Cyclin-dependent kinase 7

Protein Interactions

The similarity among Chiffon, Dbf4, and Dfp1 suggest a model for Chiffonís role in amplification: Chiffon may function in the activation of the chorion gene origins as the regulatory subunit of a kinase involved in origin firing, most likely the Drosophila homolog of Cdc7. S. cerevisiae Dbf4 contacts Cdc7 through the carboxyl terminus, where the CDDN1 domain is located. Thus it is hypothesized that, analogous to Dbf4 function in S. cerevisiae, Chiffon may contact the Drosophila Cdc7 homolog through the conserved CDDN1 domain and recruit it to the ORC via conserved ORC contact sites in the Chiffon amino terminus, perhaps the CDDN2 domain. Chiffon could also be hypothesized to recruit other, as yet unidentified proteins to the origin. Alternative and less direct models for Chiffon function during amplification cannot be ruled out. In addition to the defect in chorion gene amplification, the chiffon null phenotype also includes rough eyes and thin thoracic bristles. While there are several possibilities for how chiffon might be required for normal eye and bristle development, these phenotypes are consistent with a defect in DNA replication and/or S phase control in the cells forming these structures. For example, roughex regulates cyclin levels and entry into S phase, and roughex mutants are viable with rough eyes similar to chiffon nulls. Morula is a regulator of mitotic- and endo-cell cycles; hypomorphic morula mutants have rough eyes and thin thoracic bristles similar to chiffon null mutants. Finally, specific hypomorphic mutations in either the dDP or dE2F subunits of the Drosophila cell cycle regulator E2F cause rough eyes, thin thoracic bristles and defective chorion gene amplification nearly identical to chiffon nulls. Thus, Drosophila chorion gene amplification, eye development and thoracic bristle development appear to be processes that are particularly sensitive to defects in the cell cycle/DNA replication machinery (Landis, 1999 and references).

To analyze the biochemical activity of Drosophila Cdk7, antibodies were raised against the Cdk7 protein and used to isolate the active enzyme from tissue homogenates. On immunoblots prepared from fly tissues, monoclonal antibodies raised against the full-length recombinant Cdk7 recognize with high affinity a single polypeptide species, with a relative mobility of ~40 kD. This is the first indication that these anti-Cdk7 antibodies react specifically to Cdk7. The second indication is that Drosophila Cdc2 and Cdk2 proteins, which both share a high degree of similarity to Cdk7, cannot be detected in immunoprecipitates performed with anti-DmCdk7 antibodies. To demonstrate that the identified Drosophila protein possesses CAK activity, Cdk7 immunoprecipitated from embryos 0-4 hr old was used to activate recombinant human HA-Cdk2/Cyclin A complexes. HA-Cdk2/Cyclin A is strongly phosphorylated when incubated with a Cdk7 immunoprecipitate, indicating that DmCdk7 can act as a Cdk kinase. The Cdk7-mediated phosphorylation of Cdk2 seems to occur specifically at threonine residue 160. This is demonstrated by the ability of Cdk7 to phosphorylate the wild-type Cdk2 but not the Cdk2T160A mutant protein. Cdk7 also acts as a CAK, since it can stimulate the histone H1 kinase activity of Cdk2/Cyclin A. These results confirm that Drosophila Cdk7 codes for a protein that exhibits CAK activity in vitro and likely represents a functional homolog of the vertebrate cdk7 genes (Larochelle, 1998).

To test whether cdk7 is essential for CAK activity, cdk7^ts animals were incubated at the restrictive temperature for different amounts of time and the CAK activity in total cell lysates from their embryos was measured. A gradual reduction of CAK activity, down to background level, was observed. This indicates a genetic requirement for cdk7 for most or all of the cellular CAK activity that can be measured in vitro. Because immunodepletion of Cdk7 protein from embryonic homogenates can effectively eliminate CAK activity from wild-type extracts, it can be concluded that the Cdk7 protein itself provides all of the measurable CAK activity in Drosophila embryos (Larochelle, 1998).

If Cdk7 also acts in vivo as a CAK, levels of Cdk T-loop phosphorylation and Cdk activity would be expected to be reduced in cdk7 mutant tissues. Therefore, different Cdk/Cyclin complexes were isolated from mutant and wild-type embryos using antibodies directed against Cyclins A, B, and E. In Drosophila, Cyclin A (as well as Cyclin B) associates uniquely with Cdc2 and not with Cdk2. Although Cyclin A can be precipitated equally from either wild-type or mutant embryos, the amount of Cdc2 protein recovered in the Cyclin A immunoprecipitates from mutant embryos is severely decreased. In both wild-type and mutant embryos, only the fast migrating isoform of Cdc2 can be found associated with Cyclin A in a stable complex. This indicates that Cdk7 activity is required for the formation of a stable Cdc2/Cyclin A complex in vivo. In contrast, Cdc2 can still form a stable complex with Cyclin B in cdk7 mutant embryos, but the amount of Thr-161 phosphorylated isoform of Cdc2 associated with Cyclin B is reduced. The addition of recombinant Cdk7/Cyclin H to the mutant extracts before immunoprecipitation results in an increase in the amount of a fast migrating isoform of Cdc2, confirming Cdk's identity as the T-loop phosphorylating agent (Larochelle, 1998).

After immunoprecipitation from both wild-type and cdk7^ts embryos, the kinase activity toward histone H1 of Cdc2/Cyclin A, Cdc2/Cyclin B, and Cdk2/Cyclin E complexes was measured. Although the total amount of Cdc2 associated with Cyclin B is similar in both mutant and wild-type embryos, the Cdc2/Cyclin B complex isolated from mutant embryos is less active than the one isolated from wild-type embryos. This loss of activity correlates with the observed decrease in Thr-161 phosphorylation of Cdc2. If the reduction in activity of Cyclin B-bound Cdc2 isolated from cdk7^ts embryos is attributable uniquely to reduced Thr-161 phosphorylation, normal activity should be restored by treatment of this complex with CAK. To test this, the Cyclin B immunoprecipitates were incubated with active human recombinant Cdk7/Cyclin H after the initial measurement of the histone H1 kinase activity. This treatment results in the restoration of the activity and Thr-161 phosphorylation of the Cdc2 isolated from mutant embryos to a level equivalent to the one isolated from wild-type embryos. Therefore, it appears that the major reason why the activity of the Cyclin B-bound Cdc2 is lower in cdk7 mutant embryos (as compared to the control) is that this Cdc2 is hypophosphorylated on Thr-161. These results indicate that cdk7^ts embryos are deficient in physiological CAK activity. The slight delay that is observed between the time at which there is apparently no active Cdk7 protein remaining and the loss of Thr-161 phosphorylation of Cdc2 (and the appearance of early arrest phenotype) may be attributable to the fact that Cdc2 is phosphorylated maternally starting from mid-oogenesis. Therefore, this pool of active Cdc2 must be used up before the effect of lack of Cdk7 can be clearly observed (Larochelle, 1998). In the wild-type situation, Cdc2 Thr-161 does not appear to be significantly dephosphorylated until nuclear cycle 11 (Edgar, 1994).

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).

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