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

Lucia E Rameh,Ann-Kristin Arvidsson,Kermit L Carraway,Anthony D Couvillon,Gary Rathbun,Anne Crompton,Barbara Vanrenterghem,Michael P Czech,Kodimangalam S Ravichandran,Steven J Burakoff,Da-Sheng Wang,Ching-Shih Chen,Lewis C Cantley

doi:10.1074/jbc.272.35.22059

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 beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and 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.

Highlights

¶ Supported by the Swedish Cancer Society. ‡‡ Supported by a postdoctoral fellowship grant from the Juvenile Diabetes Foundation International
3,4-P2 poorly compared with PtdIns-4,5-P2 or PtdIns-3,4,5-P3, indicating specificity to the binding. These results indicate that distinct PH domains have evolved selectivity for different phosphoinositides to provide discriminatory regulation
A control phosphopeptide (10 ␮M) had little effect on PtdIns-3,4,5-P3 binding. These results suggest that, like PtdIns-4,5-P2, PtdIns3,4,5-P3 can directly bind to the Shc-PTB domain, and this interaction can be competed with a phosphopeptide that binds with high affinity to this domain

Summary

Introduction

¶ Supported by the Swedish Cancer Society. ‡‡ Supported by a postdoctoral fellowship grant from the Juvenile Diabetes Foundation International. PH domains were first identified as protein regions that share similarities with pleckstrin, the major protein kinase C substrate in platelets [6] They contain approximately 120 amino acids and are found in several proteins involved in signaling, such as protein kinases (Btk, Akt, and ␤ark), phospholipases (PLC), and proteins that act as exchange factors or GTPase-activating proteins for small G-proteins (Ras-GRF, Dbl, SOS, Ras-GAP, Tiam-1) [7]. In most of the studies referenced above, the possibility that the various PH domains might bind to products of PI 3-kinase was not investigated This is an important question since unlike PtdIns-4,5-P2 and PtdIns-4-P, which are constitutively produced in cells, PtdIns-3,4-P2 and PtdIns3,4,5-P3 are nominally absent in quiescent cells and only appear in response to cell stimulation [25].

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Results

Conclusion