BIOLOGICAL OVERVIEW

The activity of cyclin dependent kinases (Cdks) is regulated by their association with regulatory subunits (cyclins), and by multiple phosphorylation events. Because threonine phosphorylation of the different Cdks is a crucial step in their activation, much effort has been directed toward identifying and characterizing the kinases responsible for this event. In a strange twist of biological logic, the activator of G2 Cdks is itself another cyclin/Cdk dimer. An enzyme complex has been identified that is able to phosphorylate a number of different Cdks on their activating threonine residue and is known as Cdk-activating kinase (CAK). CAK itself is a Cdk/Cyclin complex: Cdk7/Cyclin H. A third subunit, MAT1, has also been found to associate with Cdk7 and cyclin H and to serve as an assembly factor. However, unlike most other Cdks, Cdk7 has been found to be active throughout the cell cycle with no detectable oscillation in its activity. These results suggest that the CAK activity of Cdk7 could be sufficient to provide the activating Thr-161 phosphorylation to all Cdks throughout the cell cycle (Larochelle, 1998 and references).

In another twist of biological logic, Cdk7 has a second biochemical role that adds to its mystery. Cdk7 is also able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) as part of the TFIIH basic transcription factor complex (Roy, 1994, Serizawa, 1995 and Shiekhattar, 1995). Thus two questions plague every discussion of Cdk7: (1) why did Cdk function evolve to require activation by CAK in the first place? (2) Why the necessity for a dual biological role for Cdk7, as both an activator for Cdks and a phosphorylator of Pol II? This second role may be necessitated by a feedback loop, in which activation of mitotic cyclins feeds back to result in a global inactivation of Pol II mediated transcription, involving TFIIH as the target (Long, 1998). Adding to the controversy Cdk7 raises is the fact that in the budding yeast Saccharomyces cerevisiae, CAK activity is provided by the CAK1/Civ1 protein, which is unrelated to Cdk7. Furthermore, Kin28, the budding yeast Cdk7 homolog, functions not as a CAK but only as the catalytic subunit of TFIIH. This essay will examine the role of Cdk7 in activating Cdks in Drosophila: the function of Cdk7 in a complex with Pol II will be dealt with in the Evolutionary Homologs section.

Conclusive evidence that Cdk7 acts as a Cdk-activating kinase (CAK) in vivo has remained elusive. In the absence of better genetic evidence, it has been proposed that the CAK activity of Cdk7 may be an in vitro artifact. In an attempt to resolve this issue, the Drosophila Cdk7 homolog has been cloned and null and temperature-sensitive mutations have been created. It has been demonstrated that Cdk7 is necessary for CAK activity in vivo in Drosophila. It has also been shown that Cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division. These results suggest that there may be a fundamental difference in the way metazoans and budding yeast effect a key modification of Cdks (Larochelle, 1998).

In Cdk7 mutant fly embryos, the level of Thr-161 phosphorylation and activity of the Cyclin B-bound Cdc2 was shown to be reduced, and both activities are restored by incubation with purified Cdk7/Cyclin H. This indicates that the major difference between Cdc2 isolated from wild-type and Cdk7 mutant embryos is the extent of Thr-161 phosphorylation. Therefore, Cdk7 is essential for in vivo CAK activity. Although Cdc2/Cyclin B complexes form normally in Cdk7^ts mutant embryos, Cdc2 and Cyclin A fail to form a stable complex in the Cdk7 mutant. This is likely attributable to the fact that this event requires the phosphorylation of Cdc2 on Thr-161, as even in the wild type only the phosphorylated form is associated with Cyclin A. These in vivo results correlate well with the finding that human Cdc2 needs to be phosphorylated by CAK to form a stable complex with Cyclin A in vitro, whereas stable Cdc2/Cyclin B and Cdk2/Cyclin E complexes can form in the absence of Thr-161 (or 160) phosphorylation (Desai, 1995). The Cdc2/Cyclin A complex seems to be more sensitive to a reduction in CAK activity than the Cdc2/Cyclin B complex, as the loss of Cyclin A binding occurs more rapidly than the reduction of Thr-161 phosphorylation of Cyclin B-associated Cdc2 (Larochelle, 1998).

