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PLP2 inhibits IFN signaling by inactivating the kinase activity of TBK1.

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posted on 2011-02-18, 01:53 authored by Gang Wang, Gang Chen, Dahai Zheng, Genhong Cheng, Hong Tang

(A) PLP2 inhibits IRF3 phosphorylation by inactivating TBK1. Plasmids (800 ng) expressing Flag-TBK1 (WT or kinase dead mutant K38A) and Myc-PLP2 (WT or C106A) were co-expressed in HEK293T cells (in 35 mm plates). At 36 h post transfection, cells were lysed and Flag-TBK1 was immunoprecipitated with anti-Flag antibody. Immunoabsorbed Flag-TBK1 was incubation with recombinant GST-IRF3131–426 (1 µg) and γ-32P-ATP at 25°C for 30 min. Phosphorylation of proteins was resolved by SDS-PAGE and autoradiography. Expression of the exogenous proteins was verified with the indicated antibodies (WCL). (B) PLP2 inhibits TBK1 kinase activity in Traf3−/− MEF cells. The similar in vitro kinase assays were carried out as in (A) except that 8 µg of each plasmid expressing Flag-TBK1(WT or K38A) and Myc-PLP2 (WT or C106A) were co-transfected into Traf3−/− MEF cells (in 10 cm plates). (C) PLP2 inactivates the recombinant TBK1 by deubiquitination. An equal amount of recombinant TBK1 (500 ng) was incubated with Myc-PLP2 (WT or C106A) immunopurified from HEK293T cells at 37°C for 2 h. The kinase activities were measured as in (A). The deubiquitination efficiency of TBK1 was examined with anti-ubiquitin antibody and the amount of Myc-PLP2 (WT or C106A) used in each reaction was measured by anti-Myc antibody. Data are representative of at least three independent experiments.