Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4.
The product (pRb) of the retinoblastoma gene (RB-1) prevents S-phase entry during the cell cycle, and inactivation of this growth-suppressive function is presumed to result from pRb hyperphosphorylation during late G1 phase. Complexes of the cyclin-dependent kinase, cdk4, and each of three different D-type cyclins, assembled in insect Sf9 cells, phosphorylated a pRb fusion protein in vitro at sites identical to those phosphorylated in human T cells. Only D-type cyclins activated cdk4 enzyme activity, whereas cyclins A, B1, and E did not. When Sf9 cells were coinfected with baculovirus vectors encoding human pRb and murine D-type cyclins, cyclins D2 and D3, but not D1, bound pRb with high stoichiometry in intact cells. Introduction of a vector encoding cdk4, together with those expressing pRb and D-type cyclins, induced pRb hyperphosphorylation and dissociation of cyclins D2 and D3, whereas expression of a kinase-defective cdk4 mutant in lieu of the wild-type catalytic subunit yielded ternary complexes. The transcription factor E2F-1 also bound to pRb in insect cells, and coexpression of cyclin D-cdk4 complexes, but neither subunit alone, triggered pRb phosphorylation and prevented its interaction with E2F-1. The D-type cyclins may play dual roles as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates.
Kato
J
J
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Matsushime
H
H
Hiebert
S W
SW
Ewen
M E
ME
Sherr
C J
CJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D019938:Cyclin D1; D016213:Cyclins / Q000378:metabolism*; D016193:G1 Phase; D016161:Genes, Retinoblastoma; D006801:Humans; D007313:Insecta; D015513:Oncogene Proteins / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D016213:Cyclins; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D000255:Adenosine Triphosphate