Mapping kinase-substrate interactions demands robust methods to rapidly and unequivocally identify substrates from complex protein mixtures. Towards this goal we present a method in which a kinase, engineered to utilize synthetic ATPγS analogs, specifically thiophosphorylates its substrates in a complex lysate. The thiophosphate label provides a bio-orthogonal tag that can be used to affinity purify and identify labeled proteins. Following the labeling reaction proteins are digested with trypsin, thiol containing peptides are then covalently captured and non-thiol containing peptides are washed from the resin. Oxidation promoted hydrolysis, at sites of thiophosphorylation, releases phosphopeptides for analysis by tandem mass spectrometry. By incorporating two specificity gates: kinase engineering and peptide affinity purification, this method yields high confidence substrate identifications. This method gives both the identity of the substrates and phosphorylation site localization. With this information investigators can analyze the biological significance of the phosphorylation mark immediately following confirmation of the kinase-substrate relationship. Here we provide an optimized version of this technique to further enable widespread utilization of this technology.
phosphorylation; chemical genetics; analog specific kinase; kinase substrate identification; thiophosphate labeling
Current approaches for identifying synergistic targets use cell culture models with combinations of clinically available drugs to see if the combined effect of the combination is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that have a high potential as synergistic targets. In this study, we create a tool to screen and identify molecular targets that may synergize with new inhibitors of TOR (Target of Rapamycin), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. While clinical results from TORC1 inhibition using rapamycin analogs (that only inhibit TORC1) have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand the molecular basis for this increased therapeutic efficacy and to discover secondary targets that may have potential in targeted combination therapy, we engineered TOR2 in S. cerevisiae to accept an orthogonal inhibitor in order to create the first chemical tool to selectively inhibit TORC2. We used this tool to create a Chemical Epistasis Mini-Array Profile, or ChE-MAP, by measuring interactions between the chemically inhibited TOR2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1 (assessed using rapamycin) and TORC2 dependent functions. Results showed a novel TORC2-specific interaction with the pentose phosphate pathway (PPP). We used global metabolic profiling to show that that TORC2 inhibition led to decreases in metabolites specific to the PPP and confirmed that TOR2 was regulating this process using metabolic flux analysis. Regulation of the PPP is a previously unappreciated role for TORC2 that may suggest a role for the complex in balancing the high energy demand required for ribosome biogenesis.
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.
Toxoplasma gondii is sensitive to bulky pyrazolo [3,4-d] pyrimidine (PP) inhibitors due to the presence of a Gly gatekeeper in the essential calcium dependent protein kinase 1 (CDPK1). Here we synthesized a number of new derivatives of 3-methyl-benzyl-PP (3-MB-PP, or 1). The potency of PP analogs in inhibiting CDPK1 enzyme activity in vitro (low nM IC50 values) and blocking parasite growth in host cell monolayers in vitro (low μM EC50 values) were highly correlated and occurred in a CDPK1-specific manner. Chemical modification of the PP scaffold to increase half-life in the presence of microsomes in vitro led to identification of compounds with enhanced stability while retaining activity. Several of these more potent compounds were able to prevent lethal infection with T. gondii in the mouse model. Collectively the strategies outlined here provide a route for development of more effective compounds for treatment of toxoplasmosis, and perhaps related parasitic diseases.
Serine/threonine protein kinase; gatekeeper; calcium signaling; toxoplasmosis; chemotherapy
PI3Kδ and PI3Kγ regulate immune cell signaling, while the related PI3Kα and PI3Kβ regulate cell survival and metabolism. Selective inhibitors of PI3Kδ/γ represent a potential class of anti-inflammatory agents lacking the anti-proliferative effects associated with PI3Kα/β inhibition. Here we report the discovery of PI3Kδ/γ inhibitors that display up to 1,000-fold selectivity over PI3Kα/β and evaluate these compounds in a high-content inflammation assay using mixtures of primary human cells. We find selective inhibition of only PI3Kδ is weakly anti-inflammatory, but PI3Kδ/γ inhibitors show superior inflammatory marker suppression through suppression of LPS-induced TNFα production and T-cell activation. Moreover, PI3Kδ/γ inhibition yields an anti-inflammatory signature distinct from pan-PI3K inhibition and known anti-inflammatory drugs, yet bears striking similarities to glucocorticoid receptor agonists. These results highlight the potential of selectively designing drugs that target kinases with shared biological function.
