RESUMO
Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed "helicase with zinc-finger," or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4-NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system.
Assuntos
RNA Helicases/genética , RNA Helicases/metabolismo , Receptores CCR4/química , Receptores CCR4/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estabilidade de RNA , TATA BoxRESUMO
XRN1 is the major cytoplasmic exoribonuclease in eukaryotes, which degrades deadenylated and decapped mRNAs in the last step of the 5'-3' mRNA decay pathway. Metazoan XRN1 interacts with decapping factors coupling the final stages of decay. Here, we reveal a direct interaction between XRN1 and the CCR4-NOT deadenylase complex mediated by a low-complexity region in XRN1, which we term the 'C-terminal interacting region' or CIR. The CIR represses reporter mRNA deadenylation in human cells when overexpressed and inhibits CCR4-NOT and isolated CAF1 deadenylase activity in vitro. Through complementation studies in an XRN1-null cell line, we dissect the specific contributions of XRN1 domains and regions toward decay of an mRNA reporter. We observe that XRN1 binding to the decapping activator EDC4 counteracts the dominant negative effect of CIR overexpression on decay. Another decapping activator PatL1 directly interacts with CIR and alleviates the CIR-mediated inhibition of CCR4-NOT activity in vitro. Ribosome profiling revealed that XRN1 loss impacts not only on mRNA levels but also on the translational efficiency of many cellular transcripts likely as a consequence of incomplete decay. Our findings reveal an additional layer of direct interactions in a tightly integrated network of factors mediating deadenylation, decapping and 5'-3' exonucleolytic decay.
Assuntos
Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Proteínas Associadas aos Microtúbulos/genética , Capuzes de RNA/genética , Estabilidade de RNA/genética , Endorribonucleases/genética , Humanos , Complexos Multiproteicos/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Receptores CCR4/genética , Proteínas Repressoras/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5' cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP-GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , RNA Mensageiro/genética , Proteínas de Transporte/genética , Linhagem Celular , Cristalização , Fator de Iniciação 4E em Eucariotos , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Ligação Proteica/genética , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas de Ligação ao Cap de RNA/químicaRESUMO
Eukaryotic initiation factor 4G (eIF4G) plays a central role in translation initiation through its interactions with the cap-binding protein eIF4E. This interaction is a major drug target for repressing translation and is naturally regulated by 4E-binding proteins (4E-BPs). 4E-BPs and eIF4G compete for binding to the eIF4E dorsal surface via a shared canonical 4E-binding motif, but also contain auxiliary eIF4E-binding sequences, which were assumed to contact non-overlapping eIF4E surfaces. However, it is unknown how metazoan eIF4G auxiliary sequences bind eIF4E. Here, we describe crystal structures of human and Drosophila melanogaster eIF4E-eIF4G complexes, which unexpectedly reveal that the eIF4G auxiliary sequences bind to the lateral surface of eIF4E, using a similar mode to that of 4E-BPs. Our studies provide a molecular model of the eIF4E-eIF4G complex, shed light on the competition mechanism of 4E-BPs, and enable the rational design of selective eIF4G inhibitors to dampen dysregulated translation in disease.
Assuntos
Drosophila melanogaster/metabolismo , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação Eucariótico 4G/química , Iniciação Traducional da Cadeia Peptídica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Drosophila melanogaster/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
The eIF4E-binding proteins (4E-BPs) represent a diverse class of translation inhibitors that are often deregulated in cancer cells. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E-binding motifs connected by a linker. The lack of high-resolution structures including the linkers, which contain phosphorylation sites, limits our understanding of how phosphorylation inhibits complex formation. Furthermore, the binding mechanism of the non-canonical motifs is poorly understood. Here, we present structures of human eIF4E bound to 4E-BP1 and fly eIF4E bound to Thor, 4E-T, and eIF4G. These structures reveal architectural elements that are unique to 4E-BPs and provide insight into the consequences of phosphorylation. Guided by these structures, we designed and crystallized a 4E-BP mimic that shows increased repressive activity. Our studies pave the way for the rational design of 4E-BP mimics as therapeutic tools to decrease translation during oncogenic transformation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Drosophila/química , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Fatores de Iniciação de Peptídeos/química , Fosfoproteínas/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Cristalografia por Raios X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Mimetismo Molecular , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets.
