Novel phosphorylated forms of E2F-1 transcription factor bind to the retinoblastoma protein in cells overexpressing...

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.