Phosphorylation of E2F-1 by cyclin A-cdk2.
Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.
Kitagawa
M
M
Banyu Tsukuba Research Institute, Merck Research Laboratories, Japan.
Higashi
H
H
Suzuki-Takahashi
I
I
Segawa
K
K
Hanks
S K
SK
Taya
Y
Y
Nishimura
S
S
Okuyama
A
A
D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D046911:Macromolecular Substances; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases