The cyclin-dependent kinases (Cdks) are a family of serine/threonine protein kinases whose members are small proteins (~34–40 kDa) composed of little more than the catalytic core shared by all protein kinases. By definition, all Cdks share the feature that their enzymatic activation requires the binding of a regulatory cyclin subunit. In most cases, full activation also requires phosphorylation of a threonine residue near the kinase active site.

Although originally identified as enzymes that control cell-cycle events, members of the Cdk family are involved in other cellular processes as well. Animal cells, for example, contain at least nine Cdks, only four of which (Cdk1, 2, 4 and 6) are involved directly in cell-cycle control (Figure 3-2). Another family member (Cdk7) contributes indirectly by acting as a Cdk-activating kinase (CAK) that phosphorylates other Cdks, as we discuss in section 3-3. Cdks are also components of the machinery that controls basal gene transcription by RNA polymerase II (Cdk7, 8 and 9) and are involved in controlling the differentiation of nerve cells (Cdk5).

We will focus on the small number of Cdks for which there is clear evidence of a direct role in cell-cycle control (see Figure 3-2). In the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae (see section 2-1), all cell-cycle events are controlled by a single essential Cdk called Cdk1. Cell-cycle events in multicellular eukaryotes are controlled by two Cdks, known as Cdk1 and Cdk2, which operate primarily in M phase and S phase, respectively. Animal cells also contain two Cdks (Cdk4 and Cdk6) that are important in regulating entry into the cell cycle in response to extracellular factors.

Cdk function has been remarkably well conserved during evolution. It is possible, for example, for yeast cells to proliferate normally when their gene for CDK1 is replaced with the human one. This and other evidence clearly illustrates that Cdk function, and thus the function of the cell-cycle control system, has remained fundamentally unchanged over hundreds of millions of years of eukaryotic evolution.

Cdks exert their effects on cell-cycle events by phosphorylating a large number of proteins in the cell. During mitosis in particular, when many aspects of cellular architecture and metabolism are altered, Cdks phosphorylate hundreds of distinct proteins. These Cdk substrates are phosphorylated at serine or threonine residues in a specific sequence context that is recognized by the active site of the Cdk protein. In most cases, the target serine (S) or threonine (T) residue is followed by a proline (P); it is also highly favorable for the target residue to have a basic amino acid two positions after the target residue. The typical phosphorylation sequence for Cdks is [S/T*]PX [K/R], where S/T* indicates the phosphorylated serine or threonine, X represents any amino acid and K/R represents the basic amino acid lysine (K) or arginine (R).

All protein kinases have a tertiary structure comprising a small amino-terminal lobe and a larger carboxy-terminal lobe. ATP fits snugly in the cleft between the lobes, in such a way that the phosphates are oriented outwards, toward the mouth of the cleft. The protein substrate binds at the entrance of the cleft, interacting mainly with the surface of the carboxy-terminal lobe. Nearby residues catalyze the transfer of the terminal γ-phosphate of ATP to a hydroxyl oxygen in the protein substrate.

Cdks have the same two-lobed structure as other protein kinases (Figure 3-3), but with two modifications that make them inactive in the absence of cyclin. These modifications have been revealed by detailed crystallographic studies of the structure of human Cdk2. First, a large, flexible loop – the T-loop or activation loop – rises from the carboxy-terminal lobe to block the binding of protein substrate at the entrance of the active-site cleft. Second, in the inactive Cdk several important amino-acid side chains in the active site are incorrectly positioned, so that the phosphates of ATP are not ideally oriented for the kinase reaction. Cdk activation therefore requires extensive structural changes in the Cdk active site.

Two alpha helices make a particularly important contribution to the control of Cdk activity. The highly conserved PSTAIRE helix of the upper kinase lobe (also known as the α1 helix) interacts directly with cyclin and moves inward upon cyclin binding, causing the reorientation of residues that interact with the phosphates of ATP. The small L12 helix, just before the T-loop in the primary sequence, changes structure to become a beta strand upon cyclin binding, also contributing to reconfiguration of the active site and T-loop. We discuss the structural basis of Cdk activation in more detail in section 3-4. First, we will describe the cyclins and other regulators that influence activation.

L12 helix: small alpha helix adjacent to the T-loop in the active site of Cdk2 (residues 147–151), which changes structure to a beta strand upon cyclin binding.

PSTAIRE helix: alpha helix in the amino-terminal lobe of Cdks (also known as the α1 helix), which interacts with cyclin and is moved inward upon cyclin binding, resulting in reorientation of key active-site residues. The name of this helix comes from its amino-acid sequence, which is conserved among all major Cdks.

T-loop: flexible loop adjacent to the active site of Cdks, named for the threonine whose phosphorylation is required for maximal activity. Sometimes called the activation loop.