ERK and JNK control cPLA₂ activation and NKG2D-mediated cytolysis. (A) JNK and ERK both regulate cPLA₂ phosphorylation and cytolysis. Results shown were obtained with TALL-104 CTLs. Similar results were obtained with IE-CTLs. (top left) Phosphorylation time course. TALL-104 CTLs were serum-starved for 16 h before stimulation with anti-NKG2D antibodies for the indicated time. Cell lysates were subjected to Western blot analysis by anti–phospho-ERK, -JNKK1, -JNK, and -cPLA₂–specific antibodies. (bottom left) ERK and JNK inhibitors block, in an additive manner, cPLA₂ phosphorylation. TALL-104 CTLs were pretreated with vehicle or suboptimal doses (10 µM) of specific kinase inhibitors. PD 98059 (PD) and SP600125 (SP) are MEK1/2 and JNK inhibitors, respectively. cPLA₂ phosphorylation was determined by immunoblot with anti-phospho cPLA₂ after 15 min of stimulation with anti-NKG2D mAbs. Total cPLA₂ is shown as loading control. (right) AA significantly restored NKG2D-mediated cytolysis against C1R/MIC transfectants upon JNK, but not ERK, inhibition. TALL-104 CTLs were pretreated with vehicle and inhibitors as indicated in the top right graph. The rescue assay was done by addition of AA 1 h after co-culture of TALL-104 with ⁵¹Cr-labeled C1R/MIC transfectants. Data are means ± SD of three independent cytolytic assays. (B) The Vav→JNKK1/2→JNK pathway controls cPLA₂ phosphorylation. Phosphorylation experiments were performed on cells starved overnight and stimulated for the indicated time with anti-NKG2D antibodies. Western blots for phospho-JNK and -cPLA₂ were performed on total cell lysates using anti–phospho-JNK and -cPLA₂ antibodies. Vav phosphorylation was evaluated after immunoprecipitation with anti-Vav1 mAb and immunoblotting with anti-phosphotyrosine mAb. Equal loading for total lysates and for Vav 1 immunoprecipitation were assessed using anti-cPLA₂ and -Vav antibody, respectively. (top left) Time course of anti-NKG2D–induced Vav phosphorylation. (top right) Dominant-negative Vav-1 (Vav 1-ΔC) blocked JNK and cPLA₂ activation. TALL-104 CTLs were transfected with control vector and Vav-ΔC and cultured for 48 h. Cells were then analyzed by Western blot as indicated. (bottom left) Dominant-negative JNKK2 blocks cPLA₂ phosphorylation. TALL-104 CTLs transfected with control vector and dominant-negative JNKK2(KM) were cultured for 48h and analyzed by Western blot. (bottom right) Constitutive JNK activation induced cPLA₂ phosphorylation. TALL-104 CTLs were transfected with JNKK2-JNK1 (which encodes a constitutively active form of JNK) or with JNKK2(KM)-JNK1 (which encodes a kinase-deficient mutant), in addition to GFP plasmids. After 40 h of transfection, cells were sorted by flow cytometry into GFP⁺ and GFP⁻ cells. Presence of p-cPLA₂ was evaluated by immunoblot in the absence of NKG2D stimulation. NKG2D-stimulated and nonstimulated TALL-104 CTLs, transfected with a control vector, served as positive and negative control for JNK and cPLA₂ phosphorylation. Equal loading was assessed by immunoblotting with total cPLA₂. (C) ERK pathway regulates cPLA₂ phosphorylation. Dominant-negative and overexpression experiments were performed as in B. ERK and cPLA₂ phosphorylation were determined by Western blot. (left) Dominant-negative MEK-1 (MEK[2A]) significantly blocked cPLA₂ phosphorylation. (right) Constitutively active MEK-1 (MEK[2E]) induced cPLA₂ phosphorylation in the absence of NKG2D stimulation.