Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4.

Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ
Genes Dev. 1993 7 (3): 331-42

PMID: 8449399 · DOI:10.1101/gad.7.3.331

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.

MeSH Terms (15)

Adenosine Triphosphate Animals Baculoviridae Cell Line Cyclin D1 Cyclins G1 Phase Genes, Retinoblastoma Humans Insecta Oncogene Proteins Phosphorylation Recombinant Fusion Proteins Retinoblastoma Protein Transfection

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