Nucleic Acids Research, Vol 27, Issue 4 1168-1175, Copyright © 1999 by Oxford University Press
GA Partington and RK Patient
We have investigated by electrophoretic mobility shift assay (EMSA) the
level of GATA-1 DNA-binding activity in nuclear extracts prepared from the
human erythroleukaemic cell line, K562, after erythroid induction by hemin,
sodium butyrate (NaB) or Trichostatin A or treatment with N -
acetylcysteine (NAC). 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 erythroleukaemic cell line MEL was induced
with dimethylsulphoxide (DMSO), NaB or NAC, 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 phosphoryl-ation and
DNA binding of GATA-1 are already optimalin 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.
ARTICLES
Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells
Developmental Biology Research Centre, School of Biomedical Sciences, The Randall Institute, King's College London, 26-29 Drury Lane, London WC2B 5RL, UK.
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