Hama N, Paliogianni F, Fessler B J, Boumpas D T
Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Exp Med. 1995 Mar 1;181(3):1217-22. doi: 10.1084/jem.181.3.1217.
Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma BCaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma BCaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma BCaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma BCaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC-dependent signaling systems.
抗原与T细胞受体的结合会激活磷脂酶C，导致细胞内游离钙浓度（[Ca2+]i）升高并激活蛋白激酶C（PKC）。[Ca2+]i升高会激活包括多功能Ca2+/钙调蛋白依赖性蛋白激酶II（CaM-K II）以及钙调神经磷酸酶（一种2B型蛋白磷酸酶）在内的Ca2+/钙调蛋白依赖性激酶。最近的研究已将钙调神经磷酸酶确定为白细胞介素（IL）-2和IL-4启动子激活的关键酶。然而，CaM-K II的作用仍然未知。我们使用了这些激酶和磷酸酶的突变体（分别为γBCaM-K和δCaM-AI）来探究它们在细胞因子基因转录中的相对作用以及它们与PKC依赖性信号系统的相互作用。已知γBCaM-K和δCaM-AI具有组成性的不依赖钙的活性，它们（单独或组合）与含有驱动氯霉素乙酰转移酶报告基因表达的完整IL-2启动子的质粒共转染到Jurkat T细胞中。γBCaM-K与IL-2启动子构建体共转染会下调其在离子霉素和佛波酯肉豆蔻酸酯（PMA）刺激下的转录。CaM-K II对IL-2启动子的抑制作用与其AP-1和NF-AT反式激活途径转录减少有关。在相同条件下，δCaM-AI超诱导IL-2启动子活性（增加约两倍）。当同时使用这两种突变体时，γBCaM-K抑制了δCaM-AI对IL-2启动子的诱导。当使用含有IL-4启动子的构建体时也获得了类似结果。γB*CaM-K还下调了在转染组成性活性PKC突变体或用PMA刺激后AP-1的激活。这些结果表明，CaM-K II可能对人T细胞中的细胞因子基因转录产生负面影响，并为与钙调神经磷酸酶和PKC依赖性信号系统的负性相互作用提供了初步证据。
