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).

T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity

Cyclin-dependent kinase (CDK)7-cyclin H, the CDK-activating kinase (CAK) and TFIIH-associated kinase in metazoans can be activated in vitro through T-loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, in a physiological setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes temperature-dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7-cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate-specific increase in activity caused by T-loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH-associated CDK7, and thereby regulate transcription (Larochelle, 2001).

The only component of CAK described to date in Drosophila is the catalytic subunit, CDK7. Drosophila genes coding for proteins homologous to the known partners of vertebrate CDK7, cyclin H and MAT1, have now been identified and corresponding cDNAs have been isolated from an embryonic library. The putative Drosophila cyclin H is 42% identical to human cyclin H, and the candidate Drosophila MAT1 protein shares 52% amino acid identity with human MAT1. To determine the composition of physiological Drosophila CAK complexes, CDK7 was immunoprecipitated from embryonic extracts and the associated proteins were identified by mass spectrometry of tryptic peptide fragments. CDK7 complexes contain the products of the cycH and MAT1 cDNAs. Therefore, Drosophila CAK, like its vertebrate counterpart, contains the three subunits: CDK7, cyclin H and MAT1. A fraction of CDK7 is also bound to Xerodema pigmentosum D (XPD: Reynaud, 1999), which is found along with CAK in TFIIH. A quaternary complex composed of CDK7, cyclin H, MAT1 and XPD has also been described in mammalian cell extracts (Larochelle, 2001 and references therein).

Mass spectrometric analysis of the Drosophila CAK peptides indicates that both Ser164 and Thr170 are phosphorylated in vivo, as are the corresponding residues in vertebrate CDK7. CDK7 is the only member of the CDK family with two documented phosphorylations within the T-loop (Larochelle, 2001).

Ser164 and Thr170, individually (S164A, T170A) and in combination (S164A/T170A), were mutated to alanine. A third mutation, Ser180 to alanine (S180A), was a control. Ser180 is part of the conserved WYR(A/S)PE motif of protein kinases and is an alanine in most other CDKs, including mammalian CDK7. The activity of CDK7^S180A is identical to that of wild-type CDK7 (Larochelle, 2001).

The ability of a given allele of cdk7 to rescue the lethality associated with the cdk7^null mutation was assessed by crossing males carrying the mutant transgene on the third chromosome to balanced cdk7^null females. The presence of any males carrying the cdk7^null chromosome in the progeny from this cross indicates that the transgene rescued the lack of cdk7. All mutations tested were able to rescue the lethality of the null mutation at 18°C. Although relative viability varied somewhat among individual transgenic lines, stocks of each line could be established and maintained at 18°C. Thus, CDK7 T-loop phosphorylation is not absolutely essential in vivo. However, the cdk7^S164A/T170A double mutant transgene is unable to rescue viability at 25°C, and the T170A transgene, when present as a single copy, can only rescue viability consistently at temperatures below 29°C (Larochelle, 2001).

These results are in contrast to a recent report suggesting that the T170A mutation causes CDK7 to behave in a dominant-negative fashion. These data indicate that CDK7^T170A is less active than wild-type CDK7 towards at least one substrate, possibly explaining why CDK7^T170A fails to rescue viability of the null mutation when expressed at levels much lower than that of the endogenous protein. It is therefore concluded that cdk7^T170A behaves genetically as a weak loss-of-function, rather than a dominant-negative, mutation at expression levels near that of wild-type cdk7 (Larochelle, 2001).

In contrast to the effects of the previously described conditional allele of cdk7, the temperature sensitivity of the cdk7^S164A/T170A allele is expressed almost immediately upon transfer to the restrictive temperature, resulting in a rapid arrest of egg laying by adults, and in embryonic lethality at 29°C. Furthermore, the cdk7^S164A/T170A adult flies die after 48-72 h at 29°C, also in contrast to the cdk7^P140S mutant, in which viability at high temperatures is not compromised after animals reach adulthood. Moreover, S164A/T170A larvae, do not survive a 60 min heat shock at 37°C, probably due to a failure to induce a normal heat-shock response. This suggests a more complete loss of CDK7 activity in vivo upon temperature shift when the T-loop cannot be phosphorylated (Larochelle, 2001).

Various CDK7 phospho-isoforms are observed in ovaries of mutant animals. Phosphorylation of CDK7 on the T-loop increases electrophoretic mobility, as has been observed for other CDKs. At least three phospho-isoforms can be resolved under optimal conditions. In wild-type (or S180A) adults, the fastest migrating, doubly phosphorylated isoform predominates, but significant amounts of the slowest migrating, unphosphorylated form are observed. In the S164A mutant animals, the doubly phosphorylated form disappears, and an isoform appears with intermediate electrophoretic mobility, presumably representing CDK7 singly phosphorylated on Thr170. (Larochelle, 2001).

