Disruption of IB Kinase (IKK)-mediated RelA Serine 536 Phosphorylation Sensitizes Human Multiple Myeloma Cells to

Post-translational modifications of RelA play an importantrole in regulation of NF- B activation. We previously demon-strated that in malignant hematopoietic cells, histone deacety-lase inhibitors (HDACIs) induced RelA hyperacetylation andNF- B activation, attenuating lethality. We now present evi-dencethatI Bkinase(IKK) activationofNF--mediatedRelASer-536phosphor-ylation plays a significant functional role in promoting RelAacetylation, inducing NF- B activation, and limiting HDACIlethality in human multiple myeloma (MM) cells. Immunoblotprofiling revealed that although basal RelA phosphorylationvaried in MM cells, Ser-536 phosphorylation correlated withIKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589inducedphosphorylationofIKK / (Ser-180/Ser-181)andRelA (Ser-536) in MM cells, including cells expressing an I B“super-repressor,” accompanied by increased RelA nucleartranslocation, acetylation, DNA binding, and transactivationactivity. These events were substantially blocked by either pan-IKK or IKK -selective inhibitors, resulting in marked apopto-sis. Consistent with these events, inhibitory peptides targetingeithertheNF- Bessentialmodulator(NEMO)bindingdomainfor IKK complex formation or RelA phosphorylation sites alsosignificantly increased HDACI lethality. Moreover, IKKknockdown by shRNA prevented Ser-536 phosphory-lation and significantly enhanced HDACI susceptibility.Finally, introduction of a nonphosphorylatable RelA mutantS536A, which failed to undergo acetylation in response toHDACIs, impaired NF- B activation and increased celldeath. These findings indicate that HDACIs induce Ser-536phosphorylation of the NF- B subunit RelA through anIKK -dependent mechanism, an action that is functionallyinvolved in activation of the cytoprotective NF- B signalingcascade primarily through facilitation of RelA acetylationrather than nuclear translocation.