Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein’s C-Terminal Helix

Abstract

SUMMARY The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb’s tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.

Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

“…Tumor sections were obtained from a previous study 35 Clover-3xFLAG-Rb-negative cells were compared using a two-tailed t-test. Each tumor was then treated as a biological replicate (n = 3).…”

“…We therefore sought to first determine if there was an effect of Rb on cell size in vivo. To test this, we integrated a vector containing a doxycycline-inducible Clover-3xFLAG-RB allele into a Kras +/G12D ;Trp53 -/mouse pancreatic ductal adenocarcinoma cell line, and then allografted these cell lines into NSG mice by subcutaneous implantation 35,36 . Tumors were allowed to engraft and grow for five days before we induced expression of the exogenous Rb allele (Fig.…”

“…Lentivirus-containing medium from transfected HEK 293T cells was used to infect HMEC-hTERT1 cells. At least 2-3 days after infection, positive cells were sorted by FACS and clonally expanded.Rb overexpression and knockout cell linesFor inducible Rb expression, we cloned an Rb expression cassette, driven by the TRE3G doxycycline-inducible promoter (Clontech 631168), into a PiggyBac integration plasmid containing 5′ and 3′ PiggyBac homology arms36 . The expression cassette contains the human RB1 or mouse Rb1 genes fused to fluorescent Clover and 3xFLAG affinity tag sequences, a zeocin resistance gene, and a Tet-On 3G transactivator gene driven by the EF1α promoter.…”

Inhibiting CDK6 Activity by Quercetin Is an Attractive Strategy for Cancer Therapy

“…When E2F enters the nucleus, it initiates the synthesis of different proteins required for DNA replication (S phase). 14 , 15 However, during cancer conditions, the upregulation of CDK6 leads to the permanent activation of the Rb-E 2 F pathway, which results in uncontrolled cell growth. 14 , 16 CDK6 not only regulates the cell cycle but also controls the metabolic alterations in the cancer cells.…”

“… 14 , 15 However, during cancer conditions, the upregulation of CDK6 leads to the permanent activation of the Rb-E 2 F pathway, which results in uncontrolled cell growth. 14 , 16 CDK6 not only regulates the cell cycle but also controls the metabolic alterations in the cancer cells. 17 It regulates the production of reactive oxygen species (ROS), by inhibiting the activity of phosphofructokinase (PFK) (EC 2.7.1.11) and other protein kinases of the glycolytic pathways.…”