Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells

Abstract

We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythro-leukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N-acetylcysteine (NAG). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythro-leukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAG, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphorylation and DNA binding of GATA-1 are already optimal in these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.