Gene name - Cyclin dependent kinase 9
Cytological map position - 58F1-2
Function - signaling
Symbol - Cdk9
FlyBase ID: FBgn0019949
Genetic map position -
Classification - cyclin dependent protein kinase
Cellular location - nuclear
The elongation potential of RNA polymerase II has been proposed to be controlled by negative transcription elongation factors and positive transcription elongation factors. All initiated RNA polymerase II molecules enter abortive elongation in which only short transcripts are generated due to the function of negative transcription elongation factors. Upon the action of positive factors such as positive transcription elongation factor b (P-TEFb, a dimer of Cyclin dependent kinase 9 and Cyclin T), the RNA polymerase II molecules overcome the promoter-proximal pausing and premature termination, and as a consequence, enter productive elongation. After the initial switch into a productive mode, the efficiency of elongation can be further increased by other factors to generate long transcripts (Peng, 1998b and references therein).
Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is a regulatory event in transcription. The unphosphorylated RNA polymerase II (IIA) has been found in preinitiation complexes and in early elongation complexes in vitro, whereas the hyperphosphorylated polymerase II (IIO) has been observed in productive elongation. The phosphorylation state of the CTD is controlled by the action of kinases and phosphatases. Drosophila P-TEFb has been identified as a kinase with subunits of 124 and 43 kDa that can phosphorylate the CTD of RNA polymerase II (Marshall, 1996). The kinase activity of P-TEFb is very sensitive to the purine analog DRB (Marshall, 1996). Consistently, the transition from abortive to productive elongation can be inhibited by DRB in vitro and in vivo. The 43-kDa subunit of Drosophila P-TEFb has been cloned and found to be similar to Cdc2, a cyclin-dependent kinase (CDK) (Zhu, 1997; Peng, 1998b and references therein).
Most known CDKs play a role in regulating the cell cycle, but some CDKs have been found to be involved in other cellular events. A CDK can be activated by binding of a cyclin and phosphorylation of a conserved threonine residue in the T-loop of the catalytic subunit. Conversely, a CDK/cyclin complex can be inactivated by phosphorylation of a threonine residue and a tyrosine residue near the ATP binding site or by binding of a family of small proteins termed CKIs. The catalytic subunits are well conserved in the CDK family, but the cyclins are not conserved except for the cyclin box that is predicted as a helix-rich structure. Drosophila P-TEFb has been shown to be composed of a CDK/cyclin pair (Peng, 1998b and references therein).
The Drosophila P-TEFb possesses a CTD kinase activity that is very sensitive to DRB (Marshall, 1996). To examine the kinase activity of recombinate Drosophila P-TEFb, Drosophila RNA polymerase II was incubated with increasing amounts of either native P-TEFb or recombinant P-TEFb in the presence of 32P labelled ATP for 5 min. The reactions were analyzed by 6%-15% SDS-PAGE. The protein gel was silver-stained and then subjected to autoradiography. As expected, increasing the amount of native Drosophila P-TEFb increases the fraction of the hyperphosphorylated IIO form of the large subunit of RNA polymerase II as well as the total amount of label incorporated into the IIO form. The activity of recombinant P-TEFb was indistinguishable from that of native Drosophila P-TEFb. The sensitivity of recombinant P-TEFb to DRB was compared with that of native P-TEFb using a similar assay. The radioactivity incorporated into the large subunit of RNA polymerase II was quantitated, normalized to the no DRB point (100%), and plotted. The 50% inhibition point of recombinant P-TEFb (0.5 µM) is similar to that of P-TEFb (Peng, 1998a).
The function of recombinant P-TEFb in elongation control was analysed. To do this it was first necessary to generate a P-TEFb-dependent transcription system. P-TEFb allows the generation of long DRB-sensitive transcripts (Marshall, 1996), and in a human transcription system depletion of P-TEFb eliminates DRB-sensitive transcription (Zhu, 1997). To deplete Drosophila P-TEFb from Kc cell nuclear extract (KcN), antibodies to Drosophila P-TEFb were produced. A glutathione S-transferase fusion protein containing the carboxyl-terminal half of the cyclin T subunit was used to immunize rabbits. A Western blot indicated that the resulting antiserum recognized both the native and recombinant P-TEFb. Moreover, the antibodies in the antiserum were able to deplete the cyclin subunit of Drosophila P-TEFb (Peng, 1998a).
