Tyrosine kinase p56lck regulates cell motility and nuclear factor κB-mediated secretion of urokinase type plasminogen activator through tyrosine phosphorylation of IκBα following hypoxia/reoxygenation

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 p56(lck)-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 p56(lck) 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 p56(lck), 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 p56(lck) 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 p56(lck) 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.