Abstract
The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.
Highlights
A rapid cellular response to growth factor binding to cell surface receptors is the hydrolysis of phosphatidylinositol 4,5bisphosphate to produce two second messengers: inositol 1,4,5trisphosphate and diacylglycerol [1]
One analysis of SH2 fusion protein binding to activated epidermal growth factor (EGF) receptors showed binding by the N-SH2 domain and, based on the synergistic binding of an construct containing both N-SH2 and C-SH2 domains, concluded that the C-SH2 domain bound to the EGF receptor [23]
To assess the function of phospholipase C-␥1 (PLC-␥1) SH2 domains within a larger segment of the native protein, we constructed the fusion proteins shown in Fig. 1A to contain the entire central region of phospholipase C (PLC)-␥1, which contains two SH2 domains and one SH3 domain
Summary
Materials—BIAcore streptavidin chips were obtained from BIAcore (Uppsala, Sweden). The 96-well ELISA microtiter plates (Falcon 3912) were products of Becton Dickinson Labware. Surface Plasmon Resonance Spectroscopy—Real time binding kinetics of PLC-␥1 SH2 fusion proteins to immobilized EGF receptor phosphotyrosine peptides were measured using a BIAcore instrument (BIAcore 2000). The GST fusion proteins at 100, 200, 300, 400, or 500 nM concentrations were passed over the immobilized phosphotyrosine peptides or the blank chip surface at a flow rate of 10 l/min for 10 min at 25 °C. To determine the binding affinities of GST-PLC-␥1 SH2 proteins toward EGF receptor-phosphotyrosine peptides, the individual wells of avidincoated plates were filled with 50 l of a biotinylated peptide (150 nM), incubated for 1 h at room temperature and washed with PBS containing 0.05% Tween 20. Horseradish peroxidase-conjugated GST antibodies, prepared as described elsewhere [42], in PBS supplemented with 0.05% Tween 20 were added into each well and incubated for an additional 1 h at room temperature.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
