The p.Ser707Tyr Substitution Enhances PLCγ2 Activity and Intracellular Ca2+ Flux via the IP3 Signaling Pathway
(A) PLCγ2 activity was assayed in COS-7 cells transfected with wild-type (WT) or p.Ser707Tyr altered PLCγ2 constructs. [3H]inositol phosphate production was measured after stimulation with 10 ng/μl epidermal growth factor (EGF). PLCγ2 levels were analyzed by immunoblot assay (insets).
(B) The WT PLCγ2 construct (#RC200442, Origene, Rockville, MD) and the p.Ser707Tyr altered PLCγ2 construct (Site-Directed Mutagenesis Kit, QuikChange II, Stratagene-Agilent Technologies, Santa Clara, CA) were transiently introduced into 293T cells with Lipofectamine 2000 according to the manufacturers’ protocols. The transfected 293T cells were treated for 1 hr with EGF (150 ng/ml, #CN02, Cytoskeleton, Denver, CO) for Rac-mediated PLCγ2 activation and were subjected to the IP-one assay. The IP1 (surrogate for IP3) level in the cell lysate was measured by IP-One ELISA (#72IP1PEA, Cisbio, Bedford, MA) according to the manufacturer’s instructions.
(C) Increasing concentrations of PLCγ2 WT and p.Ser707Tyr altered constructs (0, 1.5, or 3.0 μg/106 cells) were transiently introduced into 293T cells for an assay of the intracellular Ca2+ level. The transfected 293T cells were plated onto 96-well plates (70,000 cells per well) and treated with EGF (150 ng/ml) and the calcium-indicating dye (Calcium Assay Kit 640176, BD Biosciences, San Diego, CA). The plates were cooled down to room temperature for 15 min, and the relative fluorescence was measured every 10 s for the duration of 6 min with the use of Victor (PerkinElmer, Waltham, MA) according the manufacturers’ manuals.
(D) The protein levels of PLCγ2 WT and p.Ser707Tyr altered constructs in 293T cells were confirmed by immunoblot assay.
All graphs represent three or four independent experiments, and error bard indicate the mean ± the standard error of the mean (SEM).