Mahabeleshwar Ganapati H, Kundu Gopal C
National Centre for Cell Science (NCCS), NCCS Complex, Pune, Marahashtra 411 007, India.
J Biol Chem. 2003 Dec 26;278(52):52598-612. doi: 10.1074/jbc.M308941200. Epub 2003 Oct 8.
Nuclear factor kappaB (NFkappaB) plays major role in regulating cellular responses as a result of environmental injuries. The molecular mechanism(s) by which hypoxia/reoxygenation (H/R) regulates p56lck-dependent activation of NFkappaB through tyrosine phosphorylation of IkappaBalpha and modulates the expression of downstream genes that are involved in cell migration in human breast cancer cells are not well defined. In this paper, we investigated the involvement of protein-tyrosine kinase p56lck in the redox-regulated activation of NFkappaB following H/R in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. We demonstrated that H/R induces tyrosine phosphorylation of p56lck, nuclear translocation of NFkappaB, NFkappaB-DNA binding, and transactivation of NFkappaB through tyrosine phosphorylation of IkappaBalpha. Transfection of these cells with wild type Lck but not with mutant Lck F394 followed by H/R induces the tyrosine phosphorylation of inhibitor of nuclear factor kappaB (IkappaBalpha) and transcriptional activation of NFkappaB, and these are inhibited by Lck inhibitors. In vitro kinase assay demonstrated that immunoprecipitated p56lck but not Lyn or Fyn directly phosphorylate IkappaBalpha in presence of H/R. Pervanadate, H2O2, and H/R induce the interaction between Lck and tyrosine-phosphorylated IkappaBalpha, and this interaction is inhibited by Src homology 2 domain inhibitory peptide, suggesting that tyrosine-phosphorylated IkappaBalpha interacts with Src homology 2 domain of Lck. Luciferase reporter gene assay indicated that Lck induces NFkappaB-dependent urokinase type plasminogen activator (uPA) promoter activity in presence of H/R. Furthermore, H/R stimulates the cell motility through secretion of uPA. To our knowledge, this is the first report that p56lck in presence of H/R regulates NFkappaB activation, uPA secretion, and cell motility through tyrosine phosphorylation of IkappaBalpha and further demonstrates an important redox-regulated pathway for NFkappaB activation following H/R injury that is independent of IkappaB kinase/IkappaBalpha-mediated signaling pathways.
核因子κB（NFκB）在调节因环境损伤引起的细胞反应中起主要作用。缺氧/复氧（H/R）通过IκBα的酪氨酸磷酸化调节p56lck依赖性NFκB激活并调节人乳腺癌细胞中参与细胞迁移的下游基因表达的分子机制尚未明确。在本文中，我们研究了蛋白酪氨酸激酶p56lck在高侵袭性（MDA-MB-231）和低侵袭性（MCF-7）乳腺癌细胞H/R后NFκB的氧化还原调节激活中的作用。我们证明，H/R通过IκBα的酪氨酸磷酸化诱导p56lck的酪氨酸磷酸化、NFκB的核转位、NFκB与DNA的结合以及NFκB的反式激活。用野生型Lck而非突变型Lck F394转染这些细胞后进行H/R处理，可诱导核因子κB抑制因子（IκBα）的酪氨酸磷酸化和NFκB的转录激活，而这些均被Lck抑制剂抑制。体外激酶测定表明，在H/R存在的情况下，免疫沉淀的p56lck而非Lyn或Fyn可直接磷酸化IκBα。过氧钒酸盐、H2O2和H/R诱导Lck与酪氨酸磷酸化的IκBα之间的相互作用，且这种相互作用被Src同源2结构域抑制肽抑制，这表明酪氨酸磷酸化的IκBα与Lck的Src同源2结构域相互作用。荧光素酶报告基因测定表明，在H/R存在的情况下，Lck可诱导NFκB依赖性尿激酶型纤溶酶原激活剂（uPA）启动子活性。此外，H/R通过uPA的分泌刺激细胞运动。据我们所知，这是首次报道在H/R存在的情况下，p56lck通过IκBα的酪氨酸磷酸化调节NFκB激活、uPA分泌和细胞运动，并进一步证明了H/R损伤后NFκB激活的一条重要的氧化还原调节途径，该途径独立于IκB激酶/IκBα介导的信号通路。
