Ctenopharyngodon idella IKKβ interacts with PKR and IκBα

Abstract

Inhibitor of nuclear factor kappa-B kinase β (IKKβ) is a subunit of the IKK complex. It can activate the NF-κB pathway through phosphorylating IκB in response to a wide range of stimuli. In the present study, an IKKβ gene from grass carp (Ctenopharyngodon idella; KT282114) was cloned and identified by homologous cloning and rapid-amplification of cDNA ends (RACE) technique. The complete CiIKKβ cDNA is 3428 bp in length, with the longest open reading frame (ORF) of 2337 bp encoding a polypeptide of 778 amino acids. The deduced amino acid sequence of CiIKKβ has similar domain distribution to those of mammalian. For example, CiIKKβ consists of a serine/threonine kinase domain at the N-terminal, a basic region leucin zipper (BRLZ) domain in the middle, a homeobox associated leucin zipper (HALZ) domain and an IKKβ NEMO (NF-κB essential modulator) binding domain at the C-terminal. Phylogenetic tree analysis also showed that CiIKKβ is highly homologous to zebrafish IKKβ (DrIKKβ) and clearly distinct from the mammalian and amphibian counterparts. The expression of CiIKKβ was ubiquitously found in the liver, intestine, kidney, gill, spleen, heart, and brain tissues of grass carp and significantly up-regulated in CIK cells under the stimulation with Poly I:C and UV-inactivated grass carp hemorrhagic virus. To investigate the activation mechanism of NF-κB pathway in fish and the role of CiIKKβ in the pathway, we explored the protein interactions of protein kinase R (PKR) with IKKβ and IKKβ with IκBα by co-immunoprecipitation and GST-pull down assays. The interaction between each pair was confirmed. The results suggest that CiIKKβ may be a primary member in the activation of NF-κB pathway in fish.

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