eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1
Fig 5
Endothelial Nitric Oxide Synthase (eNOS) positively regulates Protein Kinase C-θ (PKC-θ) activation.
(A) PKC-θ Thr538 phosphorylation in CH7C17, eNOS, and G2A T cells, stimulated for 10 min with cross-linked CD3 Ab. On the bottom, densitometric quantification of PKC-θ phosphorylation is shown as the mean fold induction of three independent experiments. Underlying data are provided in S1 Data. (B) PKC-θ phosphorylation in parental CH7C17 T cells, transduced with control or two independent eNOS short hairpin RNAs (shRNAs) (sh eNOS1 and 2) and stimulated 72 h later as in (A) for the time indicated. n = 2. (C) eNOS expression in primary human T lymphoblasts from staphylococcal enterotoxin E (SEE)-stimulated peripheral blood lymphocytes (PBLs), transduced for 72 h with either control (shC) or eNOS (sheNOS) shRNAs. Human umbilical vein endothelial cells (HUVEC) extracts were loaded as control. (D) PKC-θ phosphorylation in human T lymphoblasts interfered for eNOS as in (C) and subsequently stimulated with SEE-pulsed Raji antigen-presenting cells (APCs) (cell ratio = 4:1) for the time indicated. n = 3. (E) PKC-θ phosphorylation in eNOS T cells, transduced with control or eNOS shRNAs and stimulated 72 h later with SEB-pulsed Raji APCs for the time indicated. n = 4. (F) eNOS and G2A T cells were stimulated with CD3 Ab for the time indicated. eNOS, phospho-IKKα/β, and total and phosphorylated PKC-θ, CARMA1, and IκBα were detected by immunoblot. n = 3.