Inhibition of interleukin-1β-induced NF-κB activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca²⁺ signals have been implicated in NF-κB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1β (IL-1β)-induced NF-κB signaling. In human embryonic kidney 293 cells, IL-1β induces IκB kinase β (IKKβ) activation, IκBα degradation, NF-κB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1β-induced NF-κB activation. However, IL-1β-induced NF-κB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr³⁰⁸ and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1β-induced IKKβ activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKβ inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1β-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase I (IRAK1), which plays a key role in IL-1β-induced NF-κB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-κB activity and its association with MyD88 in response to IL-1β stimulation. Furthermore, CaMKKc and Akt over-expression increases IRAK1 phosphorylation at Thr¹⁰⁰, and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-κB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1β signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1β-induced NF-κB activation through interference with the coupling of IRAK1 to MyD88.