A region of the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo

1993 ◽  
Vol 13 (9) ◽  
pp. 5560-5566
Author(s):  
A Klippel ◽  
J A Escobedo ◽  
Q Hu ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Catalytic Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Region Gene ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Homology

Phosphatidylinositol (PI) 3-kinase is a heterodimer consisting of an 85-kDa subunit (p85) and 110-kDa subunit (p110). The 85-kDa noncatalytic subunit, which contains two Src homology 2 (SH2) domains, one SH3 domain, and a domain homologous to the carboxy terminus of the breakpoint cluster region gene product, is known to mediate the association of the PI 3-kinase complex with activated growth factor receptors. We previously demonstrated that the C-terminal SH2 domain of p85 is responsible for the interaction of PI 3-kinase with phosphorylated platelet-derived growth factor receptor. To define the region in p85 that directs the complex formation with the PI 3-kinase catalytic subunit, a series of truncated p85 mutants was analyzed for association with p110 in vivo. We found that a fragment of p85 containing the region between the two SH2 domains was sufficient to promote the interaction with p110 in vivo. The complex between the fragment of p85 and p110 had PI 3-kinase activity that was comparable in magnitude to the activity of p110 associated with full-length p85. The binding with p110 was abolished when this domain in p85 was disrupted. These results identify a novel structural and functional element that is responsible for localizing the catalytic subunit of PI 3-kinase.

scholarly journals A region of the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo.

1993 ◽  
Vol 13 (9) ◽  
pp. 5560-5566 ◽  
Author(s):  
A Klippel ◽  
J A Escobedo ◽  
Q Hu ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Catalytic Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Region Gene ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Homology

Phosphatidylinositol (PI) 3-kinase is a heterodimer consisting of an 85-kDa subunit (p85) and 110-kDa subunit (p110). The 85-kDa noncatalytic subunit, which contains two Src homology 2 (SH2) domains, one SH3 domain, and a domain homologous to the carboxy terminus of the breakpoint cluster region gene product, is known to mediate the association of the PI 3-kinase complex with activated growth factor receptors. We previously demonstrated that the C-terminal SH2 domain of p85 is responsible for the interaction of PI 3-kinase with phosphorylated platelet-derived growth factor receptor. To define the region in p85 that directs the complex formation with the PI 3-kinase catalytic subunit, a series of truncated p85 mutants was analyzed for association with p110 in vivo. We found that a fragment of p85 containing the region between the two SH2 domains was sufficient to promote the interaction with p110 in vivo. The complex between the fragment of p85 and p110 had PI 3-kinase activity that was comparable in magnitude to the activity of p110 associated with full-length p85. The binding with p110 was abolished when this domain in p85 was disrupted. These results identify a novel structural and functional element that is responsible for localizing the catalytic subunit of PI 3-kinase.

scholarly journals Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

1994 ◽  
Vol 14 (9) ◽  
pp. 5929-5938 ◽  
Author(s):  
M Yoakim ◽  
W Hou ◽  
Z Songyang ◽  
Y Liu ◽  
L Cantley ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
T Antigen ◽  
Site Directed Mutagenesis ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Src Homology ◽  
Polyomavirus Middle T Antigen ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

scholarly journals Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding receptors DCBLD1 and DCBLD2

2017 ◽  
Vol 474 (23) ◽  
pp. 3963-3984 ◽  
Author(s):  
Anna M. Schmoker ◽  
Jaye L. Weinert ◽  
Kyle J. Kellett ◽  
Hannah E. Johnson ◽  
Ryan M. Joy ◽  
...  
Keyword(s):  
Growth Factor ◽  
High Performance ◽  
Biological Activities ◽  
Family Members ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Src Homology 2 ◽  
Negative Role ◽  
Src Homology ◽  
Epidermal Growth

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL–SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL–SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL–SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

scholarly journals Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity

1994 ◽  
Vol 14 (9) ◽  
pp. 5929-5938
Author(s):  
M Yoakim ◽  
W Hou ◽  
Z Songyang ◽  
Y Liu ◽  
L Cantley ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
T Antigen ◽  
Site Directed Mutagenesis ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Src Homology ◽  
Polyomavirus Middle T Antigen ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

scholarly journals PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells

2013 ◽  
Vol 305 (3) ◽  
pp. C266-C275 ◽  
Author(s):  
Nicholas C. Zachos ◽  
Luke J. Lee ◽  
Olga Kovbasnjuk ◽  
Xuhang Li ◽  
Mark Donowitz
Keyword(s):  
Sh2 Domain ◽  
Signaling Proteins ◽  
Multiprotein Complexes ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Inhibitor ◽  
Intracellular Ca ◽  
Direct Binding ◽  
Src Homology

Elevated levels of intracellular Ca2+([Ca2+]i) inhibit Na+/H+exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca2+]iinhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca2+signaling proteins necessary for regulation of NHE3 activity. [Ca2+]iregulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca2+]iinhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y416phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca2+]iinhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca2+]iconditions, and 4) does not directly bind NHE3. Under elevated [Ca2+]iconditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides

1993 ◽  
Vol 13 (12) ◽  
pp. 7278-7287
Author(s):  
K B Bibbins ◽  
H Boeuf ◽  
H E Varmus
Keyword(s):  
Intramolecular Interaction ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Binding Properties ◽  
Vitro Assay ◽  
Sh2 Domains ◽  
Free Peptide ◽  
C Terminus

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

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