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ABSTRACT: 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.
Molecular cell 04/2013; 50(2):250-60. · 14.61 Impact Factor
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ABSTRACT: 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.
Nature Structural & Molecular Biology 10/2012; · 12.71 Impact Factor
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Lara Wohlbold,
Karl A Merrick,
Saurav De,
Ramon Amat,
Jun Hyun Kim,
Stéphane Larochelle,
Jasmina J Allen,
Chao Zhang, Kevan M Shokat,
John H J Petrini,
Robert P Fisher
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ABSTRACT: 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.
PLoS Genetics 08/2012; 8(8):e1002935. · 8.69 Impact Factor
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Miriam Sansó,
Karen M Lee,
Laia Viladevall,
Pierre-Étienne Jacques,
Viviane Pagé,
Stephen Nagy,
Ariane Racine,
Courtney V St Amour,
Chao Zhang, Kevan M Shokat,
Beate Schwer,
François Robert,
Robert P Fisher,
Jason C Tanny
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ABSTRACT: 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.
PLoS Genetics 08/2012; 8(8):e1002822. · 8.69 Impact Factor
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Christine K Cheng,
W Clay Gustafson,
Elizabeth Charron,
Benjamin T Houseman,
Eli Zunder,
Andrei Goga,
Nathanael S Gray,
Brian Pollok,
Scott A Oakes,
C David James, Kevan M Shokat,
William A Weiss,
Qi-Wen Fan
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ABSTRACT: Malignant glioma, the most common primary brain tumor, is generally incurable. Although phosphatidylinositol-3-kinase (PI3K) signaling features prominently in glioma, inhibitors generally block proliferation rather than induce apoptosis. Starting with an inhibitor of both lipid and protein kinases that induced prominent apoptosis and that failed early clinical development because of its broad target profile and overall toxicity, we identified protein kinase targets, the blockade of which showed selective synthetic lethality when combined with PI3K inhibitors. Prioritizing protein kinase targets for which there are clinical inhibitors, we demonstrate that cyclin-dependent kinase (CDK)1/2 inhibitors, siRNAs against CDK1/2, and the clinical CDK1/2 inhibitor roscovitine all cooperated with the PI3K inhibitor PIK-90, blocking the antiapoptotic protein Survivin and driving cell death. In addition, overexpression of CDKs partially blocked some of the apoptosis caused by PIK-75. Roscovitine and PIK-90, in combination, were well tolerated in vivo and acted in a synthetic-lethal manner to induce apoptosis in human glioblastoma xenografts. We also tested clinical Akt and CDK inhibitors, demonstrating induction of apoptosis in vitro and providing a preclinical rationale to test this combination therapy in patients.
Proceedings of the National Academy of Sciences 07/2012; 109(31):12722-7. · 9.68 Impact Factor
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Colm J Ryan,
Assen Roguev,
Kristin Patrick,
Jiewei Xu,
Harlizawati Jahari,
Zongtian Tong,
Pedro Beltrao,
Michael Shales,
Hong Qu,
Sean R Collins, [......],
Derek Greene,
Chao Tang,
Pádraig Cunningham, Kevan M Shokat,
Gerard Cagney,
J Peter Svensson,
Christine Guthrie,
Peter J Espenshade,
Trey Ideker,
Nevan J Krogan
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ABSTRACT: 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 nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized 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 crosstalk between biological processes, suggesting general design principles of genetic interactomes.
Molecular cell 06/2012; 46(5):691-704. · 14.61 Impact Factor
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ABSTRACT: 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.
Nature 06/2012; 486(7401):80-4. · 36.28 Impact Factor
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ABSTRACT: Although 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 patients with NCSLC. To understand how mutations in EGFR influence response to therapy, we generated glioma cells expressing either glioma- or NSCLC-derived alleles and quantified kinase-site occupancy by clinical inhibitors with the use of a novel affinity probe and kinetic methodology. At equivalent doses, erlotinib achieved lower kinase-site occupancy in glioma-derived EGFRvIII compared with NSCLC-derived EGFR mutants. Kinase-site occupancy correlated directly with cell-cycle arrest. EGFRvIII released erlotinib rapidly compared with wild-type EGFR, whereas NSCLC-derived mutants released erlotinib slowly. SIGNIFICANCE: These data suggest that kinase-site occupancy is a biomarker for efficacy of EGFR inhibitors, that rapid binding and release of erlotinib in glioma-derived EGFRvIII opposes the blockade of downstream signaling, and that slower cycling of erlotinib within the active site of NSCLC-derived mutants underlies their improved clinical response.
Cancer discovery. 05/2012; 2(5):450-7.
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ABSTRACT: 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.
