Abstract
Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.
Highlights
PLC-γ1 plays a pivotal role in cell growth and differentiation by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG)
COS7 cells transfected with FLAG-tagged cDNA encoding either Y509A/F510A mutant or wild-type PLC-γ1 protein were treated with 50 ng/ml EGF for 10 min, or untreated, and cell lysates were subjected to immunoprecipitation using anti-FLAG antibodies
Our results indicate that the Y509A/F510A mutations strongly promoted Y783 phosphorylation upon EGF stimulation in vivo
Summary
PLC-γ1 plays a pivotal role in cell growth and differentiation by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG). Agonist stimulation results in activation of receptor tyrosine kinases, such as the EGF receptor and the PDGF receptor, followed by association with PLC-γ1 (Rhee, 2001; Meisenhelder et al, 1989) This interaction allows receptor tyrosine kinases to phosphorylate PLC-γ1 at tyrosine residues, including Y771, Y783, and Y1254 (Kim et al, 1990, 1991; Wahl and Carpenter, 1998; Sekiya et al, 2004; Poulin et al, 2005). Y783 is proposed to be a critical phosphorylation site for enzyme activation, in view of the finding that the Y783F mutant protein is devoid of PIP2 hydrolyzing activity in agonist-treated cells (Kim et al, 1991; Sekiya et al, 2004; Poulin et al, 2005)
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