Cdk7: Dual Functions in Cell Cycle Regulation and Transcription

[Enlarge Image] Cdk7 T-loop phosphorylation is a substrate-specific activity switch In the context of the the trimeric Cdk7-cyclin H-Mat1 complex, phosphorylation of Thr-170 of the Cdk7 T-loop stimulates activity towards the Pol II CTD ~20-fold compared to both the unphosphorylated trimer and the phosphorylated Cdk7-cyclin H dimer, but has only a modest (~twofold) stimulatory effect on CAK activity towards Cdk2. For experimental details, see paper by Larochelle et al., 2001.

In vitro, Cdk7-cyclin H dimers can assemble if Cdk7 is phosphorylated on Thr-170 of its activation segment (T-loop) by an exogenous kinase. In the presence of Mat1, a trimeric Cdk7-cyclin H-Mat1 complex can form, independent of T-loop phosphorylation. In theory, the two pathways of Cdk7 activation could operate independently, and give rise to complexes with different properties and functions. In fact, however, the two assembly mechanisms cooperate in vivo to produce the fully active, phosphorylated trimer that is the predominant Cdk7-containing complex in both mammalian and Drosophila cells. Furthermore, although Mat1 was proposed to function as a specificity switch for Cdk7--turning on CTD kinase at the expense of CAK activity--the actual situation is more complicated. Phosphorylation of Thr-170 within the Cdk7 T-loop is the real switch, which, in the context of the fully assembled trimeric complex, stimulates enzyme turnover with CTD as substrate ~20-fold, while having only a modest (~twofold) effect on CAK activity towards Cdk2. T-loop phosphorylation thus provides a mechanism for modulating the transcriptional activity of Cdk7 over a 20-fold range without affecting its function as a CAK required for cell cycle progression. Consistent with this model, mutating Thr-170 of Drosophila Cdk7 to alanine caused temperature-sensitive lethality and derangement of Pol II phosphorylation patterns in vivo.

	[Enlarge Image] Reciprocal activation by Cdks 1, 2 and 7 Schematic diagram of a potential positive feedback loop of CDK-activating T-loop phosphorylation. Mammalian Cdk7 activates Cdk1 and Cdk2 both in vivo and in vitro, and Cdk1 and Cdk2 can both activate Cdk7 in vitro. This arrangement may obviate the need for an upstream activator of Cdk7 in mammalian cells.

In fission yeast, the Cdk7 homolog Mcs6 is activated via T-loop phosphorylation by Csk1. In metazoans, however, there is no Csk1 ortholog, and an upstream kinase capable of activating Cdk7 has not been found. An interesting solution to this problem was suggested by our demonstration that Cdk7's downstream targets, Cdk1 and Cdk2, can phosphorylate and activate Cdk7 efficiently in vitro. The reciprocal activation mechanism could obviate the need for an upstream activator of Cdk7 in metazoan systems. More intriguing, however, is the possibility it raises that CDK activation by T-loop phosphorylation in vivo is sustained by a positive feedback loop, analogous to feedback mechanisms that govern inhibitory phosphorylation of Cdk1 at the G2/M boundary.

Cdk7 activates Cdks 1 and 2, which in turn can activate Cdk7, but no CDK is capable of autophosphorylation of its own T-loop. In an effort to understand the rules governing this strict specificity, we constructed chimeric CDKs with transplanted T-loops from other CDKs. Analysis of these hybrid CDKs as both enzymes and substrates (for natural CDKs) revealed that the principle mechanism by which Cdk7 recognizes Cdks 1 and 2 (and vice versa) was dependent on determinants outside of the T-loop. This may help to explain the paradoxical recognition of completely different phosphorylation sites by Cdk7 within its two major classes of substrates: CDKs and the Pol II CTD. Phosphorylation of the CTD is directed by sequences surrounding the actual site of phosphorylation, and is likely to depend on the conformation of the Cdk7 active site, whereas recognition of CDK substrates is independent of the substrate T-loop sequence, and likely to involve protein-protein interactions between the target CDK and CAK away from the latter's active site.

The wiring of the mammalian CAK-CDK network has diverged from that of fission yeast in one important respect: the absence of a monomeric CAK orthologous to Csk1. We believe that the functions of the network have been essentially conserved, however. Challenges for the future will include a dissection of Cdk7's dual functions in transcription and cell cycle control; having established that the two can be regulated independently, we are attempting to abrogate them separately in vivo. Especially intriguing is the possibility -- raised by our work in fission yeast -- that Cdk7 is critically important for transcription of genes involved in cell cycle control. We are also identifying new substrates and novel regulators of Cdk7 and its target CDKs, through a variety of biochemical and chemical-genetic approaches.