Assuntos
Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , RNA Mensageiro/metabolismo , Receptores CCR4/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genéticaRESUMO
The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotes. This complex catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing an mRNA to degradation. The conserved core of the complex is assembled by the interaction of at least two modules: the NOT module, which minimally consists of NOT1, NOT2 and NOT3, and a catalytic module comprising two deadenylases, CCR4 and POP2/CAF1. Additional complex subunits include CAF40 and two newly identified human subunits, NOT10 and C2orf29. The role of the NOT10 and C2orf29 subunits and how they are integrated into the complex are unknown. Here, we show that the Drosophila melanogaster NOT10 and C2orf29 orthologs form a complex that interacts with the N-terminal domain of NOT1 through C2orf29. These interactions are conserved in human cells, indicating that NOT10 and C2orf29 define a conserved module of the CCR4-NOT complex. We further investigated the assembly of the D. melanogaster CCR4-NOT complex, and demonstrate that the conserved armadillo repeat domain of CAF40 interacts with a region of NOT1, comprising a domain of unknown function, DUF3819. Using tethering assays, we show that each subunit of the CCR4-NOT complex causes translational repression of an unadenylated mRNA reporter and deadenylation and degradation of a polyadenylated reporter. Therefore, the recruitment of a single subunit of the complex to an mRNA target induces the assembly of the complete CCR4-NOT complex, resulting in a similar regulatory outcome.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Complexos Multiproteicos , Ribonucleases/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Sequência Conservada , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Poliadenilação , Ligação Proteica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Estabilidade de RNA , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Ribonucleases/genéticaRESUMO
Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases.
Assuntos
Proteínas de Drosophila/metabolismo , MicroRNAs/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Interferência de RNA , Ribonucleases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HeLa , Humanos , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Fatores de Transcrição/químicaRESUMO
The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotic cells. It catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing mRNAs to decay. The conserved core of the complex consists of a catalytic module comprising two deadenylases (CAF1/POP2 and CCR4a/b) and the NOT module, which contains at least NOT1, NOT2 and NOT3. NOT1 bridges the interaction between the two modules and therefore, acts as a scaffold protein for the assembly of the complex. Here, we present the crystal structures of the CAF1-binding domain of human NOT1 alone and in complex with CAF1. The NOT1 domain comprises five helical hairpins that adopt an MIF4G (middle portion of eIF4G) fold. This NOT1 MIF4G domain binds CAF1 through a pre-formed interface and leaves the CAF1 catalytic site fully accessible to RNA substrates. The conservation of critical structural and interface residues suggests that the NOT1 MIF4G domain adopts a similar fold and interacts with CAF1 in a similar manner in all eukaryotes. Our findings shed light on the assembly of the CCR4-NOT complex and provide the basis for dissecting the role of the NOT module in mRNA deadenylation.
Assuntos
Ribonucleases/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Exorribonucleases/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Dano ao DNA/efeitos da radiação , Proteínas Nucleares/metabolismo , Radiação Ionizante , Hidrolases Anidrido Ácido , Ciclo Celular , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11 , FosforilaçãoRESUMO
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.
Assuntos
Transformação Celular Neoplásica , Quinase 2 Dependente de Ciclina/fisiologia , Oncogenes , Animais , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , RNA Interferente PequenoRESUMO
mRNA export is mediated by the TAP-p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP-p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and they mediate the export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical half of the CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating the nuclear export of viral genomic RNAs, and, more generally, provide insights on cargo RNA recognition by mRNA export receptors.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/fisiologia , RNA Viral/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologiaRESUMO
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.
Assuntos
Proliferação de Células , Quinase 2 Dependente de Ciclina/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Linhagem Celular , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Humanos , Estrutura Terciária de Proteína , Fase S/efeitos dos fármacos , Fase S/fisiologiaRESUMO
Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair--homologous recombination (HR) and non-homologous end joining (NHEJ)--are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs--as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function--could be balanced to produce a coordinated and effective response to DNA damage.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Animais , Ciclo Celular , Reparo do DNA , Humanos , Recombinação Genética/genética , Telômero/metabolismoRESUMO
Cell division is controlled by cyclin-dependent kinases (CDKs). In metazoans, S phase onset coincides with activation of Cdk2, whereas Cdk1 triggers mitosis. Both Cdk1 and -2 require cyclin binding and T loop phosphorylation for full activity. The only known CDK-activating kinase (CAK) in metazoans is Cdk7, which is also part of the transcription machinery. To test the requirements for Cdk7 in vivo, we replaced wild-type Cdk7 with a version sensitive to bulky ATP analogs in human cancer cells. Selective inhibition of Cdk7 in G1 prevents activation (but not formation) of Cdk2/cyclin complexes and delays S phase. Inhibiting Cdk7 in G2 blocks entry to mitosis and disrupts Cdk1/cyclin B complex assembly, indicating that the two steps of Cdk1 activation-cyclin binding and T loop phosphorylation-are mutually dependent. Therefore, by combining chemical genetics and homologous gene replacement in somatic cells, we reveal different modes of CDK activation by Cdk7 at two distinct execution points in the cell cycle.
