Ras and Rap1 activation of PLCε lipase activity

Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in signal transduction from various receptor molecules on the plasma membrane. PLC epsilon is characterized by possession of two Ras/Rap-associating (RA) domains and a CDC25 homology domain acting as a guanine nucleotide exchange factor for Rapt. Our recent studies using PLC epsilon-deficient mice have suggested that PLC epsilon plays crucial roles in cardiac semilunar valvulogenesis downstream of the EGF receptor, as well as in chemical carcinogen-induced skin tumor development downstream of Ha-Ras. Stimulation of cultured mammalian cells with growth factors induces translocation of PLC epsilon from the cytoplasm to the plasma membrane and to the Golgi apparatus through direct association at its RA domains with the GTP-bound forms of Ras and Rap1, respectively. These results suggest that growth factor stimulation activates PLC epsilon by means of Ras and/or Rapt. However, growth factor-induced activation of the PLC epsilon lipase activity cannot be measured accurately because of simultaneous activation of PLC gamma through receptor-dependent phosphorylation. In this article, we introduce two methods to assay Ras- or Rap1-dependent activation of PLC epsilon lipase activity, with special emphasis on the use of cells expressing a mutant platelet-derived growth factor receptor lacking the PLC gamma-binding sites.