ACTIVATION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY IN U937 LEUKEMIC-CELLS INDUCES PHOSPHORYLATION OF THE AMINO-TERMINUS OF THE TATA-BINDING PROTEIN

Phorbol ester treatment of U937 leukemic cells results in the activati on of numerous protein kinase pathways, followed by cell cycle arrest and differentiation into macrophage-like cells. Because major changes in gene transcription are associated with this process, the role of ge neral transcription factors in the cell response to phorbol esters was examined. Experiments demonstrate that phorbol ester treatment of U93 7 cells stimulates the phosphorylation of the TATA-binding protein (TB P); this phosphorylation occurs within 30 min and is still apparent, a lthough greatly reduced, at 4 h, The following results demonstrate tha t TBP phosphorylation occurs as a result of activation of an extracell ular signal-regulated kinase (ERK) protein kinase: (a) overexpression of mitogen-activated protein kinase phosphatase-1 blocks phorbol 12-my ristate 13-acetate (PMA)-induced phosphorylation of TBP both in vitro and in vivo; (b) pretreatment with the ERK kinase kinase inhibitor PD0 98059 also blocks PMA-induced phosphorylation of TBP both in vitro and in vivo; and (c) phosphorylation of TBP is observed when serum-starve d NIH 3T3 cells are stimulated with fresh serum, another activator of the ERK pathway. TBP can be phosphorylated in vitro by extracts of U93 7 cells or by bacterially expressed activated ERK2; the phosphorylatio n sites were mapped to ERK kinase consensus sites in the TBP amino-ter minal domain. Using glutathione S-transferase-TBP fusion proteins, cel lular proteins that bind specifically to the TBP amino terminus have b een identified. These observations suggest that ERK-mediated phosphory lation of TBP occurs during the PMA-induced differentiation of U937 ce lls and the stimulation of the G(0)-G(1) transition in fibroblasts and could play a role in the regulation of TBP protein interactions and t hus regulate gene transcription during these two processes.