A Comparative Analysis of the Phosphoinositide Binding Specificity of Pleckstrin Homology Domains*
- Lucia E. Rameh‡§,
- Ann-kristin Arvidsson‡¶,
- Kermit L. Carraway III‡,
- Anthony D. Couvillon‡,
- Gary Rathbun‡,
- Anne Crompton‖,
- Barbara VanRenterghem**‡,
- Michael P. Czech**,
- Kodimangalam S. Ravichandran§§,
- Steven J. Burakoff¶¶,
- Da-Sheng Wang166,
- Ching-Shih Chen166 and
- Lewis C. Cantley‡
- From the ‡Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115, ‖Onyx Pharmaceuticals, Richmond, California 94806, the **Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, the ¶¶Division of Pediatric Oncology, Dana-Farber Cancer Institute and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, the166Division of Medical Chemistry Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky 50536-0082, and the §§Beirne B. Carter Center for Immunology Research and Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908
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.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM41890 and GM36624 (to L. C. C.) and GM 53448 (to C.-S. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported by The Medical Foundation of the Charles King Trust, Boston.
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↵¶ Supported by the Swedish Cancer Society.
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↵‡ Supported by a postdoctoral fellowship grant from the Juvenile Diabetes Foundation International.
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↵1 The abbreviations used are: PH, pleckstrin homology; PTB, phosphotyrosine binding; PLC, phospholipase; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; EGF, epidermal growth factor; PtdIns, phosphatidylinositol; Ins, inositol; OSBP, oxysterol-binding protein.
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↵2 A. D. Couvillon, C. L. Carpenter and P. Janmey, unpublished results.
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- Received March 28, 1997.
- Revision received May 30, 1997.
