A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins
Figure 9
Binding of Irga6 to ROP5 is required for enhanced ROP18-mediated phosphorylation of Irga6 in vitro.
Bacterially expressed and purified wt GST-Irga6 (A) or the GST-Irga6-D164A mutant (B) that is unable to bind ROP5 (see Figure 8A) and GST-ROP18-Ty were incubated in the absence or presence of purified pseudokinase domain protein ROP5BI in an in vitro kinase reaction supplemented with 1 mM ATP (left panels). Each kinase reaction was analyzed by Western blot for phosphorylation of Irga6 using anti-pT108 antibody. (A) Enhanced ROP18-mediated phosphorylation of wt Irga6 can be detected after addition of ROP5BI. (B) With Irga6-D164A mutant the presence of ROP5BI does not result in enhanced phosphorylation. Addition of BSA instead of ROP5BI served as a control for an effect of total protein in the reaction system (right panels). In the absence of ROP18 there was no detectable phosphorylation of either wt Irga6 or Irga6D164A (right panels). Vertical lines indicate excision of irrelevant tracks from the images of the two gels.