Structural and Biochemical Evaluation of the Interaction of the Phosphatidylinositol 3-Kinase p85α Src Homology 2 Domains with Phosphoinositides and Inositol Polyphosphates

Abstract

Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85␣ subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85␣ SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85␣ SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85␣ SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosinephosphorylated proteins to bind to p85␣. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85␣ SH2 domains.

Specific Binding of the C-terminal Src Homology 2 Domain of the p85α Subunit of Phosphoinositide 3-Kinase to Phosphatidylinositol 3,4,5-Trisphosphate

“…It has been proposed that PIP 3 binds selectively to the p85 Cterminal (CT-)SH2 domain of PI3K, thereby blocking the binding of p85 to tyrosine-phosphorylated proteins (3). This model, however, was refuted by a recent NMR study that showed lack of specific binding of PIP 3 to p85 SH2 domains (13). Nevertheless, this question remains outstanding, because different structural analogues of PIP 3 were used as binding ligands in these investigations, i.e.…”

“…Conceivably, understanding the mode of this unique phosphoinositide recognition will shed light on the mechanism by which PI3K regulates downstream signaling pathways. Although recent investigations demonstrate that distinct PH domains have evolved selectivity for different phosphoinositides to provide discriminatory regulation (5), data pertaining to the interactions between SH2 domains and PIP 3 are conflicting (3,13). It has been proposed that PIP 3 binds selectively to the p85 Cterminal (CT-)SH2 domain of PI3K, thereby blocking the binding of p85 to tyrosine-phosphorylated proteins (3).…”

Deposition of Laminin 5 by Keratinocytes Regulates Integrin Adhesion and Signaling

“…PI3K Assay-PI3K enzymatic activity was assayed as described previously by (49). HFKs were suspended, or adherent on immobilized anti-␣ 2 (P1H5), anti-␣ 3 (P1B5), or anti-␣ 6 (G0H3) integrin antibodies for 1 h, then extracted 1% v/v Nonidet P-40 detergent in kinase buffer (100 mM NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.1 mM sodium orthovanadate, 20 mM MgCl 2 ).…”

Six X-Linked Agammaglobulinemia-Causing Missense Mutations in the Src Homology 2 Domain of Bruton’s Tyrosine Kinase: Phosphotyrosine-Binding and Circular Dichroism Analysis

“…The ligand-binding specificity arises mainly from the nature of the ␤D5 residue and pY ϩ 1 and pY ϩ 3 binding pockets (26,34). The second-messenger phosphoinositides produced by phosphoinositide 3-OH kinase have also been shown to bind to SH2 domains (35), although the physiological significance of this interaction has been disputed recently (36).…”