Abstract
The phosphotyrosine-binding (PTB) domain of Drosophila Shc (dShc) binds in vitro to phosphopeptides containing the sequence motif NPXpY, and physically associates with the activated Drosophila epidermal growth factor receptor homologue (DER) in vivo. The structural elements, specificity and binding kinetics of the dShc PTB domain have now been characterized. The dShc PTB domain appeared similar to the insulin-like receptor substrate-1 PTB domain in secondary structure as suggested by Fourier transform infrared spectroscopy. Surface plasmon resonance measurements indicated that the dShc PTB domain bound with high affinity to phosphopeptides (Der) derived from the Tyr1228 site of the DER receptor. The kinetics of the dShc PTB domain-Der phosphopeptide interaction differed from those of a typical SH2 domain-ligand interaction, in that the PTB domain displayed slower on/off rates. Competition binding assays using truncated versions of the Der peptides revealed that high affinity binding to the dShc PTB domain requires, in addition to the NPXpY motif, the presence of hydrophobic residues at both positions -5 and -7 relative to phosphotyrosine. The dShc PTB domain showed a similar binding specificity to the human Shc (hShc) PTB domain, but subtle differences were noted; such that the hShc PTB domain bound preferentially to a phosphopeptide from the mammalian nerve growth factor receptor, whereas the dShc PTB domain bound preferentially to phosphopeptides from the Drosophila DER receptor. The invertebrate dShc PTB domain therefore possesses a binding specificity for tyrosine-phosphorylated peptides that is optimally suited for recognition of the activated DER receptor.
Highlights
The phosphotyrosine-binding (PTB) domain of Drosophila Shc binds in vitro to phosphopeptides containing the sequence motif NPXpY, and physically associates with the activated Drosophila epidermal growth factor receptor homologue (DER) in vivo
Kinetics of Drosophila Shc (dShc) PTB Binding to a Phosphopeptide Derived from the DER Receptor—A polypeptide containing residues 1–203 of dShc, and encompassing the PTB domain, was expressed in E. coli
High Affinity Binding of dShc PTB to the Der Phosphopeptides Requires Hydrophobic Residues at Both Ϫ7 and Ϫ5 Positions—Since hydrophobic residues N-terminal to the Tyr(P) have been implicated in the interactions of the Shc and IRS-1 PTB domains with their respective phosphopeptide ligands, we investigated the roles of the hydrophobic residues present at positions Ϫ5, Ϫ7, Ϫ9, and Ϫ11 of the Der1 peptide in binding to the dShc PTB domain
Summary
The phosphotyrosine-binding (PTB) domain of Drosophila Shc (dShc) binds in vitro to phosphopeptides containing the sequence motif NPXpY, and physically associates with the activated Drosophila epidermal growth factor receptor homologue (DER) in vivo. To examine if Shc function is evolutionarily conserved, we have previously cloned a Drosophila shc gene homologue, dshc [34] This gene encodes a 45-kDa protein that is closely related in organization to mammalian p52 Shc and contains a C-terminal SH2 domain, an N-terminal PTB domain, and a central CH1 region. Sequence alignment revealed 51% identity between the PTB domains of p52 Shc and dShc, and the dShc PTB domain was found to interact with phosphopeptides containing an NPXpY motif, including a phosphopeptide modeled after the Tyr1228 site in the DER receptor tyrosine kinase. Autophosphorylation of Tyr1228 in DER may create a physiological binding site for the dShc PTB domain, based on the observation that the dShc protein associates physically with activated DER in vivo [34]
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