Molecular and cellular biology 04/2012; 32(13):2372-83. · 6.06 Impact Factor
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ABSTRACT: A family of conserved serine/threonine kinases known as cyclin-dependent kinases (CDKs) drives orderly cell cycle progression in mammalian cells. Prior studies have suggested that CDK2 regulates S-phase entry and progression, and frequently shows increased activity in a wide spectrum of human tumors. Genetic KO/knockdown approaches, however, have suggested that lack of CDK2 protein does not prevent cellular proliferation, both during somatic development in mice as well as in human cancer cell lines. Here, we use an alternative, chemical-genetic approach to achieve specific inhibition of CDK2 kinase activity in cells. We directly compare small-molecule inhibition of CDK2 kinase activity with siRNA knockdown and show that small-molecule inhibition results in marked defects in proliferation of nontransformed cells, whereas siRNA knockdown does not, highlighting the differences between these two approaches. In addition, CDK2 inhibition drastically diminishes anchorage-independent growth of human cancer cells and cells transformed with various oncogenes. Our results establish that CDK2 activity is necessary for normal mammalian cell cycle progression and suggest that it might be a useful therapeutic target for treating cancer.
Proceedings of the National Academy of Sciences 04/2012; 109(17):E1019-27. · 9.68 Impact Factor
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Andrew C Hsieh,
Yi Liu,
Merritt P Edlind,
Nicholas T Ingolia,
Matthew R Janes,
Annie Sher,
Evan Y Shi,
Craig R Stumpf,
Carly Christensen,
Michael J Bonham,
Shunyou Wang,
Pingda Ren,
Michael Martin,
Katti Jessen,
Morris E Feldman,
Jonathan S Weissman, Kevan M Shokat,
Christian Rommel,
Davide Ruggero
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ABSTRACT: 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.
Nature 02/2012; 485(7396):55-61. · 36.28 Impact Factor
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ABSTRACT: ATP-competitive mammalian target of rapamycin (mTOR) inhibitors are in early phase clinical trials. These novel targeted agents, including PP242, are mechanistically distinct from the allosteric, partial mTOR inhibitor, rapamycin. The goal of this study was to evaluate how PP242 best combines with standard chemotherapies for colorectal cancer (CRC), and which subsets of patients are most likely to benefit. The combination index for PP242 plus 5-fluorouracil, oxaliplatin, or irinotecan was determined in CRC cell lines with different mutational backgrounds. In KRAS mutant CRC cell lines, sensitivity to PP242 increases with co-mutation of PIK3CA. Mutation of p53 predicts resistance to chemotherapy, but not PP242. Efficacy of PP242 was comparable to that of standard chemotherapies over the dose range tested. Sensitivity or resistance to PP242 dictates relative synergy or antagonism, respectively, when PP242 is combined with 5-fluorouracil. The same trend exists for PP242 + oxaliplatin, but with a narrower dynamic range. Conversely potency of PP242 and the combination index for PP242 + irinotecan were unrelated, but synergy exists across all dose levels in PP242 and irinotecan sensitive, p53 wild-type cell lines. Overall, our in vitro analysis predicts that mutational status can be used to rank sensitivity to PP242 and standard chemotherapies. Single agent potency can in turn be used to predict the combination index in a drug-specific manner. Our data suggest a clinical trial to determine whether ATP-competitive mTOR inhibitors provide benefit in combination with standard chemotherapies for patients with PIK3CA mutant metastatic CRC, stratified by the presence or absence of KRAS co-mutation.
Investigational New Drugs 01/2012; · 3.36 Impact Factor
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ABSTRACT: Protein phosphorylation represents the most dominant and evolutionary conserved posttranslational modification for information transfer in cells and organisms. The human genome encodes >500 protein kinases, and thousands of phosphorylation sites are present in mammalian proteome. To develop a global view of phosphorylation network, there is a need to map the connectivity between kinases and phosphoproteome. We developed a chemical kinase-substrate cross-linker 1 that converts transient kinase-substrate interactions into a covalently linked kinase-substrate complex in vitro and in the presence of cell lysates. The method can be applied to identify unknown upstream kinases responsible for phosphorylation events in cell lysates.
Methods in molecular biology (Clifton, N.J.) 01/2012; 795:179-90.
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ABSTRACT: The site-specific and degree-specific methylation of histone lysine residues is important for the regulation of chromatin. To study the biochemical roles of lysine methylation, several approaches have been developed to reconstitute chromatin fibers in vitro with well-defined methylation patterns. Here, we describe the installation of methyl-lysine analogues (MLAs) as a simple and scalable method to introduce mono-, di-, or trimethylation at specific sites of recombinantly expressed histones. In this method, a histone is engineered to harbor a lysine-to-cysteine mutation at the desired site of modification. These mutant histones are treated with halo-ethylamines that react with the cysteine side chain, providing high yields of N-methylated aminoethylcysteines, analogues of N-methylated lysine residues. These MLA histones have been used to construct well-defined chromatin templates to study the direct biochemical consequences of histone lysine methylation in a variety of contexts.
