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

Ganapati H Mahabeleshwar,Gopal C Kundu

doi:10.1074/jbc.m308941200

Abstract

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.

Highlights

Tumor invasion, malignant progression, and distant metastasis depend on complex multistep processes
Transfection of these cells with wild type Lck but not with mutant Lck F394 followed by hypoxia/ reoxygenation (H/R) induces the tyrosine phosphorylation of inhibitor of nuclear factor ␬B (I␬B␣) and transcriptional activation of Nuclear factor ␬B (NF␬B), and these are inhibited by Lck inhibitors
We present evidence for the involvement of protein-tyrosine kinase, p56lck in the redox-regulated activation of NF␬B through tyrosine phosphorylation of I␬B␣, urokinase type plasminogen activator (uPA) secretion and cell motility following H/R in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells

Summary

Introduction

Malignant progression, and distant metastasis depend on complex multistep processes. These transfected cells were individually or in combination used for Lck kinase assay, interaction studies between Lck and tyrosine-phosphorylated I␬B␣, EMSA, luciferase reporter gene assay, cell migration assay, and detection of uPA expression by Western blot analysis.

Results

Conclusion