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

Abstract

Nuclear factor κB (NFκB) 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 NFκB through tyrosine phosphorylation of IκBα 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 NFκB 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 NFκB, NFκB-DNA binding, and transactivation of NFκB through tyrosine phosphorylation of IκBα. 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 κB (IκBα) and transcriptional activation of NFκB, and these are inhibited by Lck inhibitors. In vitro kinase assay demonstrated that immunoprecipitated p56 lck, but not Lyn or Fyn directly phosphorylate IκBα in presence of H/R. Pervanadate, H2O2, and H/R induce the interaction between Lck and tyrosine-phosphorylated IκBα, and this interaction is inhibited by Src homology 2 domain inhibitory peptide, suggesting that tyrosine-phosphorylated IκBα interacts with Src homology 2 domain of Lck. Luciferase reporter gene assay indicated that Lck induces NFκB-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 NFκB activation, uPA secretion, and cell motility through tyrosine phosphorylation of IκBα and further demonstrates an important redox-regulated pathway for NFκB activation following H/R injury that is independent of IκB kinase/IκBα-mediated signaling pathways.