Chuang Samuel S, Lee Jae-Kyung, Mathew Porunelloor A
Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107-2699, USA.
Immunology. 2003 Jul;109(3):432-9. doi: 10.1046/j.1365-2567.2003.01662.x.
2B4 (CD244) is a member of the CD2 subset of the immunoglobulin superfamily and functions as a triggering molecule on natural killer (NK) cells. Previously, we have found that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways. Here we assessed the role of protein kinase C (PKC) in 2B4-mediated cytotoxicity of YT cells, a human NK cell line. Our data indicate that PKC-delta is activated upon stimulation with monoclonal antibody against 2B4. Treatment with the PKC inhibitor, bisindolylmaleimide I (Gö6850), of YT cells or YT cells depleted of Ca2+-dependent isoforms of PKC prior to 2B4 stimulation, resulted in inhibition of natural cytotoxicity and redirected antibody-dependent cellular cytotoxicity. However, inhibition of PKC failed to block 2B4 stimulation of interferon-gamma secretion as opposed to pretreatment with LY294002, a phosphoinositide 3-kinase inhibitor. We also examined the effect of phorbol 12-myristate 13-acetate (PMA) induction on 2B4 gene transcription. PMA induction resulted in a more than two-fold increase of 2B4 transcription. However, when we introduced a three-base substitution mutation to disrupt the activator protein-1 binding site at (-106 to -100) in the 2B4 promoter, we found complete loss of transcriptional activity, including the two-fold increase due to PMA induction of PKC. The present study indicated that PKC may play an important role in 2B4 signalling and activator protein-1 activation.
2B4（CD244）是免疫球蛋白超家族CD2亚群的成员，在自然杀伤（NK）细胞上作为触发分子发挥作用。此前，我们发现2B4介导的NK细胞激活涉及LAT、Ras、Raf、ERK和p38的复杂相互作用，并且细胞溶解功能和细胞因子产生可能受不同途径调节。在此，我们评估了蛋白激酶C（PKC）在2B4介导的人NK细胞系YT细胞细胞毒性中的作用。我们的数据表明，用抗2B4单克隆抗体刺激后PKC-δ被激活。在2B4刺激之前，用PKC抑制剂双吲哚基马来酰亚胺I（Gö6850）处理YT细胞或耗尽PKC钙依赖性同工型的YT细胞，导致自然细胞毒性受到抑制以及抗体依赖性细胞毒性重定向。然而，与用磷酸肌醇3-激酶抑制剂LY294002预处理相反，抑制PKC未能阻断2B4对干扰素-γ分泌的刺激。我们还研究了佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）诱导对2B4基因转录的影响。PMA诱导导致2B4转录增加两倍以上。然而，当我们引入一个三碱基取代突变以破坏2B4启动子中（-106至-100）的激活蛋白-1结合位点时，我们发现转录活性完全丧失，包括由于PKC的PMA诱导导致的两倍增加。本研究表明PKC可能在2B4信号传导和激活蛋白-1激活中起重要作用。