Mitochondria have long been implicated in the pathogenesis of Parkinson’s disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal recessive form of early onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered since no allosteric regulatory sites for PINK1 are known. Here we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD related mutant PINK1G309D and PINK1wt. Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.
The kinase Akt plays a central role as a regulator of multiple growth factor input signals, making it an attractive anti-cancer drug target. A-443654 is an ATP-competitive Akt inhibitor. Unexpectedly, treatment of cells with A-443654 causes paradoxical hyperphosphorylation of Akt at its two regulatory sites (Thr308 and Ser473). We explore whether inhibitor-induced hyperphosphorylation of Akt by A-443654 is a consequence of disrupted feedback regulation at a pathway level or whether it is a direct consequence of inhibitor binding to the ATP binding site of Akt. Catalytically inactive mutants of Akt reveal that binding of an inhibitor to the ATP site of Akt is sufficient to directly cause hyperphosphorylation of the kinase in the absence of any pathway feedback effects. We conclude that ATP-competitive Akt inhibitors impart regulatory phosphorylation of their target kinase Akt providing new insights into both natural regulation of Akt activation and Akt inhibitors entering the clinic.
Post-translational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β, hence OGT and GSK3β exhibit reciprocal regulation. Modulating OGlcNAcylation levels alter circadian period length in both mice and Drosophila, and conversely protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662–S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine tune circadian clock.
Protein phosphorylation is a ubiquitous mechanism for cellular signal propagation, and signaling network complexity presents a challenge to protein kinase substrate identification. Few targets of Polo-like kinases are known, despite their significant role in coordinating cell cycle progression. Here, we combine chemical genetic, bioinformatic, and proteomic tools for Polo-like kinase substrate identification. Monospecific pharmacological inhibition of budding yeast Polo-like kinase, Cdc5, resulted in a misaligned pre-anaphase spindle and subsequently delayed anaphase nuclear migration, revealing a novel Cdc5 function. A cellular screen for Cdc5 substrates identified Spc72, a spindle pole body (SPB) component and microtubule anchor required for nuclear positioning. Spc72 bound to the Cdc5 PBD in a mitosis-specific manner, was phosphorylated by Cdc5 in vitro, and demonstrated a loss of mitotic phosphorylation in vivo upon Cdc5 inhibition. Finally, an examination of Cdc5 binding by SPB-localized proteins expanded our knowledge of Cdc5 function at the SPB.
Maintaining adequate numbers of spermatogonial stem cells is required for the production of the millions of sperm required for male fertility. To date, however, the mechanisms that regulate the size of this pool in the adult are poorly defined. Glial cell line-derived neurotrophic factor (GDNF) is required for establishing this pool in the prepubertal animal, but its in vivo function in the normal adult testis has never been examined directly. We used a chemical-genetic approach to address this issue. We generated mice carrying a single amino acid mutation (V805A) in Ret, the kinase subunit of the GDNF receptor. This mutation does not affect normal GDNF signaling, but renders it susceptible to inhibition by the ATP competitive inhibitor, NA-PP1. When GDNF signaling was blocked in adults for 11 days, only a few cells remained that expressed the stem spermatogonial markers, Gfrα1 and Zbtb16 and testicular Ret mRNA content was reduced markedly. These decreases were associated with depletion of functional stem spermatogonia; some were lost when GDNF signaling was inhibited for only 2 days while others survived for up to 11 days. However, when signaling was restored, the remaining stem cells proliferated, initiating tissue restoration. In conclusion, these results provide the first direct proof that GDNF acutely regulates the numbers of spermatogonial stem cells in the normal adult testis. Additionally, these results demonstrate different sensitivities among subpopulations of these stem cells to inhibition of GDNF signaling.
stem spermatogonia; GDNF; Sertoli cell; Ret; spermatogenesis
Promoter-proximal pausing by RNA polymerase II (Pol II) ensures both gene-specific regulation and RNA quality control. Structural considerations suggested initiation factor eviction would be required for 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 carboxyl-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 co-transcriptional RNA maturation.
