Abstract
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.
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Funding
This work was supported by Natural Science Foundation of Guangdong Province (No. 2015A030313691), Public interest and ability construction funds in Guangdong Province (No. 2014A020212036).
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Liu, Y., Yin, W., Xu, L. et al. Identification of a Constitutively Active Mutant Mouse IRAK2 by Retroviral Expression Screening. Mol Biotechnol 60, 245–250 (2018). https://doi.org/10.1007/s12033-018-0064-9
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DOI: https://doi.org/10.1007/s12033-018-0064-9