G-protein-coupled-receptor kinases mediate TNF alpha-induced NF-kappa B signalling via direct interaction with and phosphorylation of I kappa B alpha

TNF alpha (tumour necrosis factor alpha) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNF alpha activation of the NF-kappa B (nuclear factor kappa B) signalling pathway particularly in macrophages has been implicated in many diseases. We demonstrate in the present study that GRK2 and GRK5 (G-protein-coupled-receptor kinases 2 and 5) regulate TNF alpha-induced NF-kappa B signalling in Raw 264.7 macrophages. RNAi (RNA interference) knockdown of GRK2 or GRK5 in macrophages significantly inhibited TNF alpha-induced I kappa B alpha (inhibitory kappa B alpha) phosphorylation and degradation, NF-kappa B activation and expression of the NF-kappa B-regulated gene MIP1 beta (macrophage inflammatory protein 1 beta). Consistent with these results, overexpression of GRK2 or GRK5 enhanced TNF alpha-induced NF-kappa B activity. In addition, we show that GRK2 and GRK5 interacted with I kappa B alpha via the N-terminal domain of I kappa B alpha and that I kappa B alpha is a substrate for GRK2 and GRK5 in vitro. Furthermore, we also found that GRK5, but not GRK2, phosphorylated I kappa B alpha at the same amino acid residues (Ser(32)/Ser(36)) as that of IKK beta (I kappa B kinase beta). Interestingly, associated with these results, knockdown of IKK beta in Raw 264.7 macrophages did not affect TNF alpha-induced I kappa B alpha phosphorylation. Taken together, these results demonstrate that both GRK2 and GRK5 are important and novel mediators of a non-traditional I kappa B alpha/NF-kappa B signalling pathway.