The depleted KcN was characterized and then used to compare the activity of recombinant P-TEFb to the native factor. The starting extract as well as a preimmune depleted extract both exhibit DRB-sensitive runoff transcripts indicative of the function of P-TEFb. Depletion of P-TEFb decreases these runoff transcripts. As was found in the human transcription system (Zhu, 1997), depletion of P-TEFb does not deplete other factors needed to generate DRB-sensitive transcription. Addition of increasing amounts of native P-TEFb to the depleted extract restores the appearance of runoff transcripts to levels higher than that observed with the low level of endogenous P-TEFb in the starting extract. As expected, the runoff transcripts stimulated by P-TEFb are sensitive to DRB. Incomplete inhibition by DRB is likely due to the saturating amount of P-TEFb being added. The recombinant P-TEFb gives identical results to native Drosophila P-TEFb, demonstrating that it is fully functional in transcription (Peng, 1998a).
To begin to elucidate the role of the cyclin subunit and to determine the requirement of the kinase activity, recombinant proteins containing various mutations were generated. The proteins were produced in baculovirus-infected Sf9 cells and then purified. Recombinant proteins include a kinase knockout, CDK9 alone, and two CDK9/cyclin pairs in which portions of the carboxyl-terminal region of the cyclin subunit were deleted. In each case, the CDK9 subunit was His tagged, and as long as the cyclin subunit contained an intact cyclin box, it associated strongly with the kinase subunit. This was also found with another construct that contained only amino acids 1-348 of the cyclin subunit (Peng, 1998a).
The recombinant proteins were examined for their ability to phosphorylate the CTD of RNA polymerase II as well as their ability to function during transcription. CDK9 alone has no activity in either assay, demonstrating that, indeed, its activity is cyclin dependent. The kinase knockout mutant also has no activity in either assay, demonstrating that the function of P-TEFb is through its ability to carry out phosphorylation. Deletion of the unique carboxyl-terminal domain of cyclin T causes a dramatic reduction in both activities measured. The two cyclin T truncation mutants gave only 10%-20% of the kinase activity seen with the intact protein, and this reduction was quantitatively mirrored in the transcription assay. An unusual feature of the phosphorylation of RNA polymerase II by the two truncation mutants is that the shift from IIA to IIO does not occur as readily. Drosophila P-TEFb has been shown to preferentially phosphorylate a CTD that has already been phosphorylated (Marshall, 1996), and at low levels of the kinase this leads to the hyperphosphorylation of a subset of the polymerases. Evidently, the carboxyl-terminal region of cyclin T is responsible for this preferential phosphorylation of the partially phosphorylated polymerase molecules (Peng, 1998a).
To better compare the transcriptional activity of the two cyclin truncation mutants with intact DmP-TEFb, a wider range of kinase concentrations was titrated. It was especially interesting to determine if higher levels of the truncation mutants could compensate for their lower kinase activity or if they were in some other way defective. Intact P-TEFb functioned as expected, giving rise to a high level of runoff transcripts. Comparison of the amount of runoff at intermediate levels of intact P-TEFb (4x to 16x) indicated that addition of 2-fold more kinase has a greater than 2-fold effect. A different result was obtained with the two truncation mutants. Neither showed the effect seen at intermediate levels of intact P-TEFb. Even though very high levels were used, the effect of both truncation mutants saturates before reaching the high level of runoff seen with the intact protein. It is concluded that the carboxyl-terminal region of cyclin T is important for the function of P-TEFb in transcription (Peng, 1998a).
P-TEFb must interact at least briefly with the polymerase to carry out phosphorylation, and this transient interaction may be enhanced by the positive charge of the carboxyl-terminal region of cyclin T. This could explain why P-TEFb preferentially phosphorylates a CTD that was already partially phosphorylated (Marshall, 1996). When the CTD is partially phosphorylated, it contains more negative charge and might interact more strongly with the positively charged region of cyclin T. Removal of this region decreases the kinase activity (Peng, 1998a).
