Phorbol ester treatment of K562 cells regulates the transcriptional activity of AML1c through phosphorylation.
We find that phorbol ester (PE) treatment of K562 cells greatly stimulates promoters (T cell receptor beta, myeloperoxidase, macrophage colony-stimulating factor receptor, and granulocyte macrophage colony-stimulating factor receptor) containing AML1 transcription factor binding sites. This stimulation of AML1c transcriptional activity is mediated by direct phosphorylation of the AML1c molecule on multiple phosphorylation sites. Eleven AML1c (S/T)P sites in the transcriptional activating domain are phosphorylated at a basal level in untreated K562 cells; treatment of the K562 cells with PE results in increased phosphorylation at five of these sites (serines 276, 293, 303, 462, and threonine 300). Mutation of these five sites to alanine inhibits PE-induced transcriptional activity; mutation of the sites to an acidic amino acid, aspartic acid, stimulates constitutive activity. Single mutations in four amino acids or double mutations (serines 276 and 293 or threonine 300 and serine 303) have little effect on AML1c transcriptional activity. Inhibitor assays suggest that the ERK family of protein kinases is activated by PEs to phosphorylate the (S/T)P sites within the AML1c molecule and markedly enhance the transcriptional activity of AML1c.
Hollings Cancer Center, the Medical University of South Carolina, 86 Jonathan Lucas St., Charleston, SC 29425, USA.
Youhong
Y
Zhang
Joseph R
JR
Biggs
Andrew S
AS
Kraft
D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002454:Cell Differentiation; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D047468:Immunoprecipitation; D020014:K562 Cells; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D010703:Phorbol Esters / Q000494:pharmacology*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D020298:U937 Cells
D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D010703:Phorbol Esters; D011518:Proto-Oncogene Proteins; C493728:RUNX1 protein, human; D014157:Transcription Factors; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D008156:Luciferases