol 3 Kinase ◽  
Binding Properties ◽  
Sh2 Domains ◽  
Phosphatidylinositol 3

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

1994 ◽  
Vol 14 (1) ◽  
pp. 42-49
Author(s):  
K H Holt ◽  
L Olson ◽  
W S Moye-Rowley ◽  
J E Pessin
Keyword(s):  
Gene Fusion ◽  
Kinase Activity ◽  
Expression System ◽  
Sh2 Domain ◽  
Full Length ◽  
Transactivation Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Amino Terminal ◽  
Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

scholarly journals Tyrosine Phosphorylation of p85 Relieves Its Inhibitory Activity on Phosphatidylinositol 3-Kinase

2001 ◽  
Vol 276 (29) ◽  
pp. 27455-27461 ◽  
Author(s):  
Bruce D. Cuevas ◽  
Yiling Lu ◽  
Muling Mao ◽  
Jinyi Zhang ◽  
Ruth LaPushin ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Inhibitory Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

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.

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