Tandem SH2 Domains Confer High Specificity in Tyrosine Kinase Signaling*
- From the Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 and §ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts 02139
Abstract
SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors. Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-γ1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5–3.0 nm). Alternative TAMs bind the tandem SH2 domains with 1,000- to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the ∼20–50-fold typically seen for individual SH2 domains. We conclude that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates.
Footnotes
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↵* These studies were funded in part by National Institutes of Health Grants DK43123 and DK45943 (to S. E. S.) and by ARIAD Pharmaceuticals. The Biochemistry Facility at the Joslin Diabetes Center is supported by National Institutes of Health DERC Grant DK36836.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 National Institutes of Health Fellowship DK09146.
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↵¶ To whom correspondence should be addressed. Present address: 130 Waverly St., Cambridge MA 02139. Tel.: 617-577-6375; Fax: 617-577-6400; E-mail: botfield{at}macnet.vpharm.com.
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↵‖ Recipient of a Burroughs Wellcome Fund scholar award in experimental therapeutics.
To whom correspondence should be addressed: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2528; Fax: 617-735-1970; E-mail: Shoelson{at}Joslab.Harvard.edu.
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↵1 G. Wolf and S. E. Shoelson, unpublished observations.
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↵2 The abbreviations used are: TAM, tyrosine-based activation motif; PI, phosphatidylinositol; PLC, phospholipase C; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; pY, phosphotyrosine; IRS, insulin receptor substrate; Fmoc,N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; TCR, T cell receptor; RU, resonance unit(s); cRU, corrected resonance unit(s).
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↵3 A. Proudfoot, personal communication.
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↵4 E. A. Ottinger, M. C. Botfield, and S. E. Shoelson, unpublished observation.
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↵5 M. C. Botfield, unpublished result.
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↵6 P. Hof, S. Pluskey, S. Dhe-Paganon, M. Eck, and S. E. Shoelson, submitted for publication.
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- Received September 10, 1997.
- Revision received October 23, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
