The role of individual SH2 domains in mediating association of phospholipase C-gamma1 with the activated EGF receptor.
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.
Chattopadhyay
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A
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Vecchi
M
M
Ji
Q s
Qs
Mernaugh
R
R
Carpenter
G
G
D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D010738:Type C Phospholipases; D051966:Phospholipase C gamma