Figure 4

AIP regulates optimal T cell signaling responses. (A) IL-2 mRNA levels are decreased in AIP knockdown cells. Jurkat T cells were transfected with siGFP or siAIP1 and 3 and stimulated with P/I or CD3/CD28 for 3 h. RNA was isolated and IL-2 transcript levels were analyzed by quantitative RT-PCR. Bars show average and standard deviation of three independent experiments. (B) Downregulation of AIP reduces IL-2 production. Jurkat T cells were transfected with siRNAs (siAIP1, 2 and 3) as in (A) and stimulated with P/I. Secreted IL-2 was measured by ELISA. In A and B bars show average and standard deviation of three independent experiments. Significance was evaluated using Student t-test (one star: p < 0,05; three stars p < 0,001). (C) AIP knockdown diminishes NF-κB and NF-AT activation, while AP-1 activation is only slightly affected. Jurkat T cells were transfected with siRNAs against GFP and AIP (siAIP1 and 3), respectively, and stimulated with P/I or CD3/CD28 for 3 hours. NF-κB, NF-AT and AP-1 DNA binding was assessed by EMSA. (D) Working model how AIP affects CARMA1. Upon CARMA1 linker phosphorylation by PKCθ, AIP binding to PDZ-SH3 can compete for intramolecular GUK-SH3 interaction, which may facilitate structural rearrangements to transfer the double-closed conformation of CARMA1 into an active open state.