Fig 8. Differential effects of HopZ3 acetylation on kinase activity of RIPK, PTO and FEN.

GST fusions of SlRIPK (A–C) and FEN (D) or PTO-His (E–F) were incubated with or without acetyl-CoA, IP6 and HopZ3 or HopZ3_C300A (C/A) for 2 h at RT and then washed with PBS buffer. After acetylation, the kinase activity of SlRIPK, FEN or PTO was initiated by adding γ³²P-ATP and MgSO₄ for 30 min. at RT. (A) Incubation of SlRIPK with HopZ3 in the absence of Acetyl-CoA did not affect the kinase activity of RIPK. (B) After incubation with acetyl-CoA and HopZ3, RIPK-mediated phosphorylation of itself and HopZ3 was reduced. (C) Phosphorylation of SlRIN4s was reduced after acetylation of SlRIPK by HopZ3. (D) Incubation with acetyl-CoA, IP6 and HopZ3 did not affect the ability of FEN to phosphorylate itself. (E–F) Acetylation of PTO by HopZ3 did not affect PTO activity.