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates the phosphoinositide-3-kinase (PI3K) signaling pathway. In colorectal cancer (CRC), observed frequencies of loss of PTEN expression, concordant expression in primary tumors and metastases, and the association of PTEN status with outcome vary markedly by detection method. We determined the degree to which PTEN expression is consistent in 70 matched human CRC primaries and liver metastases using a validated immunohistochemistry assay. We found loss of PTEN expression in 12.3% of assessable CRC primaries and 10.3% of assessable liver metastases. PTEN expression (positive or negative) was concordant in 98% of matched colorectal primaries and liver metastases. Next we related PTEN status to mutations in RAS and PI3K pathway genes (KRAS, NRAS, BRAF, and PIK3CA) and to overall survival (OS). PTEN expression was not significantly associated with the presence or absence of mutations in RAS or PI3K pathway genes. The median OS of patients whose tumors did not express PTEN was 9 months, compared to 49 months for patients whose tumors did express PTEN (HR = 6.25, 95% confidence intervals (CI) (1.98, 15.42), P = 0.0017). The association of absent PTEN expression with increased risk of death remained significant in multivariate analysis (HR = 6.31, 95% CI (2.03, 17.93), P = 0.0023). In summary, PTEN expression was consistent in matched CRC primaries and in liver metastases. Therefore, future investigations of PTEN in metastatic CRC can use primary tumor tissue. In patients with liver-only metastases, loss of PTEN expression predicted poor OS.
We observed concordant PTEN expression in 98% of colorectal cancer (CRC) primary and liver metastasis pairs using a validated immunohistochemistry assay. Consistent PTEN expression at both disease sites is significant because tumor tissue is usually available from CRC primaries but not metastases. Loss of PTEN expression associated with poor survival of CRC patients with liver-only metastases.
Biomarker; colorectal cancer; concordance; PTEN; survival
To investigate the role of the kinase zeta-associated protein of 70 kDa (ZAP-70) in T cells, we generated mice expressing a ZAP-70 mutant whose catalytic activity can be selectively blocked by a small molecule inhibitor. Conventional naïve, effector and memory T cells were dependent on ZAP-70 kinase activity for their activation, demonstrating a non-redundant role for ZAP-70 in TCR-induced signals. In contrast, ZAP-70 catalytic activity was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This ZAP-70 kinase-independent pathway is sufficient for regulatory T (TREG) cell suppressive activity, which was unperturbed by ZAP-70 catalytic inhibition. Our results implicate ZAP-70 as an attractive therapeutic target.
The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the ‘anti-target’ Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.
To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the non-essential genome. Using these signatures, we generated a catalogue of 297 functional modules, and assigned function to 144 previously uncharacterised genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional cross-talk between biological processes, suggesting general design principles of genetic interactomes.
Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa.
EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, as EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knockin mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly, and specifically blocked. Using these mice we demonstrate that the tyrosine kinase activity of EphBs is required for axon guidance in vivo. By contrast, EphB-mediated synaptogenesis occurs normally when the kinase activity of EphBs is inhibited suggesting that EphBs mediate synapse development by an EphB tyrosine kinase-independent mechanism. Taken together, these experiments reveal that EphBs control axon guidance and synaptogenesis by distinct mechanisms, and provide a new mouse model for dissecting EphB function in development and disease.
The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the ‘cancerous’ translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.
While mutational activation of the Epidermal Growth Factor Receptor (EGFR) features prominently in glioma and non-small-cell lung cancer (NSCLC), inhibitors of EGFR improve survival only in NCSLC. To understand how mutations in EGFR influence response to therapy, we generated glioma cells expressing either glioma- or NSCLC-derived alleles, quantifying kinase site occupancy by clinical inhibitors using novel affinity probe and kinetic methodology. At equivalent doses, erlotinib achieved lower kinase site occupancy in glioma-derived EGFRvIII, compared to NSCLC-derived EGFR mutants. Kinase site occupancy correlated directly with cell cycle arrest. EGFRvIII released erlotinib rapidly compared to wild-type EGFR, whereas NSCLC-derived mutants released erlotinib slowly.
