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Identification of a Constitutively Active Mutant Mouse IRAK2 by Retroviral Expression Screening
- Liu, Yanmei, Yin, Weilan, Xu, Lingqing, Zhang, Helin, Liu, Qian, Yin, Weiguo
- Molecular biotechnology 2018 v.60 no.4 pp. 245-250
- binding sites, enzyme activity, gain-of-function mutation, gene overexpression, genes, macrophages, mice, mutants, retroviral vectors, screening, sequence analysis, signal transduction, site-directed mutagenesis
- To identify the importance of IRAK2 kinase activity in TLR-mediated signaling pathways, we constructed a retroviral vector harboring either a mouse IRAK2 gene (IRAK2-WT) or with its mutant with loss of function of its ATP-binding site (IRAK2-KD). Further, we comparatively analyzed for the gain of function and modulations in TLR-mediated signaling pathways in IRAK2 knockout (IRAK2-KO) macrophages upon introduction of the IRAK2-WT retroviral constructs. The pBS/IRAK2-KD with the ATP-binding site mutation in IRAK2 was obtained by using site-specific mutagenesis. The recombinants were identified with appropriate double digestion and sequence analysis. The recombinant vector constructs were transfected by lipofection into phoenix packaging cells. The viral vectors (10⁷ cfu/mL) with the construct were allowed to infect IRAK2-KO macrophages. The results showed that IRAK2-WT gene overexpressed in the IRAK2-KO macrophages exhibited a modified IRAK2 expression upon LPS induction. However, the modification was absent with IRAK2-KD construct on LPS stimulation; instead, the IRAK2 protein stability was reduced considerably. The results further show that the LPS-induced effect on the stability of IRAK2 is dependent of IRAK4 stimulation.