It was asked whether the T-loop phosphorylation state of CDK7 changes in a number of physiological contexts. During embryonic development, the distribution of CDK7 between a predominant, doubly phosphorylated form and a minor, unphosphorylated form appears to be relatively constant. Likewise, CDK7 isoforms do not fluctuate appreciably in early embryos fractionated into interphase, prophase, metaphase, anaphase and telophase populations. In contrast, variations are observed when different developmental stages and different tissues are compared. In third instar larvae, the unphosphorylated isoform is virtually absent. Instead, a doublet probably corresponding to doubly and singly phosphorylated CDK7 is seen, with the singly, presumably Thr170-phosphorylated, form usually predominating. In contrast, the unphosphorylated form is a major one in imaginal disc, and is also abundant in ovaries. Although the physiological significance of these tissue-specific differences is not yet understood, it is suggested that CDK7 T-loop phosphorylation in vivo could modulate kinase activity in response to developmental or environmental signals (Larochelle, 2001).

To understand the temperature-sensitive phenotype in cdk7 mutant animals, whether the mutant proteins could be inactivated by a temperature shift was tested in vitro. The activity of CDK7 immunoprecipitated from embryos or adult flies raised at 18°C were tested towards both CDK2 and CTD after incubation at either room temperature or 33°C. Remarkably, mutation of Thr170 to alanine differentially affects activity towards the two different substrates, revealing a previously unsuspected role for this residue in determining substrate specificity. In addition, both the CAK and CTD kinase activities of all T-loop mutant forms of CDK7 are reduced after a short incubation at 33°C in vitro. Interestingly, the activity associated with CDK7 in the S164A/T170A mutant is <5% (CAK) or 1% (CTD kinase) that of wild-type CDK7, although the animals are viable. Thus, wild-type CDK7 activity vastly exceeds the level required to sustain its essential function or, alternatively, compensatory mechanisms can act to rescue a drastic drop in CAK and CTD kinase activity. T-loop phosphorylation appears to protect CDK7 from thermal inactivation. T-loop phosphorylation is an important contributor to the thermal stability of physiological CDK7 complexes, even when they contain MAT1 (Larochelle, 2001).

In vertebrates, CDK7 exists in two major complexes that can be separated by gel filtration: a >600 kDa complex corresponding to TFIIH; and an ~100 kDa heterotrimeric complex comprising CDK7, cyclin H and MAT1, which migrates aberrantly with an apparent size of ~240 kDa. Drosophila embryonic extracts were fractionated to determine the apparent size of the CDK7-containing complexes. Most endogenous Drosophila CDK7 chromatographs with the same apparent size as the mammalian trimer. Therefore, soluble CDK7 in embryos is predominantly in the form of free CAK trimer, most of which is phosphorylated on the T-loop. This is consistent with the apparently stoichiometric amounts of CDK7, cyclin H and MAT1 typically recovered in anti-CDK7 immunoprecipitates from embryonic extracts. After chromatography, the fractions were immunoprecipitated with an anti-CDK7 antibody, and kinase activity towards a recombinant CTD substrate was measured. A minor peak of both immunoreactivity and CTD kinase activity was consistently observed in fraction 18, which probably corresponds to TFIIH. Thus Drosophila CDK7 forms most or all of the same complexes as does vertebrate CDK7 (Larochelle, 2001).

The size distribution of the CDK7 proteins with T-loop mutations was examined. When extracts from either cdk7^S164A or cdk7^T170A embryos were analyzed, the majority of CDK7 remained in fractions corresponding to the trimeric form. In both cases, however, detectable amounts of CDK7 protein appeared in the smaller size fractions, possibly corresponding to free CDK7 monomer. Interestingly, the unphosphorylated CDK7 isoform is enriched in the monomer-sized fractions of the S164A lysate. In cdk7^S164A/T170A lysates, the redistribution of CDK7 protein to low molecular weight forms is even more pronounced, indicating a defect in complex formation when CDK7 cannot be phosphorylated. Consistent with this interpretation, little or no MAT1 could be detected in immunoprecipitates of fractions of the S164A/T170A lysate, although it was detected readily in wild-type and both single mutants. Because the cdk7^S164A/T170A mutation causes lethality at high temperature and alters the distribution of CDK7 between different complexes, it was asked whether the basis for temperature sensitivity might be an impaired ability to interact with cyclin H and MAT1. Indeed, after cdk7^S164A/T170A embryos were shifted from 18° to 29°C, CDK7 complexes dissociated almost completely. This correlates well with the inactivation of mutant CDK7 complexes in vitro, suggesting that the basis for thermal instability in the absence of T-loop phosphorylation is due, at least in part, to decreased affinity of CDK7 for its positive regulators, cyclin H and MAT1 (Larochelle, 2001).

