title: Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors.
abstract: Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.
authors.affiliation: Department of Medicine, Virginia Commonwealth University/Massey CancerCenter, Richmond, Virginia 23298, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Dai
authors.forename: Shuang
authors.initials: S
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Xin-Yan
authors.initials: XY
authors.lastname: Pei
authors.forename: Vanessa L
authors.initials: VL
authors.lastname: Funk
authors.forename: Lora B
authors.initials: LB
authors.lastname: Kramer
authors.forename: Paul
authors.initials: P
authors.lastname: Dent
authors.forename: Steven
authors.initials: S
authors.lastname: Grant
mesh_terms: D000107:Acetylation / Q000187:drug effects; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D056284:Histone Deacetylase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009101:Multiple Myeloma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D000077337:Vorinostat
chemical_list: D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D009363:Neoplasm Proteins; C498143:RELA protein, human; D051996:Transcription Factor RelA; D000077337:Vorinostat; D051550:I-kappa B Kinase; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1