Assuntos
Proteína Quinase CDC2/genética , Ciclina B/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclo Celular , Divisão Celular , Sobrevivência Celular , Ativação Enzimática , Humanos , Mitose , Fase S , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The cyclin-dependent kinases (CDKs) that drive the eukaryotic cell cycle must be phosphorylated within the activation segment (T-loop) by a CDK-activating kinase (CAK) to achieve full activity. Although a requirement for CDK-activating phosphorylation is conserved throughout eukaryotic evolution, CAK itself has diverged between metazoans and budding yeast, and fission yeast has two CAKs, raising the possibility that additional mammalian enzymes remain to be identified. We report here the characterization of PNQALRE (also known as CCRK or p42), a member of the mammalian CDK family most similar to the cell-cycle effectors Cdk1 and Cdk2 and to the CAK, Cdk7. Although PNQALRE/CCRK was recently proposed to activate Cdk2, we show that the monomeric protein has no intrinsic CAK activity. Depletion of PNQALRE by >80% due to RNA interference (RNAi) impairs cell proliferation, but fails to arrest the cell cycle at a discrete point. Instead, both the fraction of cells with a sub-G(1) DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) increase. PNQALRE knockdown did not diminish Cdk2 T-loop phosphorylation in vivo or decrease CAK activity of a cell extract. In contrast, depletion of Cdk7 by RNAi causes a proportional decrease in the ability of an extract to activate recombinant Cdk2. Our data do not support the proposed function of PNQALRE/CCRK in activating CDKs, but instead reinforce the notion of Cdk7 as the major, and to date the only, CAK in mammalian cells.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular , Proliferação de Células , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/deficiência , Quinases Ciclina-Dependentes/genética , Éxons/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Testículo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
A number of highly specific small molecule inhibitors of oncogenic tyrosine kinases have been developed and may potentially improve the treatment of different malignant diseases. However, it became rapidly evident that multiple resistance mechanisms compromise the successful clinical application of these inhibitors, particularly in advanced solid tumors. To develop efficient therapeutic strategies with small molecule inhibitors, one must understand the causes for treatment failure. Three different types of resistance to small molecule inhibitors of oncogenic tyrosine kinases have been observed. The malignant phenotype may be independent of the activity of the target kinase (target-independent resistance). Alternatively, overexpression or mutation of the target kinase can counteract the inhibition of oncogenic tyrosine kinases (target-dependent resistance). Finally, alterations of drug transporters or drug-metabolizing pathways may block the bioavailability of the tyrosine kinase inhibitors (drug-dependent resistance). This article reviews the current knowledge of clinical resistance to small molecule inhibitors approved for treatment of cancer patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Benzamidas , Humanos , Mesilato de Imatinib , Mutação , Neoplasias/genética , Piperazinas/uso terapêutico , Proteínas Tirosina Quinases/genética , Pirimidinas/uso terapêuticoRESUMO
Progression of CML from chronic phase to blast crisis is accompanied by accumulating genetic alterations. To analyze whether this abnormality can be prevented by inhibition of Bcr-Abl, we measured the frequency of spontaneous and irradiation-induced HPRT mutations in cells treated with or without imatinib mesylate (Gleevec, STI571). Imatinib treatment of cells expressing Bcr-Abl reversed the mutation frequency to a value comparable to that of Bcr-Abl negative cells. Experiments with a Bcr-Abl deletion mutant indicate that in addition to the kinase activity, protein-protein interactions are required for induction of the mutator phenotype by Bcr-Abl.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas , Linhagem Celular , Sistema Hematopoético/citologia , Sistema Hematopoético/efeitos dos fármacos , Humanos , Hipoxantina Fosforribosiltransferase/genética , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Mutação , FenótipoRESUMO
Bcr-Abl proteins are effective inducers of the leukemic phenotype in chronic myeloid leukemia (CML) and distinct variants of acute lymphoblastic leukemia (ALL). Targeting bcr-abl by treatment with the selective tyrosine kinase inhibitor imatinib has proved to be highly efficient for controlling leukemic growth. However, it is unclear whether imatinib is sufficient to eradicate the disease because of primary or secondary resistance of leukemic cells. Therefore, targeting Bcr-Abl with an alternative approach is of great interest. We demonstrate that RNA interference (RNAi) with a breakpoint-specific short-interfering RNA (siRNA) is capable of decreasing Bcr-Abl protein expression and of antagonizing Bcr-Abl-induced biochemical activities. RNAi selectively inhibited Bcr-Abl-dependent cell growth. Furthermore, bcr-abl-homologous siRNA increased sensitivity to imatinib in Bcr-Abl-overexpressing cells and in a cell line expressing the imatinib-resistant Bcr-Abl kinase domain mutation His396Pro, thereby antagonizing 2 of the major mechanisms of resistance to imatinib.