Abstract
Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin β(3), Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated β(3)integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr(759), which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr(747) of the highly conserved NPXY motif of β(3). The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
One of the three isoforms, the p52 Shc contains three distinct domains: phosphotyrosine-binding (PTB) domain, a poorly characterized glycine/proline-rich region termed as collagen homology domain (CH1), and the SH2 domain
PTB domains comprise a large family of protein binding modules, which exhibit a conserved structural architecture similar to the pleckstrin homology (PH) domains consisting of a core -sandwich made of two anti-parallel  sheets flanked by a C-terminal helix
Summary
Previous studies have shown that two of these domains, PTB and SH2, could potentially interact with 3CT containing phosphorylated tyrosines [12, 13]. Phosphorylated tyrosine is required for high affinity binding in case of proteins such as Shc PTB, IRS-1/IRS-2/IRS-3, Dok, and SNT/FRS2, the PTB domains of Dab1/Dab, ARH, Fe65, ICAP1␣, JIP-1/JIP-1b, Numb, Talin, and X11␣ exhibit similar or in some cases even higher affinity for non-phosphorylated peptides [15]. For Shc PTB-integrin interaction, a bi-phosphorylated (pY747 and pY759) peptide has been shown to have greater binding affinity than a mono-phosphorylated (pY759) peptide [8]. We present the NMR-derived atomic view of how tyrosine phosphorylation affects 3CT interaction with Shc PTB, and we show for the first time a high resolution three-dimensional structure of Shc PTB domain in complex with bi-phosphorylated integrin 3CT peptide
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