Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1.
Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following deltaRaf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.
McCarthy
S A
SA
Department of Cell Signaling, DNAX Research Institute, Palo Alto, California 94304, USA.
Chen
D
D
Yang
B S
BS
Garcia Ramirez
J J
JJ
Cherwinski
H
H
Chen
X R
XR
Klagsbrun
M
M
Hauser
C A
CA
Ostrowski
M C
MC
McMahon
M
M
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006493:Heparin / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology*; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf; D012097:Repressor Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism* / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494069:ETS2 protein, human; C494070:Ets2 protein, mouse; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D006493:Heparin; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases