P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼ 100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5'-to-3' "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.

A Cdk7-Cdk4 T-loop phosphorylation cascade promotes G1 progression.

Eukaryotic cell division is controlled by cyclin-dependent kinases (CDKs), which require phosphorylation by a CDK-activating kinase (CAK) for full activity. Chemical genetics uncovered requirements for the metazoan CAK Cdk7 in determining cyclin specificity and activation order of Cdk2 and Cdk1 during S and G2 phases. It was unknown if Cdk7 also activates Cdk4 and Cdk6 to promote passage of the restriction (R) point, when continued cell-cycle progression becomes mitogen independent, or if CDK-activating phosphorylation regulates G1 progression. Here we show that Cdk7 is a Cdk4- and Cdk6-activating kinase in human cells, required to maintain activity, not just to establish the active state, as is the case for Cdk1 and Cdk2. Activating phosphorylation of Cdk7 rises concurrently with that of Cdk4 as cells exit quiescence and accelerates Cdk4 activation in vitro. Therefore, mitogen signaling drives a CDK-activation cascade during G1 progression, and CAK might be rate-limiting for R point passage.

Distinct activation pathways confer cyclin-binding specificity on Cdk1 and Cdk2 in human cells.

In metazoans, different cyclin-dependent kinases (CDKs) bind preferred cyclin partners to coordinate cell division. Here, we investigate these preferences in human cells and show that cyclin A assembles with Cdk1 only after complex formation with Cdk2 reaches a plateau during late S and G2 phases. To understand the basis for Cdk2's competitive advantage, despite Cdk1's greater abundance, we dissect their activation pathways by chemical genetics. Cdk1 and Cdk2 are activated by kinetically distinct mechanisms, even though they share the same CDK-activating kinase (CAK), Cdk7. We recapitulate cyclin A's selectivity for Cdk2 in extracts and override it with a yeast CAK that phosphorylates monomeric Cdk1, redirecting Cdk1 into a pathway normally restricted to Cdk2. Conversely, upon Cdk7 inhibition in vivo, cyclin B, which normally binds Cdk1 nearly exclusively, is diverted to Cdk2. Therefore, differential ordering of common activation steps promotes CDK-cyclin specificity, with Cdk7 acting catalytically to enforce fidelity.

Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.

The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as) phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.

Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics.

Cdk7 performs two essential but distinct functions as a CDK-activating kinase (CAK) required for cell-cycle progression and as the RNA polymerase II (Pol II) CTD kinase of general transcription factor IIH. To investigate the substrate specificity underlying this dual function, we created an analog-sensitive (AS) Cdk7 able to use bulky ATP derivatives. Cdk7-AS-cyclin H-Mat1 phosphorylates approximately 10-15 endogenous polypeptides in nuclear extracts. We identify seven of these as known and previously unknown Cdk7 substrates that define two classes: proteins such as Pol II and transcription elongation factor Spt5, recognized efficiently only by the fully activated Cdk7 complex, through sequences surrounding the site of phosphorylation; and CDKs, targeted equivalently by all active forms of Cdk7, dependent on substrate motifs remote from the phosphoacceptor residue. Thus, Cdk7 accomplishes dual functions in cell-cycle control and transcription not through promiscuity but through distinct, stringent modes of substrate recognition.

Xpd/Ercc2 regulates CAK activity and mitotic progression.

General transcription factor IIH (TFIIH) consists of nine subunits: cyclin-dependent kinase 7 (Cdk7), cyclin H and MAT1 (forming the Cdk-activating-kinase or CAK complex), the two helicases Xpb/Hay and Xpd, and p34, p44, p52 and p62 (refs 1-3). As the kinase subunit of TFIIH, Cdk7 participates in basal transcription by phosphorylating the carboxy-terminal domain of the largest subunit of RNA polymerase II. As part of CAK, Cdk7 also phosphorylates other Cdks, an essential step for their activation. Here we show that the Drosophila TFIIH component Xpd negatively regulates the cell cycle function of Cdk7, the CAK activity. Excess Xpd titrates CAK activity, resulting in decreased Cdk T-loop phosphorylation, mitotic defects and lethality, whereas a decrease in Xpd results in increased CAK activity and cell proliferation. Moreover, Xpd is downregulated at the beginning of mitosis when Cdk1, a cell cycle target of Cdk7, is most active. Downregulation of Xpd thus seems to contribute to the upregulation of mitotic CAK activity and to regulate mitotic progression positively. Simultaneously, the downregulation of Xpd might be a major mechanism of mitotic silencing of basal transcription.

Cdk7 is required for full activation of Drosophila heat shock genes and RNA polymerase II phosphorylation in vivo.

TFIIH has been implicated in several fundamental cellular processes, including DNA repair, cell cycle progression, and transcription. In transcription, the helicase activity of TFIIH functions to melt promoter DNA; however, the in vivo function of the Cdk7 kinase subunit of TFIIH, which has been hypothesized to be involved in RNA polymerase II (Pol II) phosphorylation, is not clearly understood. Using temperature-sensitive and null alleles of cdk7, we have examined the role of Cdk7 in the activation of Drosophila heat shock genes. Several in vivo approaches, including polytene chromosome immunofluorescence, nuclear run-on assays, and, in particular, a protein-DNA cross-linking assay customized for adults, revealed that Cdk7 kinase activity is required for full activation of heat shock genes, promoter-proximal Pol II pausing, and Pol II-dependent chromatin decondensation. The requirement for Cdk7 occurs very early in the transcription cycle. Furthermore, we provide evidence that TFIIH associates with the elongation complex much longer than previously suspected.

Activation of the p53 Transcriptional Program Sensitizes Cancer Cells to Cdk7 Inhibitors.

Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

CDK-activating kinases: detection and activity measurements.

All cyclin-dependent kinases (CDKs) involved in eukaryotic cell cycle control require phosphorylation at a conserved threonine (or serine) residue within the activation- or T-loop to attain full enzymatic activity. The enzyme responsible for this activating phosphorylation, the CDK-activating kinase (CAK), is therefore essential for proliferation of all eukaryotic cells. We describe methods to assess the T-loop phosphorylation state of the major mammalian CDKs in vivo; to measure the levels of CAK activity in cell-free extracts; and to analyze the abundance, subunit composition, and phosphorylation state of CAK complexes in metazoan cells. When derangement of normal CDK regulation is suspected as a cause of disturbed cell cycle progression, the combination of these methodologies can ascertain whether a primary CAK defect is the explanation.

Separate domains of fission yeast Cdk9 (P-TEFb) are required for capping enzyme recruitment and primed (Ser7-phosphorylated) Rpb1 carboxyl-terminal domain substrate recognition.

In fission yeast, discrete steps in mRNA maturation and synthesis depend on a complex containing the 5'-cap methyltransferase Pcm1 and Cdk9, which phosphorylates the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) and the processivity factor Spt5 to promote transcript elongation. Here we show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to both Pcm1 and the Pol II CTD. Removal of this segment diminishes Cdk9/Pcm1 chromatin recruitment and Spt5 phosphorylation in vivo and leads to slow growth and hypersensitivity to cold temperature, nutrient limitation, and the IMP dehydrogenase inhibitor mycophenolic acid (MPA). These phenotypes, and the Spt5 phosphorylation defect, are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition of CTD substrates "primed" by Mcs6 (Cdk7). On defined peptide substrates in vitro, Cdk9 prefers CTD repeats phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a conserved mechanism, independent of chromatin recruitment, to order transcriptional CDK functions. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition and a multivalent recruitment module that couples transcription with capping.