The direct interaction of phospholipase C-γ1 with phospholipase D2 is important for epidermal growth factor signaling

Abstract

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-γ1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-γ1 and phospholipase D2 (PLD2). PLC-γ1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and coimmunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-γ1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-γ1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-γ1 and PLD2 and fails to potentiate EGF-induced IP3 formation and Ca2+ increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-γ1. These findings suggest that, upon EGF stimulation, PLC-γ1 directly interacts with PLD2 and this interaction is important for PLC-γ1 activity.