Figure 4.

Phosphorylation and acetylation cooperate to cause the degradation of acetylated GATA-1. (A) Each individual phosphorylation mutant is more stable than wild-type protein. Western blot showing GATA-1 phosphorylation mutant proteins immunoprecipitated from transfected Cos 7 cells. Transfection efficiency was normalised to β-galactosidase. (B) Each phosphorylation mutant is less ubiquitinated than wild-type protein. The levels of ubiquitination were determined via immunoprecipitation from transfected 293T cells as described in Figure 2. The same Western blot was re-probed with anti-GATA-1 antibody (lower panel) and confirms that protein is present in all lanes. Higher ubiquitination of the total phosphorylation mutant and S26A (which is similar between the two proteins but notably less than wild type) correlates with their lower protein levels (Figure 4A). (C) Phosphorylation is needed for efficient degradation of acetylated GATA-1. Western blot showing GATA-1 levels in extracts from BM-SCF cells that stably express GATA-1 mutated in serine 26 (Flag-26A). Endogenous GATA-1 in the same cell provides an internal control for degradation upon TSA treatment. β-Tubulin levels verify that equivalent cell numbers were taken (lower panel). (D) MEK inhibitor prevents degradation of acetylated GATA-1. Haemopoietic (BM-SCF) cells were treated with or without TSA in the presence or absence of the MEK inhibitor U0126. Acetylated GATA-1 (Ac-GATA-1; upper panel) and total GATA-1 (GATA-1; lower panel) were detected as in Figure 3. The graph shows quantification of the level of acetylated GATA-1 divided by the amount of total GATA-1 in two further independent experiments where a higher level of acetylated GATA-1 in untreated cells was observed.