Cyclin-dependent kinase 7

Protein Interactions

The similarity among Chiffon, Dbf4, and Dfp1 suggest a model for Chiffon’s 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).

CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid

The mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. This study reports that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. cdk7 was identified through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppresses apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid fails to localize to the mitochondria and fails to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid (Morishita, 2013).

This study reports a mechanism of cell death regulation in Drosophila in which the mitochondrial localization of a proapoptotic TA protein is regulated by CDK7. Moreover, the mitochondrial localization of Hid is coupled with its ability to bind to DIAP1. These finding provides an explanation for the mitochondrial requirement of IAP antagonists (Morishita, 2013).

Future studies are required to elucidate the structural nature of these Hid subspecies, and how they can be generated in a CDK7-dependent manner. Since only the faster-migrating form binds to DIAP1, the idea is favored that the two isoforms differ in their N terminus. In one speculative model, the faster-migrating form represents the proteolytically processed form that exposes the critical N-terminal alanine, which is responsible for DIAP1 binding. Alternatively, it is also possible that the slower-migrating form undergoes a modification that inhibits DIAP1 binding (Morishita, 2013).

Recent studies indicated that dedicated trafficking machinery exists for other TA proteins destined for the endoplasmic reticulum. However, the equivalent trafficking factors for mitochondria-destined TA proteins have not yet been found, and it is widely assumed that these TA proteins insert into the mitochondrial outer membrane without active assistance. By contrast, the finding of this study indicates that Hid's mitochondrial localization can be regulated in cells, suggesting the existence of an active trafficking machinery for the mitochondrial TA protein (Morishita, 2013).

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