The kinase activity of TFIIH has been implicated in CTD phosphorylation and in elongation control. Therefore, it was of interest to determine if the CDK-activating kinase associated with TFIIH could affect the CTD kinase activity of P-TEFb. Kinase reactions were set up with RNA polymerase II as substrate, limiting amounts of P-TEFb or lower levels of Drosophila TFIIH. As expected, CTD phosphorylation by P-TEFb but not TFIIH was sensitive to DRB. In reactions containing both kinases, the signals were apparently the sum of the two activities. The phosphorylation of cyclin T and CDK9 was also unaffected by the presence of TFIIH. This suggests that neither kinase has a dramatic effect on the other (Peng, 1998a).
These results suggest that Drosophila P-TEFb is not part of a larger complex and does not stably associate with RNA polymerase II or the elongation complex. Depletion of P-TEFb even under low salt conditions does not remove other required factors because addition of pure DmP-TEFb is able to restore DRB-sensitive transcription. It is possible that other required factors are in excess over the P-TEFb. This however is thought not to be the case because when the kinase knockout mutant was added in more than 10-fold excess over the wild type kinase, no reduction in kinase activity or DRB-sensitive transcription was observed. Consistent with this, no more than one chromatographic form of P-TEFb has been detected (Peng, 1998a).
The timing of phosphorylation of the polymerase in an early elongation complex is important because of the termination activity of factor 2 (Xie, 1996). Essentially, there is a functional competition between factor 2 and P-TEFb. During a short period of time after initiation, P-TEFb must phosphorylate the CTD to an appropriate extent to cause the transition into productive elongation. Otherwise factor 2 will cause premature termination. This may be why the cyclin mutants lacking the carboxyl-terminal region are able to generate only low levels of runoff transcripts. The reduced ability of the cyclin mutants to recognize partially phosphorylated RNA polymerase II in an early elongation complex would lead to a reduced rate of phosphorylation and, therefore, ultimately a reduced number of hyperphosphorylated polymerases that generate runoff (Peng, 1998a).
As a cyclin-dependent kinase, P-TEFb is likely to be activated by phosphorylation of the catalytic subunit. The TFIIH-associated kinase is a CDK-activating kinase termed CDK7 (see Drosophila Cdk7) that has been shown to activate CDC2, CDK2, and CDK4. TFIIH has also been shown to function in transcription elongation. Although it is possible that TFIIH plays a role in activating P-TEFb, the results presented here do not support this hypothesis. Using pure RNA polymerase II as a substrate, TFIIH has no effect on the kinase activity of P-TEFb. It is possible that in the context of transcription with other factors involved, TFIIH might play a role in activating P-TEFb. It is also possible that P-TEFb purified from eucaryotic cells is already activated. Further work in vitro and in vivo will be required to understand how P-TEFb activity is regulated (Peng, 1998a).
A cDNA encoding the small subunit of the Drosophila factor has been cloned. The two subunits of purified P-TEFb were separated by gel electrophoresis, and the small subunit was excised and subjected to protein sequencing. The peptide sequence information was used in the cloning of full-length cDNA. The deduced amino acid sequence identified the small subunit of Drosophila P-TEFb as a member of the Cdc2-like cyclin dependent kinase family with >40% identity to Schizosaccharomyces pombe Cdc2. A search of the protein database revealed a human protein, PITALRE, that exhibits 72% identity and 83% similarity to the Drosophila protein. The high level of sequence similarity indicates that PITALRE is a potential homolog of the small subunit of Drosophila P-TEFb and therefore may be a component of human P-TEFb. Two kinases from Saccharomyces cerevisiae, SGV1 and CTK1, each share 43% identity with PITALRE and the small subunit of Drosophila P-TEFb. Although sequence similarity does not allow the prediction of a potential yeast homolog, CTK1 has recently been demonstrated to increase the elongation efficiency of RNA polymerase II (Zhu, 1997 and references therein).
date revised: 25 March 2001
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