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 activation 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 general transcription factors in the cell response to phorbol esters was examined. Experiments demonstrate that phorbol ester treatment of U937 cells stimulates the phosphorylation of the TATA-binding protein (TBP); this phosphorylation occurs within 30 min and is still apparent, although greatly reduced, at 4 h. The following results demonstrate that TBP phosphorylation occurs as a result of activation of an extracellular signal-regulated kinase (ERK) protein kinase: (a) overexpression of mitogen-activated protein kinase phosphatase-1 blocks phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of TBP both in vitro and in vivo; (b) pretreatment with the ERK kinase kinase inhibitor PD098059 also blocks PMA-induced phosphorylation of TBP both in vitro and in vivo; and (c) phosphorylation of TBP is observed when serum-starved NIH 3T3 cells are stimulated with fresh serum, another activator of the ERK pathway. TBP can be phosphorylated in vitro by extracts of U937 cells or by bacterially expressed activated ERK2; the phosphorylation sites were mapped to ERK kinase consensus sites in the TBP amino-terminal domain. Using glutathione S-transferase-TBP fusion proteins, cellular proteins that bind specifically to the TBP amino terminus have been identified. These observations suggest that ERK-mediated phosphorylation of TBP occurs during the PMA-induced differentiation of U937 cells and the stimulation of the G0-G1 transition in fibroblasts and could play a role in the regulation of TBP protein interactions and thus regulate gene transcription during these two processes.
Department of Medical Oncology, University of Colorado Health Sciences Center, Denver 80262, USA.
J R
JR
Biggs
N G
NG
Ahn
A S
AS
Kraft
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism* / Q000502:physiology; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D054638:Dual Specificity Phosphatase 1; D006801:Humans; D017874:Immediate-Early Proteins / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D011233:Precipitin Tests; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D035181:TATA-Box Binding Protein; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D020298:U937 Cells
D018797:Cell Cycle Proteins; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; D017027:Protein Tyrosine Phosphatases; D013755:Tetradecanoylphorbol Acetate