Methods in enzymology 01/2012; 512:57-69. · 1.90 Impact Factor
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Max R Banko,
Jasmina J Allen,
Bethany E Schaffer,
Erik W Wilker,
Peiling Tsou,
Jamie L White,
Judit Villén,
Beatrice Wang,
Sara R Kim,
Kei Sakamoto,
Steven P Gygi,
Lewis C Cantley,
Michael B Yaffe, Kevan M Shokat,
Anne Brunet
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ABSTRACT: 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.
Molecular cell 11/2011; 44(6):878-92. · 14.61 Impact Factor
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ABSTRACT: The Src family kinase Lck is crucial for the initiation of TCR signaling. The activity of Lck is tightly controlled to prevent erroneous immune activation, yet it enables rapid cellular responses over a range of sensitivities to antigens. Here, in experiments with an analog-sensitive variant of the tyrosine kinase Csk, we report that Lck in T cells is dynamically controlled by an equilibrium between Csk and the tyrosine phosphatase CD45. By rapidly inhibiting Csk, we showed that changes in this equilibrium were sufficient to activate canonical TCR signaling pathways independently of ligand binding to the TCR. The activated signaling pathways showed sustained and enhanced phosphorylation compared to that in TCR-stimulated cells, revealing a feedback circuit that was sensitive to the basal signaling machinery. We identified the inhibitory adaptor molecule Dok-1 (downstream of kinase 1) as a candidate that may respond to alterations in basal signaling activity. Our results also suggest a role for Csk in the termination or dampening of TCR signals.
Science Signaling 09/2011; 4(190):ra59. · 7.50 Impact Factor
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ABSTRACT: The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes, so there is great interest in understanding the complete spectrum of mTOR-regulated networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin inhibits only a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. A number of studies have highlighted mTORC1 as a regulator of lipid homeostasis. We show that the ATP-competitive inhibitor PP242, but not rapamycin, significantly down-regulates cholesterol biosynthesis genes in a 4E-BP1-dependent manner in NIH 3T3 cells, whereas S6 kinase 1 is the dominant regulator in hepatocellular carcinoma cells. To identify other rapamycin-resistant transcriptional outputs of mTOR, we compared the expression profiles of NIH 3T3 cells treated with rapamycin versus PP242. PP242 caused 1,666 genes to be differentially expressed whereas rapamycin affected only 88 genes. Our analysis provides a genomewide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.
Proceedings of the National Academy of Sciences 08/2011; 108(37):15201-6. · 9.68 Impact Factor
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ABSTRACT: The conserved nature of the ATP-binding site of the > 500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few off-targets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases.
Proceedings of the National Academy of Sciences 08/2011; 108(37):15046-52. · 9.68 Impact Factor
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ABSTRACT: Postsynaptic density 95 (PSD-95), the major scaffold at excitatory synapses, is critical for synapse maturation and learning. In rodents, eye opening, the onset of pattern vision, triggers a rapid movement of PSD-95 from visual neuron somata to synapses. We showed previously that the PI3 kinase-Akt pathway downstream of BDNF/TrkB signaling stimulates synaptic delivery of PSD-95 via vesicular transport. However, vesicular transport requires PSD-95 palmitoylation to attach it to a lipid membrane. Also, PSD-95 insertion at synapses is known to require this lipid modification. Here, we show that BDNF/TrkB signaling is also necessary for PSD-95 palmitoylation and its transport to synapses in mouse visual cortical layer 2/3 neurons. However, palmitoylation of PSD-95 requires the activation of another pathway downstream of BDNF/TrkB, namely, signaling through phospholipase Cγ and the brain-specific PKC variant protein kinase M ζ (PKMζ). We find that PKMζ selectively regulates phosphorylation of the palmitoylation enzyme ZDHHC8. Inhibition of PKMζ results in a reduction of synaptic PSD-95 accumulation in vivo, which can be rescued by overexpressing ZDHHC8. Therefore, TrkB and PKMζ, two critical regulators of synaptic plasticity, facilitate PSD-95 targeting to synapses. These results also indicate that palmitoylation can be regulated by a trophic factor. Our findings have implications for neurodevelopmental disorders as well as aging brains.
Journal of Neuroscience 08/2011; 31(33):11894-904. · 7.11 Impact Factor
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ABSTRACT: Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and nonphysiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a noncatalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and noninhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and nonredundant, rate-limiting roles in restriction point passage and S phase entry.
Molecular cell 06/2011; 42(5):624-36. · 14.61 Impact Factor