If Cdk7 is required specifically for mitosis, it would be expected that the ovarian phenotype resulting from lack of cdk7 would be similar to the one resulting from lack of Cdc2. Therefore, the Cdc2 phenotype was analyzed using the temperature-sensitive transgene Dmcdc2^A171T (Sigrist, 1995). Females carrying two copies of this temperature-sensitive allele in the Dmcdc2^B47 background show a rapid depletion of follicle cells when transferred to the restrictive temperature after eclosion. This depletion of follicle cells is identical to the one observed in Cdk7^ts ovaries. Also, as noted for the Cdk7^ts mutant ovaries, mitotic proliferation of the germ line stops but the capacity of the germ-line cells to replicate their DNA is not affected by the loss of Cdc2 activity. Polyploidization of the germ cells usually occurs only when the mitotic division program is terminated and the 16-cell cyst is formed. In both cdc2 and cdk7 mutants the polyploidization of the germ line occurs prematurely. Because Cdc2 mediates this block of endoreplication in mitotic tissues, these results also suggest that the premature endoreplication observed in cdk7 mutant ovaries may be attributable to lack of Cdc2 activity (Larochelle, 1998).

The basic components of the cell cycle regulatory machinery are, for the most part, shared by both yeast and higher eukaryotes. It has been shown in numerous cases that the mechanisms, as well as molecules, that regulate the cell cycle in yeast are usually also conserved in higher eukaryotes. Therefore, it may come as a surprise that yeast and metazoans would use entirely different molecules, such as yeast CAK1 and higher eukaryote Cdk7, to carry out identical enzymatic reactions in such a basic mechanism as the activating phosphorylation of Cdks. Perhaps even more surprising is that a "complex" multicellular organism would use a single enzyme to carry out two very distinct functions, whereas the apparently much simpler unicellular yeast would use two different ones. However, the analysis of all the data obtained in this study and previously with Cdk7, Kin28, and CAK1 clearly point in this direction (Larochelle, 1998).

Now that the sequence of the whole genome of S. cerevisiae is known, it is clear that of all yeast proteins, Kin28 is the one with the highest sequence similarity to Cdk7. At the functional level, both proteins can be found as subunits of TFIIH and are known to interact physically with related cyclin-like molecules. Both Cdk7 and Kin28 can use the CTD of RNA Pol II as substrate in vitro. From these data it seems clear that Cdk7 and Kin28 are not only related by sequence, but also that each protein carries out similar cellular functions in its respective organism. However, there is a major difference between the two molecules; Cdk7 is a very efficient CAK in vitro, whereas Kin28 has no detectable CAK activity either in vitro or in vivo (Cismowski, 1995; Valay, 1995). The present work underlines another major difference between Cdk7 and Kin28, this time at the level of a genetic requirement. It was clearly demonstrated that Drosophila Cdk7 activity is required for the production of CAK activity in vivo, whereas Yeast Kin28 is not. Evidently unicellular organisms have continued to evolve, just as have metazoans. Maybe it was advantageous for S. cerevisiae to use two distinct proteins to carry out functions for which only one has remained necessary in other organisms. The emergence of yeast CAK1 may then have lead to the evolution of Kin28, a Cdk7 that has lost its ability to act as a CAK. In this context it would be interesting to know whether metazoans have a CAK1 homolog. Thus far, none have been reported, but whether there is or is not a CAK1 homolog in multicellular organisms will be resolved only by its discovery or with the completion of the sequencing of a metazoan genome (Larochelle, 1998).

GENE STRUCTURE

The Cdk7 gene is located in cytological interval 4F and is separated by ~0.4 and 3 kb from its proximal neighbors sans fille (snf) and deadhead (dhd), respectively. snf and Cdk7 are oriented head to head (Larochelle, 1998).

Exons - 2

PROTEIN STRUCTURE

Amino Acids - 353

Structural Domains

A Drosophila sequence homologous to the vertebrate cdk7 genes was isolated using a degenerate PCR-based approach. Drosophila and human Cdk7 proteins share 65% identity over the entire polypeptide, a sequence similarity higher than to any other Cdk (Larochelle, 1998).

date revised: 5 July 98

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