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.

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.

Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.

Promoter-proximal pausing by RNA polymerase II (Pol II) ensures gene-specific regulation and RNA quality control. Structural considerations suggested a requirement for initiation-factor eviction in elongation-factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7--part of TFIIH--increases TFIIE retention, prevents DRB sensitivity-inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9-cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events--Pol II C-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation--in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3'-end formation. Cdk7 thus acts through TFIIE and DSIF to establish, and through P-TEFb to relieve, barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and cotranscriptional RNA maturation.

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.

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.

TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II.

The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7(as/as) mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5' ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase "leaking" into the gene. Consistent with a 5' pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36-two marks of elongation-within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3' ends, it was detected within genes and 3'-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

p27Kip1 inhibits cyclin D-cyclin-dependent kinase 4 by two independent modes.

Cell cycle progression is regulated by cyclin-dependent kinases (cdk's), which in turn are regulated by their interactions with stoichiometric inhibitors, such as p27(Kip1). Although p27 associates with cyclin D-cyclin-dependent kinase 4 (cdk4) constitutively, whether or not it inhibits this complex is dependent on the absence or presence of a specific tyrosine phosphorylation that converts p27 from a bound inhibitor to a bound noninhibitor under different growth conditions. This phosphorylation occurs within the 3-10 helix of p27 and may dislodge the helix from cdk4's active site to allow ATP binding. Here we show that the interaction of nonphosphorylated p27 with cdk4 also prevents the activating phosphorylation of the T-loop by cyclin H-cdk7, the cdk-activating kinase (CAK). Even though the cyclin H-cdk7 complex is present and active in contact-arrested cells, p27's association with cyclin D-cdk4 prevents T-loop phosphorylation. When p27 is tyrosine phosphorylated in proliferating cells or in vitro with the tyrosine Y kinase Abl, phosphorylation of cdk4 by cyclin H-cdk7 is permitted, even without dissociation of p27. This suggests that upon release from the contact-arrested state, a temporal order for the reactivation of inactive p27-cyclin D-cdk4 complexes must exist: p27 must be Y phosphorylated first, directly permitting cyclin H-cdk7 phosphorylation of residue T172 and the consequent restoration of kinase activity. The non-Y-phosphorylated p27-cyclin D-cdk4 complex could be phosphorylated by purified Csk1, a single-subunit CAK from fission yeast, but was still inactive due to p27's occlusion of the active site. Thus, the two modes by which p27 inhibits cyclin D-cdk4 are independent and may reinforce one another to inhibit kinase activity in contact-arrested cells, while maintaining a reservoir of preformed complex that can be activated rapidly upon cell cycle reentry.

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.

Hyperphosphorylation of RNA polymerase II in response to topoisomerase I cleavage complexes and its association with transcription- and BRCA1-dependent degradation of topoisomerase I.

The progression of RNA polymerase II can be blocked by lesions on the DNA template. In this study, we focused on the modifications of the largest subunit of RNA polymerase II, Rpb1, in response to stabilized topoisomerase I (Top1)-DNA cleavage complexes. In addition to DNA modifications (base damages and strand breaks), Top1 cleavage complexes can be trapped by camptothecin (CPT) and its derivatives used in cancer treatment. We found that, within a few minutes, CPT produces the complete hyperphosphorylation of Rpb1 in both primary and transformed cancer cells. Hyperphosphorylation is rapidly reversible following CPT removal. Hyperphosphorylation occurs selectively on the serine 5 residue of the conserved heptapeptide repeats in the Rpb1 carboxy-terminal domain and is mediated principally by the transcription factor IIH-associated cyclin-dependent kinase Cdk7. Hyperphosphorylated Rpb1 is not primarily targeted for proteosomal degradation and instead is subjected to cycles of phosphorylation and dephosphorylation as long as Top1 cleavage complexes are trapped by CPT. Finally, we show that transcription-induced degradation of Top1 is Brca1 dependent, suggesting a role for Brca1 in the repair or removal of transcription-blocking Top1-DNA cleavage complexes.

Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells.

Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single Plk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single Plk family member, Plk1, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1's role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human cells through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase (Plk1(wt)) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs (Plk1(as)). When cultured in the presence of these analogs, Plk1(as) cells accumulate in prometaphase with defects that parallel those found in PLK1(Delta/Delta) cells. In addition, acute treatment of Plk1(as) cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics.