A Comparative Analysis of the Phosphoinositide Binding Specificity of Pleckstrin Homology Domains

TLDR

Pleckstrin homology and phosphotyrosine binding domains are regulatory modules in signal transduction that mediate protein interactions and bind phosphoinositides, yet most research has focused on PtdIns‑4‑P and PtdIns‑4,5‑P2, leaving PI3K‑derived lipids understudied. The authors compared the phosphoinositide binding specificity of six PH domains and the Shc PTB domain across all five phosphoinositides. The study found that Btk PH binds PtdIns‑3,4,5‑P3 more strongly than other phosphoinositides, a selectivity reduced by the xid mutation, while Tiam‑1, Sos, and β‑adrenergic receptor kinase PH domains also preferentially bind PtdIns‑3,4,5‑P3, whereas oxysterol binding protein, β‑spectrin PH, and Shc PTB domains bind PtdIns‑3,4,5‑P3 and PtdIns‑4,5‑P2 with comparable affinities, demonstrating that different PH domains exhibit distinct phosphoinositide preferences.

Abstract

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P₂ and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P₂, and PtdIns-3,4,5-P₃. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4,5-P₃ with higher affinity than to PtdIns-4,5-P₂, PtdIns-3,4-P₂ or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P₄). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P₃ over PtdIns-4,5-P₂ or PtdIns-3,4-P₂ was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the β-adrenergic receptor kinase. The oxysterol binding protein and β-spectrin PH domains bound PtdIns-3,4,5-P₃and PtdIns-4,5-P₂ with similar affinities. PtdIns-3,4,5-P₃ and PtdIns-4,5-P₂ also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.