Cell Cycle Machinery

The cyclin-dependent kinases (Cdks) act as switches that control cell-cycle progression by alternating between active and inactive states. Cdks are activated by binding to cyclin proteins and also by phosphorylation. Cdks are inactivated by the binding of Kip/Cip or INK4 tumor suppressor proteins, and by additional phosphorylation.

Our studies of this system have included partially activated CyclinA-bound Cdk2, the fully activated phosphorylated CyclinA-Cdk2 complex, the latter complex inactivated by p27 (Kip1) binding, and the Cdk6 kinase with the bound p16 (INK4a) and p19 (INK4d) inhibitors.

[Enlarge Image] CyclinA-Cdk2 Complex

A ribbon representation of the CyclinA-Cdk2 complex is shown above. Cdk2 is shown in cyan, with the helix containing the Cdk sequence motif (PSTAIRE) in red and the activation loop in yellow. CyclinA is shown in purple.

[Enlarge Image] CyclinA-Cdk2 Phosphorylated on Thr160

Above: A view of the phosphorylated CyclinA-Cdk2 complex superimposed on the unphosphorylated complex in the same viewpoint as above. The rearrangement of the T-loop (activation loop) leads to a fully active kinase.

[Enlarge Image] p27(Kip1)-CyclinA-Cdk2 Complex

The phosphorylated CyclinA-Cdk2 complex can be completely inhibited by the Cip/Kip family of inhibitors. Above, p27 (Kip1) is shown bound to the CyclinA-Cdk2 complex, provoking profound changes in the kinase active site and rendering it inactive. p27 also interacts with the secondary substrate recognition site on the cyclin.

[Enlarge Image] p19 (INK4d)-Cdk6 Complex

The INK4 Cdk inhibitors bear no sequence or structural similarity to the Cip/Kip inhibitors but are able to bind to Cdk4 and Cdk6 and abolish the activity of the kinase. Here, we show the structure of p19 bound to the Cdk6 kinase in its monomeric form. p16 (INK4a) and p19 (INK4d) bind to the Cdk in a completely different manner to the p27 (Kip1) inhibitor. The INK4 inhibitors inhibit Cdks allosterically. They induce conformational changes that propagate to the cyclin-binding site and interfere with cyclin binding. The INK4 inhibitors also distort the kinase active site and interfere with ATP binding.

These studies reveal the extensive conformational changes undertaken by the Cdk subunit in response to activation and inhibition, and suggest that this flexibility is evolution's response to the need for Cdks to respond to a diverse set of growth regulatory signals affecting the cell cycle.

The Rb tumor suppressor protein connects the activity of the Cdks to transcriptional control of cell-cycle regulatory proteins via its interactions with E2F-DP. Rb binds to E2F-DP transcription factors, altering the expression of E2F-responsive genes. Phosphorylation of Rb by Cdk4 leads to release of bound E2F-DP and ultimately to the expression of genes required for S-phase entry and DNA replication.

[Enlarge Image] Retinoblastoma Pocket Domain Bound to E7 Viral Protein Peptide

The pocket domain of Rb is shown bound to a peptide of the E7 human papilloma virus oncoprotein via the LxCxE motif shared by other Rb-binding cellular and viral proteins. The A- and B- boxes that form the pocket domain have the same fold as CyclinA.

[Enlarge Image] E2F4/DP2 Dimer Bound to DNA

The structure of the E2F4/DP2 heterodimer in complex with DNA appears here. E2F4 and DP2 share a common fold -- the winged helix fold found in other DNA-binding proteins -- and make symmetric interactions with the DNA helix via the RRXXD motifs on the third helix of both proteins.