Ouellet Michel, Roy Jocelyn, Barbeau Benoit, Geleziunas Romas, Tremblay Michel J
Centre de Recherche en Infectiologie, Hôpital CHUL, Centre Hospitalier Universitaire de Québec, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Quebéc, Canada.
Biochemistry. 2003 Jul 15;42(27):8260-71. doi: 10.1021/bi034013b.
We have previously shown that bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors can potently activate NF-kappaB. We have already determined that p56(lck), ZAP-70, SLP-76, capacitative entry of calcium, and calcium-regulated effectors are important in bpV-induced NF-kappaB activation. In this study, we evaluated whether other signal transducers previously reported in NF-kappaB induction by T cell activating stimuli are also activated by bpV compounds. Nuclear translocation of NF-kappaB was evaluated in cell lines deficient for either CD45 or p36(LAT) to assess the role of these signal transducers in bpV-mediated NF-kappaB activation. A deficiency of either protein greatly reduced the extent of NF-kappaB nuclear translocation following bpV treatment. Isoform-specific PKC inhibitors were then used to show that bpV compounds activate NF-kappaB through both calcium-sensitive and -insensitive PKC isoforms. The implication of the IkappaB-kinase complex was then investigated through the use of an IkappaBalpha-specific kinase assay and plasmids expressing catalytically inactive forms of IKKalpha and IKKbeta. Upstream kinases involved in IKK complex activation such as TPL-2/COT, NIK, and IKKepsilon were also shown to play an important role in bpV-mediated NF-kappaB activation. Finally, reporter gene transcriptional assays and gel shift assays were performed to compare the kinetics of activation of NF-kappaB by bpV with those of antigenic and TNFalpha stimulation. We demonstrate, both in Jurkat cells and in primary T cells, that bpV-mediated NF-kappaB activation kinetics are comparable to those of an antigenic stimulation but occur much slower than the kinetics seen upon TNFalpha treatment.
我们之前已经表明，双过氧钒（bpV）磷酸酪氨酸磷酸酶抑制剂能够有效激活核因子κB（NF-κB）。我们已经确定，p56(lck)、ζ链相关蛋白70（ZAP-70）、SH2结构域含酪氨酸磷酸酶-76（SLP-76）、钙的容量性内流以及钙调节效应器在bpV诱导的NF-κB激活中起重要作用。在本研究中，我们评估了先前报道的在T细胞激活刺激诱导NF-κB过程中起作用的其他信号转导分子是否也被bpV化合物激活。在缺乏CD45或p36(LAT)的细胞系中评估NF-κB的核转位，以评估这些信号转导分子在bpV介导的NF-κB激活中的作用。这两种蛋白的缺乏都极大地降低了bpV处理后NF-κB核转位的程度。然后使用亚型特异性蛋白激酶C（PKC）抑制剂表明，bpV化合物通过钙敏感和钙不敏感的PKC亚型激活NF-κB。然后通过使用κB抑制蛋白（IkappaB）激酶复合物特异性激酶测定法以及表达无催化活性形式的IKKα和IKKβ的质粒来研究IkappaB激酶复合物的作用。还显示参与IKK复合物激活的上游激酶如TPL-2/COT、NF-κB诱导激酶（NIK）和IKKε在bpV介导的NF-κB激活中起重要作用。最后，进行报告基因转录测定和凝胶迁移测定，以比较bpV激活NF-κB的动力学与抗原和肿瘤坏死因子α（TNFα）刺激的动力学。我们证明，在Jurkat细胞和原代T细胞中，bpV介导的NF-κB激活动力学与抗原刺激的动力学相当，但比TNFα处理时的动力学慢得多
