Tip60 acetylates PRAK in a phosphorylation dependent manner.
(A) His-PRAK was incubated with Tip60α, p38α, MKK6E, ATP and acetyl-CoA for an indicated period.
(B) His-PRAK was incubated with increasing amounts of Tip60α or buffer (−), p38α, MKK6E and acetyl-CoA with or without ATP.
(C) His-PRAK was incubated first with p38α, MKK6E and ATP, and then with wild type Tip60α (WT), Tip60α-A158 or buffer (−) and acetyl-CoA with or without SB203580.
(D) Wild type PRAK or PRAK-A182 was incubated with increasing amounts of Tip60α or buffer (−), p38α, MKK6E, ATP and acetyl-CoA.
(E) PRAK was incubated first with p38α and MKK6E with or without ATP, and then with Tip60α and acetyl-CoA with or without SB203580.
(A–E) Acetylation of PRAK and phosphorylation of PRAK and Tip60 were detected by Western blotting using anti-acetyl-Lys, anti-pPRAK-T182 and anti-pTip60-T158 antibodies, respectively. Input of recombinant proteins was stained by Ponceau.
See also Figure S5.