Novel phosphorylated forms of E2F-1 transcription factor bind to the retinoblastoma protein in cells overexpressing an E2F-1 cDNA.
In cells overexpressing an exogenous cDNA encoding the E2F-1 transcription factor, as many as eight closely-migrating protein bands were detected after denaturing protein gel electrophoresis and immunoblotting with an E2F-1-specific antibody. Control cells, not overexpressing an E2F-1 cDNA, contained only four E2F-1 specific bands. Pretreatment of protein extracts, from both control and E2F-1 overexpressing cells, with lambda-phosphatase eliminated all E2F-1-specific bands except the single band migrating most rapidly on the gels. These data demonstrated that the multiple protein bands were differentially-phosphorylated forms of E2F-1 protein and showed that novel, more highly phosphorylated E2F-1 forms were present in cells overexpressing the E2F-1 protein. In addition, immunoprecipitation of the retinoblastoma (pRb) protein from cells overexpressing the E2F-1 cDNA showed that the novel, highly phosphorylated E2F-1 forms were preferentially coimmunoprecipitated, indicating that pRb bound preferentially to these E2F-1 forms.
Yang
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XH
Department of Microbiology and Immunology, Finch University of Health Sciences, Chicago Medical School, Illinois 60064-3095, USA.
Sladek
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D016475:3T3 Cells; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors