Analysis of phospholipase C-γ activation by a truncated receptor tyrosine kinase in vitro

Phospholipase C-γ (PLC-γ) activity increases rapidly upon activation of many receptor tyrosine kinases. To clarify the mechanism of PLC-γ regulation we measured the effect of the intracellular domain of the epidermal growth factor receptor (EGFR-ID), the core tyrosine kinase domain that lacks self-phosphorylation sites (EGFR-KD) and the core self-phosphorylation sites alone (EGFR-CT) on PLC-γ activity in vitro. Tyrosine phosphorylation of PLC-γ was catalyzed by either the cytoplasmic domain or the kinase domain of the EGFR but PLC-γ activity was increased only by the complete cytoplasmic domain of the receptor. Addition of the self-phosphorylation domain alone or in conjunction with the kinase domain did not increase PLC-γ activity. However, both the cytoplasmic domain of the EGFR and the carboxy terminal tail of the EGFR coimmunoprecipitate with PLC-γ. These results suggest that both tyrosine phosphorylation of PLC-γ and binding of PLC-γ to a tyrosine phosphorylated protein are coordinately required for enzyme activation.