CKIP-1 Is an Intrinsic Negative Regulator of T-Cell Activation through an Interaction with CARMA1

<div><p>The transcription factor NF-κB plays a key regulatory role in lymphocyte activation and generation of immune response. Stimulation of T cell receptor (TCR) induces phosphorylation of CARMA1 by PKCθ, resulting in formation of CARMA1-Bcl10-MALT1 (CBM) complex at lipid rafts and subsequently leading to NF-κB activation. While many molecular events leading to NF-κB activation have been reported, it is less understood how this activation is negatively regulated. We performed a cell-based screening for negative regulators of TCR-mediated NF-κB activation, using mutagenesis and complementation cloning strategies. Here we show that casein kinase-2 interacting protein-1 (CKIP-1) suppresses PKCθ-CBM-NF-κB signaling. We found that CKIP-1 interacts with CARMA1 and competes with PKCθ for association. We further confirmed that a PH domain of CKIP-1 is required for association with CARMA1 and its inhibitory effect. CKIP-1 represses NF-κB activity in unstimulated cells, and inhibits NF-κB activation induced by stimulation with PMA or constitutively active PKCθ, but not by stimulation with TNFα. Interestingly, CKIP-1 does not inhibit NF-κB activation induced by CD3/CD28 costimulation, which caused dissociation of CKIP-1 from lipid rafts. These data suggest that CKIP-1 contributes maintenance of a resting state on NF-κB activity or prevents T cells from being activated by inadequate signaling. In conclusion, we demonstrate that CKIP-1 interacts with CARMA1 and has an inhibitory effect on PKCθ-CBM-NF-κB signaling.</p></div

PH domain of CKIP-1 is essential not only for the interaction with CARMA1 but also for the inhibitory effect on NF-κB activation.

<p>(A) Jurkat T cells were electroporated with 5 µg of each CKIP-1 truncated form together with 5 µg of κB-Luc and 0.1 µg of <i>Renilla</i>-Luc. Nineteen hours later, cells were stimulated for 5 hr upon PMA (10 ng/ml) or CD3/CD28 (2 µg/ml each). The expressed protein levels were analyzed by Western blotting. (B) Jurkat T cells were electroporated with 5 µg of each CKIP-1 truncated form together with 5 µg of PKCθ AE or Myc-CARMA1, 5 µg of κB-Luc and 0.1 µg of <i>Renilla</i>-Luc. After 24 hr, cells were lysed and luciferase activity was assessed. The expressed protein levels were analyzed by Western blotting. Values represent the average of three independent experiments and error bars represent the SD from the average.</p

Identification of CKIP-1 as a negative regulator in NF-κB activation.

<p>(A) 400 pmol of human CKIP-1-specific siRNA or non-targeting siRNA together with 5 µg of κB-Luc, 0.1 µg of <i>Renilla</i>-Luc were electroporated into Jurkat T cells. Luciferase activity was assayed after 48 hr. The reduction of endogenous CKIP-1 protein levels was analyzed by Western blotting. (B) Jurkat T cells were electroporated with human CKIP-1-specific siRNA or non-targeting siRNA using AMAXA Nucleofector System (Lonza). Thirty hours later, nuclear protein extracts were harvested and NF-κB activity was measured by TransAM NF-κB p65 chemi kit (Active Motif). The reduction of endogenous CKIP-1 protein levels was analyzed by Western blotting. (C) Jurkat T cells were transfected with 5 µg of CKIP-1 or empty vector (mock) together with 5 µg of κB-Luc and 0.1 µg of <i>Renilla</i>-Luc. Nineteen hours later, cells were stimulated for 5 hr upon CD3 (2 µg/ml), CD3/CD28 (2 µg/ml each), TNFα (20 ng/ml), PMA (10 ng/ml) or PMA (10 ng/ml) + CD28 (2 µg/ml). The expressed protein levels were analyzed by Western blotting. (D) Jurkat T cells were transfected with 5 µg of CKIP-1 or empty vector (mock). Twenty-four hours later, cells were stimulated for 30 min upon PMA (10 ng/ml). Then cells were harvested and NF-κB activity was measured by TransAM NF-κB p65 chemi kit. The expressed protein levels were analyzed by Western blotting. Values represent the average of three independent experiments and error bars represent the SD from the average.</p

Lipid rafts accumulated by CD3/CD28 costimulation do not contain CKIP-1.

<p>Jurkat T cells were stimulated for 15-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml), together with 15 µg of mouse IgG. The cells were then lysed and subjected to OptiPrep density gradient centrifugation to isolate lipid rafts. Lysates were subjected to SDS-PAGE and analyzed by Western blotting.</p

CKIP-1 inhibits the interaction between PKCθ and CARMA1.

<p>(A) HEK293T cells were transfected with CKIP-1 or empty vector (mock) together with PKCθ and FLAG-CARMA1 (left panel), or together with HA-Bcl10 and FLAG-CARMA1 (right panel). Cell lysates were immunoprecipitated by anti-FLAG antibody, followed by Western blotting with indicated antibodies. (B) HEK293T cells were transfected with CKIP-1 truncated form together with PKCθ and FLAG-CARMA1. Cell lysates were immunoprecipitated by anti-FLAG antibody, followed by Western blotting with indicated antibodies.</p