The post-receptor pathway that leads to nuclear factor κB (NF-κB) activation begins with the assembly of a membrane-proximal complex among the interleukin 1 (IL-1) receptors and the adaptor molecules, myeloid differentiation protein 88 (MyD88), IL-1-receptor-associated kinases (IRAKs) and tumour-necrosis-factor-receptor-associated factor 6. Eventually, phosphorylation of the inhibitor of NF-κB (IκB) by the IκB kinases releases NF-κB, which translocates to the nucleus and modulates gene expression. In this paper, we report that IRAK2 and MyD88, but not IRAK1, interact physically with Akt, as demonstrated by co-immunoprecipitation and pull-down experiments. Interestingly, the association of Akt with recombinant IRAK2 is decreased by stimulation with IL-1, and is favoured by pre-treatment with phosphatase. Likewise, Akt association with IRAK2 is increased considerably by overexpression of PTEN (phosphatase and tensin homologue deleted on chromosome 10), while it is completely abrogated by overexpression of phosphoinositide-dependent protein kinase 1. These data indicate that Akt takes part in the formation of the signalling complex that conveys the signal from the IL-1 receptors to NF-κB, a step that is much more membrane-proximal than was reported previously. We also demonstrate that Akt activity is necessary for IL-1-dependent NF-κB transactivation, since a kinase-defective mutant of Akt impairs IRAK2- and MyD88-dependent, but not IRAK1-dependent, NF-κB activity, as monitored by a gene reporter assay. Accordingly, IRAK2 failed to trigger inducible nitric oxide synthase and IL-1β production in cells expressing dominant-negative Akt. However, NF-κB binding to DNA was not affected by inhibition of Akt, indicating that Akt regulates NF-κB at a level distinct from the dissociation of p65 from IκBα and its translocation to the nucleus, possibly involving phosphorylation of the p65 transactivation domain.
- Akt/protein kinase B kinase
- interleukin-1-associated-kinase 2 (IRAK2)
- interleukin 1 signalling
- nuclear factor κB (NF-κB)
- Saos2 osteosarcoma cell line
↵2 Present address: Unit of Leukocyte Biology, Scientific Institute of San Raffaele-DIBIT, Via Olgettina 58, 20132 Milan, Italy.
Abbreviations used: DN, dominant negative; DTT, dithiothreitol; EMSA, electrophoretic mobility-shift assay; GST, glutathione S-transferase; HA, haemagluttinin; HEK, human embryonic kidney; IKK, IκB kinase; IκB, inhibitor of NF-κB; IL-1, interleukin 1; IL-1RI, IL-1 receptor I; iNOS, inducible nitric oxide synthase; IRAK, IL-1 receptor associated kinase; MyD88, myeloid differentiation protein 88; NF-κB, nuclear factor κB; PARP, poly(ADP-ribose) polymerase; PDK, phosphoinositide-dependent protein kinase; PI 3-kinase, phopsphoinositide 3-kinase; λ-PPase, λ-protein phosphatase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; TNF, tumour necrosis factor; Tollip, Toll-interacting protein; TRAF, TNF-receptor-associated factor.
- The Biochemical Society, London ©2003