The interactions of E2F with pRB and with p107 are regulated via the phosphorylation of pRB and p107 by a cyclin-dependent kinase.
It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.
Suzuki-Takahashi
I
I
Banyu Tsukuba Research Institute, Merck Research Laboratories, Okubo, Japan.
Kitagawa
M
M
Saijo
M
M
Higashi
H
H
Ogino
H
H
Matsumoto
H
H
Taya
Y
Y
Nishimura
S
S
Okuyama
A
A
D001483:Base Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050720:Retinoblastoma-Like Protein p107; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D046911:Macromolecular Substances; D009687:Nuclear Proteins; C493844:RBL1 protein, human; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050720:Retinoblastoma-Like Protein p107; D050680:Transcription Factor DP1; D014157:Transcription Factors; D018844:Cyclin-Dependent Kinases