Co-relation with novel phosphorylation sites of IκBα and necroptosis in breast cancer cells

Abstract Background: The phosphorylation of NF-kappaB inhibitor alpha (IκBα) protein is pivotal to the regulation of NF-κB transcription factor activity in the cell. The phosphorylation of IκBα by IκB kinase family have been so many identified, but the phosphorylation sites of IκBα by other kinases remain poorly understood. We investigated the new phosphorylation site for IκBα and identified its biological function in breast cancer cells. Methods: Previously we observed that aurora kinase (AURK) binds IκBα in the cell. To identify the essential domains of IκBα for phosphorylation of IκBα by AURK, kinase assay was performed with a series of IκBα truncation mutants. AURK significantly promotes activation of IκBα at serine 32, but not serine 36 residues, unlike IκB kinase (IKK) family proteins activate both IκBα serine residues. We also confirmed phosphorylation of IκBα by the 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). Results: We identified two novel sites of serine phosphorylation at S63 and S262. Alanine transition of S63 and S262 (S63A and S262A) of IκBα induced inhibition of cell proliferation and suppression of p65 transcription activity. Besides, S63A and/or S262A of IκBα regulated apoptotic and necroptic effects in breast cancer cells. Conclusions: Therefore, we identified novel phosphorylation site of IκBα by AURK, and its site was related to apoptosis and necroptosis pathway in breast cancer cells.