Erlotinib; EGFR; malignant glioma; EGFRvIII; kinase site occupancy
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.
The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21 -activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism’s response to low nutrients during development, or in adult stem and cancer cells.
Dendrite arborization and synapse formation are essential for wiring the neural circuitry. The evolutionarily conserved NDR1/2 kinase pathway, important for polarized growth from yeast to mammals, controls dendrite growth and morphology in worm and fly. Whether NDR1/2 kinases regulate dendrite and synapse development in mammals was not known. Nor have their phosphorylation targets been identified. Here we show that expression of dominant negative (kinase dead) NDR1/2 mutants or siRNA increase dendrite length and proximal branching of mammalian pyramidal neurons in cultures and in vivo, whereas expression of constitutively active NDR1/2 has the opposite effects. Moreover, NDR1/2 contributes to dendritic spine development and excitatory synaptic function. We further employed chemical genetics and identified NDR1/2 substrates in the brain, including two proteins involved in intracellular vesicle trafficking: AAK1 (AP-2 associated kinase) and Rabin8, a GDP/GTP exchange factor (GEF) of Rab8 GTPase. We finally show that AAK1 contributes to dendrite growth regulation and Rabin8 regulates spine development.
This study reveals the basis for how temporal phosphoregulation of Orm protein controls sphingolipid production in response to stress. Orm protein phosphorylation is highly responsive to sphingoid bases, and Ypk1 protein kinase transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation.
Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55–protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.
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, Cdk2as 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.
Multiple cyclin-dependent kinases (CDKs) control human cell proliferation, but it remains unclear how functions of different CDKs are coordinated during unperturbed cell division or after dividing cells incur DNA damage. DNA lesions activate checkpoint signaling pathways to inhibit CDK activity, arrest the cell division cycle, and thus prevent loss of genetic information; but an effective response to damage also requires CDK activity to modify components of repair and checkpoint pathways. We took a chemical-genetic approach to ask if a specific CDK, Cdk2, played a specialized, non-redundant role in protecting genomic integrity of human cells. By sensitizing Cdk2 to chemical inhibition, we were able to detect a specific requirement for its catalytic activity in survival of cells after exposure to ionizing radiation (IR). We identified Nbs1, product of the gene mutated in the cancer-predisposing Nijmegen Breakage Syndrome, as a Cdk2 substrate and showed that mutant forms of Nbs1 that cannot be modified by Cdk2 are defective in protecting cells from death due to IR–induced DNA damage. Therefore, our work defines a DNA damage response pathway that depends on catalytic activity of a specific CDK in human cells and suggests a mechanism to promote efficient repair without triggering inappropriate cell division.
Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.
Modification of histone proteins is an important transcriptional regulatory mechanism in eukaryotic cells. Although various histone modifications are found primarily within the coding regions of transcribed genes, how they influence transcription elongation remains unclear. Among these modifications is mono-ubiquitylation of histone H2B (H2Bub1), which is needed for co-transcriptional methylation of histone H3 at specific sites. Here we show that H2Bub1 and Cdk9, the kinase component of positive transcription elongation factor b (P-TEFb), are jointly regulated by a positive feedback loop: Cdk9 activity is needed for co-transcriptional H2Bub1, and H2Bub1 in turn stimulates Cdk9 activity toward one of its major substrates, the conserved elongation factor Spt5. We provide genetic evidence that the combined action of H2Bub1 on Spt5 phosphorylation and histone methylation accounts for the gene-regulatory effects of this modification. Surprisingly, our genetic and genome-wide studies indicate that P-TEFb and H2Bub1 act in opposition on elongating RNA polymerase. We suggest that the positive feedback linking P-TEFb and H2Bub1 helps to maintain a balance between their opposing actions. These results highlight a novel regulatory role for a conserved histone modification during transcription elongation.