Cell cycle

Cyclin guides the way

The main enzymes that drive cell division can work on numerous substrates, but how is their specificity ensured? Regulatory subunits show the way, using various tricks to guide enzymes to their targets.

Even before Walther Flemming coined the term ‘mitosis’ in the 1880s, the choreography of cell division fascinated scientists. Since then it has become clear that the events that define different phases of the cell-division cycle are driven by distinct forms of an enzyme known as cyclin-dependent protein kinase.

Protein kinases facilitate the transfer of phosphate to protein substrates, generally altering their function or fate. As their name suggests, the cyclin-dependent kinases (CDKs) depend for their activity upon the binding of a regulatory subunit called a cyclin to the catalytic subunit. Many organisms use numerous cyclins (and in some cases numerous CDKs) to drive the cell cycle. Different cyclin–CDK complexes phosphorylate different substrates and so have different effects. But how do cyclins influence the capacity of their catalytic partners to recognize substrates? On page 104 of this issue, Loog and Morgan1 report that they can do so by altering the affinity of CDKs for their targets.

Structural complementarity between substrates and the active sites of enzymes — first proposed more than a hundred years ago by Emil Fischer in his ‘lock-and-key’ model — is in theory sufficient to account for the ability of the enzymes to discriminate between potential substrates (Fig. 1a, b). But enzymes that modify proteins and other macromolecules need to distinguish between similar (or even identical) sites within larger, dissimilar molecules. To do so, they must recognize the differences between substrates. That problem has been solved by diversifying the task of target recognition (Fig. 1c). Whereas the motif to be modified (one or a few amino acids in a protein, for instance) is recognized by the enzyme's active site, discrimination between different substrates bearing that motif is often accomplished through specific interactions between other sites on the enzyme and substrate.

Figure 1: How enzymes select their substrates.
figure1

a, b, In general, enzymes recognize their targets through structural complementarity between the substrate and the enzyme's active site (indicated here by the shape of the ‘pocket’). Small substrates (a) and relatively small modification sites on proteins (b) can be recognized by this mechanism. c, Some enzymes make additional, specific contacts with the substrate that enable them to distinguish between proteins that have identical or related sites of modification. d, Loog and Morgan1 have compelling new evidence that cyclin-dependent protein kinases (CDKs) have relegated that function to the exchangeable cyclin subunit, enabling a single CDK catalytic subunit to exist in numerous forms with different specificities.

Cyclin-dependent kinases have apparently broken down this process even further. Whereas responsibility for recognizing the target motif (a serine or threonine followed by a proline) is delegated to a catalytic subunit (the CDK), both genetic and biochemical studies suggest that exchangeable regulatory subunits (the cyclins) have a role in discriminating between distinct protein substrates (Fig. 1d). This is, perhaps, best illustrated by baker's yeast (Saccharomyces cerevisiae), where the cell-cycle-regulatory CDK, called Cdk1, can associate with nine distinct cyclins — three G1 cyclins (Cln1–3) and six B-type cyclins (Clb1–6). These cyclins, in addition to activating Cdk1, direct it towards distinct biological outcomes.

But although cyclins had been implicated in substrate recognition, Loog and Morgan's paper1 describes the first comprehensive study to compare the substrate specificity of purified CDK complexes that differ only in their cyclin. Their findings show that Clb5–Cdk1 and Clb2–Cdk1 complexes phosphorylate most members of a group of 150 previously confirmed Cdk1 substrates2 with roughly equal efficiency. However, 26 of those substrates are phosphorylated 2.5–800 times as efficiently by Clb5–Cdk1. In contrast, Clb2–Cdk1 does not preferentially phosphorylate any of the proteins.

The authors go on to extend previous studies3,4,5,6,7 showing that a structural motif on the surface of some cyclins, referred to as the hydrophobic patch (HP), specifically interacts with a so-called RXL or Cy motif found on some CDK substrates and inhibitors. The HP motif is important for the biological activity of Clb5 (ref. 7). Loog and Morgan1 now establish that this motif is essential for enhancing the activity of Clb5–Cdk1 towards its preferred substrates. Moreover, inactivating the Cy motif in the preferred Clb5–Cdk1 substrates eliminates their preferred status.

Strikingly, similar mutations in the Clb2 HP motif do not affect the efficiency with which Clb2–Cdk1 phosphorylates any of the substrates, regardless of the presence or absence of a Cy motif. That observation suggests that Clb2 does not use the HP motif for substrate recognition. In fact, Clb2 may not confer substrate specificity upon Cdk1. It may simply activate it and leave substrate recognition entirely to the active site. In keeping with that interpretation, Archambault et al.8 have found that Cy-containing substrates depend upon the HP motif to interact with Clb5 in an in vivo assay, but that those lacking Cy motifs interact equally well with HP-deficient Clb5 and Clb2.

So what is the role of the HP motif in Clb2? Analysis of the relationship between the six yeast B-type cyclins reveals that, although Clb5 and Clb2 are closely related in terms of their overall sequence, their HP motifs appear to be significantly different8. Given the known structure of a complex between human cyclin A3 and a Cy-motif peptide3, the Clb2 HP motif seems to be incompatible with binding to the Cy motif8. Nevertheless, it has been well conserved between different organisms, suggesting that it is still important to Clb2's function. One possibility is that it regulates a function of Clb2–Cdk1 other than its enzymatic activity. Indeed, mutation of the HP motif in Clb2 impairs the protein's export from the nucleus and its localization to at least one site in the cytoplasm9. Because Loog and Morgan's analysis was performed largely in vitro, using purified proteins, the importance of subcellular localization in substrate selection was not evaluated.

Loog and Morgan's study1 underlines the importance of cyclins in recognizing appropriate CDK substrates. The extent to which similar mechanisms are exploited by other cyclins remains to be fully examined, but there is ample evidence that other properties of cyclins are also important in substrate selection. Subcellular localization, already mentioned in the context of Clb2, is a well-established determinant of the biological function of yeast G1 cyclins10,11. Of equal or even greater importance is the hallmark of the cyclin proteins — their periodic accumulation during the cell cycle. Clearly, for a substrate to be phosphorylated it must be present in the cell along with the specific form of CDK that phosphorylates it.

So cyclins have a substantial role in directing CDKs to specific substrates. But there are numerous mechanisms for doing so, more than one of which may be used by a single cyclin. Ultimately, it is the combined action of these mechanisms that orchestrates the orderly progression of events leading to the faithful duplication of cells.

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Wittenberg, C. Cyclin guides the way. Nature 434, 34–35 (2005). https://doi.org/10.1038/434034a

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