PHORBOL ESTER-STIMULATED PHOSPHORYLATION OF PU.1 - ASSOCIATION WITH LEUKEMIC-CELL GROWTH-INHIBITION

PU.1, a member of the ets transcription factor family, has been previo usly shown to be necessary for tetradecanoylphorbol-13 acetate (TPA)-i nduced U937 leukemic cell maturation. We examined the effects of TPA o n PU.1 content and PU.1 DNA binding activity in U937 cells. Unstimulat ed cells expressed PU.1 mRNA transcripts and TPA did not increase thes e levels. However, TPA treatment induced phosphorylation of PU.1. Gel- shift analysis using a labeled PU.1 oligomer showed that TPA induced a unique PU.1 binding activity. This binding activity was phosphorylati on-dependent, as indicated by the ability of phosphatase treatment to abolish its detection. The PU.1 binding activity was generated at TPA- 13 concentrations stimulating growth arrest and was blocked by the PKC inhibitor GF109203X, which antagonized TPA-induced growth inhibition, Bryostatin 1, another protein kinase C activator, induced only a mode st degree of U937 growth inhibition and antagonized TPA-stimulated gro wth arrest. Bryostatin 1 was unable to induce this TPA-generated PU.1 binding activity. High bryostatin 1 concentrations inhibited generatio n of this TPA-induced band shift, These data suggest that TPA-induced growth inhibition is associated with phosphorylation of PU.1 and gener ation of a unique PU.1 binding activity. (C) 1996 by The American Soci ety of Hematology.