Phosphorylation of a specific cdk site in E2F-1 affects its electrophoretic mobility and promotes pRB-binding in vitro.
The E2F transcription factor family participates in growth control presumably through transcriptional activation of genes that promote entry into S phase. E2F activity is believed to be controlled across the cell cycle by association with various cellular proteins, including the product of the retinoblastoma gene (pRB). We find that E2F-1 proteins are heterogeneously phosphorylated in insect cells, as a result of which they migrate as a doublet on SDS-polyacrylamide gels. This electrophoretic shift is shown to be dependent upon specific phosphorylation of E2F-1 on serine-375 (S375), near the pRB-binding site. Phosphorylation on S375 also occurs in human cells. E2F-1 was most efficiently phosphorylated on this residue by cyclin A/cdk2 kinase, and to a lesser extent by cyclin A/cdk2, irrespective of the presence of the pRB-related p107 protein. Phosphorylation of E2F-1 on S375 greatly enhanced its affinity of pRB in vitro. These results suggest a novel way of regulating E2F-1 activity, namely by cell-cycle-dependent phosphorylation of this transcription factor.
Peeper
D S
DS
Department of Molecular Carcinogenesis, Sylvius Laboratory, University of Leiden, The Netherlands.
Keblusek
P
P
Helin
K
K
Toebes
M
M
van der Eb
A J
AJ
Zantema
A
A
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011485:Protein Binding; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D018411:Spodoptera; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D009838:Oligodeoxyribonucleotides; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D018844:Cyclin-Dependent Kinases