Co-relation with novel phosphorylation sites of I kappa B alpha and necroptosis in breast cancer cells

Abstract

BackgroundPhosphorylation of NF-kappaB inhibitor alpha (I kappa B alpha) is key to regulation of NF-kappa B transcription factor activity in the cell. Several sites of I kappa B alpha phosphorylation by members of the I kappa B kinase family have been identified, but phosphorylation of the protein by other kinases remains poorly understood. We investigated a new phosphorylation site on I kappa B alpha and identified its biological function in breast cancer cells.MethodsPreviously, we observed that aurora kinase (AURK) binds I kappa B alpha in the cell. To identify the domains of I kappa B alpha essential for phosphorylation by AURK, we performed kinase assays with a series of I kappa B alpha truncation mutants. AURK significantly promoted activation of I kappa B alpha at serine 32 but not serine 36; by contrast, I kappa B kinase (IKK) family proteins activated both of these residues. We also confirmed phosphorylation of I kappa B alpha by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and nano-liquid chromatography hybrid quadrupole orbitrap mass spectrometer (nanoLC-MS/MS; Q-Exactive).ResultsWe identified two novel sites of serine phosphorylation, S63 and S262. Alanine substitution of S63 and S262 (S63A and S262A) of I kappa B alpha inhibited proliferation and suppressed p65 transcription activity. In addition, S63A and/or S262A of I kappa B alpha regulated apoptotic and necroptotic effects in breast cancer cells.ConclusionsPhosphorylation of I kappa B alpha by AURK at novel sites is related to the apoptosis and necroptosis pathways in breast cancer cells.