The retinoblastoma gene family members pRB and p107 coactivate the AP-1-dependent mouse tissue factor promoter in fibroblasts.
Serum-stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of blood coagulation. This requires the rapid entry of c-Fos into specific AP-1 DNA-binding complexes and can be strongly inhibited by the adenovirus EIA 12S gene product. In this study, we utilized a panel of E1A mutants deficient in cellular protein binding to analyse the molecular basis for EIA inhibition of a minimal, c-Fos-dependent TF promoter/ reporter construct in mouse AKR-2B fibroblasts. Mutations which impaired binding of the retinoblastoma tumor suppressor protein family members pRB, p107, and p130 relieved E1A-mediated inhibition of transcription in response to serum-stimulation or c-Fos overexpression. Inhibition was restricted to the G0 to G1 transition, consistent with the specificity of E1A for hypophosphorylated forms of RB proteins. Although E1A mutants deficient in CBP/p300 binding retained the ability to inhibit TF transcription, deletion of the amino-terminal portion of the CBP/p300 interaction domain was required to permit rescue of TF promoter activity by coexpression of pRB. Moreover, ectopic p107 could effectively substitute for pRB in relieving E1A-mediated repression. In primary mouse embryo fibroblasts, activity of the minimal AP-1-dependent TF promoter was suppressed in Rb(-/-) cells compared to parallel Rb(+/-) and Rb(+/+) transfectants. Ectopic expression of either pRB or p107 markedly enhanced TF promoter activity in Rb(-/-) fibroblasts. Collectively, these data imply that pRB and p107 can cooperate with c-Fos to activate TF gene transcription in fibroblasts and suggest a requirement for another, as yet unidentified, E1A-binding protein.
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
S L
SL
Liu
A
A
Rand
R J
RJ
Kelm
M J
MJ
Getz
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011506:Proteins; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D013925:Thromboplastin / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D017500:Adenovirus E1A Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D016760:Proto-Oncogene Proteins c-fos; C489485:Rbl1 protein, mouse; C493838:Rbl2 protein, mouse; D011956:Receptors, Cell Surface; D016160:Retinoblastoma Protein; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D013925:Thromboplastin