In the absence of heat treatment, little difference was observed between wild-type Drosophila CDK7 and the single phosphorylation site mutants in activity towards a CDK2 substrate. However, the T170A mutant protein has dramatically reduced activity towards CTD, compared with wild-type. To measure the relative effects of Ser164 and Thr170 phosphorylation on CDK7 activity towards CDK2 and the CTD, anti-CDK7 immunoprecipitates were divided in half and assayed with both substrates. The immunoprecipitations were done under conditions that minimized CDK7 complex dissociation in vitro. The CDK7^S164A protein is ~50% as active as wild-type CDK7 with either substrate, possibly because it is more prone than wild-type and T170A proteins to dephosphorylation and consequent destabilization of the complex. Indeed, the CDK7^S164A immunoprecipitate shows a reduced amount of MAT1 relative to the wild-type, which correlates with the presence of completely unphosphorylated CDK7. The CDK7^T170A protein, in contrast, is nearly identical to wild-type CDK7 in activity towards CDK2, but only 4% as active towards the CTD. Moreover, complexes containing CDK7^T170A remain intact throughout the immunoprecipitation and the kinase assays, as judged by the stable association of MAT1. Thus, phosphorylation of Thr170 stimulates CTD kinase activity ~25-fold under these assay conditions without significantly affecting CAK activity (Larochelle, 2001).

This study has shown that a major role in vivo for T-loop phosphorylation of CDK7 is the stabilization of the predominant physiological form of the kinase: the CDK7-cyclin H-MAT1 trimer. The results suggest that the two mechanisms for CDK7 complex stabilization and activation -- MAT1 addition and T-loop phosphorylation -- which can operate independently in vitro, actually cooperate under physiological conditions to maintain complex integrity. With prolonged exposure to elevated temperature, dissociation to monomeric subunits occurs in vivo when CDK7 is dephosphorylated, even in the presence of MAT1 (Larochelle, 2001).

Since its discovery as a component of both CAK and TFIIH in metazoans, CDK7 has been studied as a possible link between the cell cycle and transcriptional machinery. Those investigations have uncovered several potential regulatory mechanisms, but no clear evidence for their usefulness in vivo. T-loop phosphorylation is an example of such a mechanism in search of a biological context. This study has uncovered two important functions of CDK7 phosphorylation: stable complex assembly and modulation of CTD kinase activity. Whereas neither function is absolutely essential, impairment of either may cause temperature-sensitive loss of viability (Larochelle, 2001).

It has been reported that the addition of MAT1 to the CDK7-cyclin H complex alters its substrate specificity, favoring CTD phosphorylation at the expense of CAK activity. An ~20-fold stimulation of the CTD kinase activity of trimeric CDK7-cyclin H-MAT1 when Thr170 is phosphorylated is observed, with no loss (or gain) of CAK activity, under conditions where neither substrate is in limiting concentration. MAT1 is required for this effect; the phosphorylated dimeric complex is no more active than the unphosphorylated trimer. Indeed, the modest lowering of the K_m for CTD when MAT1 joins the complex could explain the apparent stimulation observed previously. It is suggested, however, that MAT1 merely serves to facilitate substrate-specific stimulation by Thr170 phosphorylation, and that cycles of phosphorylation and dephosphorylation of the T-loop are more likely to regulate the function of CDK7 in vivo than are association and dissociation of MAT1 (Larochelle, 2001).

The CTD of RNA pol II undergoes a cycle of phosphorylation and dephosphorylation during the process of transcription. RNA pol II with a hypophosphorylated CTD initiates transcription, the CTD becomes phosphorylated as the enzyme proceeds from initiation to elongation and, finally, the CTD is dephosphorylated as it completes the transcription cycle. Phosphorylation of Thr170 uniquely regulates the activity of CDK7 towards the CTD. The mechanism is direct acceleration of the catalytic rate of the enzyme, and so would provide a way to increase CTD phosphorylation rates and thereby favor promotor clearance, perhaps in opposition to dephosphorylation by a CTD phosphatase. Whether this modulation is critical to regulation of gene expression has yet to be tested thoroughly. However, these studies raise the intriguing possibility that a kinase cascade or network regulates transcription through changes in the state of CDK7 T-loop phosphorylation. The failure to observe any changes in the steady-state levels of CTD phosphorylation in cdk7 mutants may reflect the complex network of kinases and phosphatases that act in concert on the CTD. Regulation of CDK7 T-loop phosphorylation may be critical, however, when rapid changes in gene expression are induced, for example by heat shock (Larochelle, 2001).

The dual function of metazoan CDK7 in control of cell cycle and transcription programs remains a puzzle. Although the notion that CDK7 coordinates gene expression with cell division in some fashion is intriguing, it has received little experimental support, and so the question of why two seemingly disparate functions are combined in one enzyme is still unanswered. There is now increased insight into how CDK7 can phosphorylate both the T-loops of CDKs and the CTD of RNA pol II, despite the complete lack of sequence homology between its two physiological substrates, by adopting different strategies for substrate recognition. Moreover, the CTD kinase activity of CDK7 can be regulated by Thr170 phosphorylation, independent of CAK activity. Strikingly, Thr170 phosphorylation of trimeric CDK7 enables the enzyme to catalyze CTD phosphorylation at ~100 times the maximal rate for CDK2 phosphorylation. Because the CTD contains many (~52) target sites for CDK7-mediated phosphorylation, whereas CDK2 contains only one, this rate enhancement could allow the major physiological form of CDK7, the phosphorylated trimer, to catalyze CDK activation and CTD hyperphosphorylation at very similar rates (Larochelle, 2001).

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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