Ras and Rap1 activation of PLCepsilon lipase activity.
Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in signal transduction from various receptor molecules on the plasma membrane. PLCepsilon is characterized by possession of two Ras/Rap-associating (RA) domains and a CDC25 homology domain acting as a guanine nucleotide exchange factor for Rap1. Our recent studies using PLCepsilon-deficient mice have suggested that PLCepsilon 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 PLCepsilon 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 PLCepsilon by means of Ras and/or Rap1. However, growth factor-induced activation of the PLCepsilon lipase activity cannot be measured accurately because of simultaneous activation of PLCgamma through receptor-dependent phosphorylation. In this article, we introduce two methods to assay Ras- or Rap1-dependent activation of PLCepsilon lipase activity, with special emphasis on the use of cells expressing a mutant platelet-derived growth factor receptor lacking the PLCgamma-binding sites.
Edamatsu
Hironori
H
Division of Molecular Biology, Department of Molecular and Cellular Biology, Kobe University, Graduate School of Medicine, Kobe, Japan.
Satoh
Takaya
T
Kataoka
Tohru
T
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D009687:Nuclear Proteins; D054801:Phosphoinositide Phospholipase C / Q000032:analysis / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000502:physiology; D011869:Radioligand Assay; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D007295:Inositol Phosphates; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; C490403:SIPA1 protein, human; D017479:Receptors, Platelet-Derived Growth Factor; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins