RESUMEN
IKKß plays a central role in the canonical NF-kB pathway, which has been extensively characterized. The role of IKKα in the noncanonical NF-kB pathway, and indeed in the canonical pathway as a complex with IKKß, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKß. Herein, we report for the first time a series of novel, potent, and selective inhibitors of IKKα. We demonstrate effective target engagement and selectivity with IKKα in U2OS cells through inhibition of IKKα-driven p100 phosphorylation in the noncanonical NF-kB pathway without affecting IKKß-dependent IKappa-Bα loss in the canonical pathway. These compounds represent the first chemical tools that can be used to further characterize the role of IKKα in cellular signaling, to dissect this from IKKß and to validate it in its own right as a target in inflammatory diseases.
Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Animales , Biomarcadores Farmacológicos/metabolismo , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Quinasa I-kappa B/química , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Subunidad p52 de NF-kappa B/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Pirroles/síntesis química , Pirroles/química , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Cyclin-dependent kinases (CDKs) play important roles in the control of fundamental cellular processes. Some of the most characterized CDKs are considered to be pertinent therapeutic targets for cancers and other diseases, and first clinical successes have recently been obtained with CDK inhibitors. Although discovered in the pre-genomic era, CDK10 attracted little attention until it was identified as a major determinant of resistance to endocrine therapy for breast cancer. In some studies, CDK10 has been shown to promote cell proliferation whereas other studies have revealed a tumor suppressor function. The recent discovery of Cyclin M as a CDK10 activating partner has allowed the unveiling of a protein kinase activity against the ETS2 oncoprotein, whose degradation is activated by CDK10/Cyclin M-mediated phosphorylation. CDK10/Cyclin M has also been shown to repress ciliogenesis and to maintain actin network architecture, through the phoshorylation of the PKN2 protein kinase and the control of RhoA stability. These findings shed light on the molecular mechanisms underlying STAR syndrome, a severe human developmental genetic disorder caused by mutations in the Cyclin M coding gene. They also pave the way to a better understanding of the role of CDK10/Cyclin M in cancer.
Asunto(s)
Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Proteína Quinasa C/metabolismo , HumanosRESUMEN
CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2's core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.
Asunto(s)
Actinas/metabolismo , Canal Anal/anomalías , Cilios/fisiología , Quinasas Ciclina-Dependientes/fisiología , Hipertelorismo/enzimología , Riñón/anomalías , Sindactilia/enzimología , Dedos del Pie/anomalías , Anomalías Urogenitales/enzimología , Actinas/ultraestructura , Canal Anal/enzimología , Línea Celular , Estabilidad de Enzimas , Humanos , Hipertelorismo/genética , Riñón/enzimología , Fosforilación , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Sindactilia/genética , Anomalías Urogenitales/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome.
Asunto(s)
Canal Anal/anomalías , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Hipertelorismo/genética , Riñón/anomalías , Proteolisis , Proteína Proto-Oncogénica c-ets-2/metabolismo , Sindactilia/genética , Dedos del Pie/anomalías , Anomalías Urogenitales/genética , Canal Anal/metabolismo , Western Blotting , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/deficiencia , Ciclinas/genética , Células HEK293 , Humanos , Hipertelorismo/metabolismo , Inmunoprecipitación , Riñón/metabolismo , Células MCF-7 , Complejo de la Endopetidasa Proteasomal/metabolismo , Sindactilia/metabolismo , Técnicas del Sistema de Dos Híbridos , Anomalías Urogenitales/metabolismoRESUMEN
The inhibitory kappa B kinases (IKKs) are well recognized as key regulators of the nuclear factor kappa B (NF-κB) cascade and as such represent a point of convergence for many extracellular agents that activate this pathway. The IKKs generally serve to transduce pro-inflammatory and growth stimulating signals that contribute to major cellular processes but also play a key role in the pathogenesis of a number of human diseases. Therefore, the catalytic IKKs represent attractive targets for intervention with small molecule kinase inhibitors. IKK isoforms are assembled as variable multi-subunit IKK complexes that regulate not only NF-κB dimers, but also protein substrates out-with this cascade. Consequently, close consideration of how these individual complexes transduce extracellular signals and more importantly what impact small molecule inhibitors of the IKKs have on functional outcomes are demanded. A number of adenosine triphosphate (ATP)-competitive IKKß-selective inhibitors have been developed but have demonstrated a lack of activity against IKKα. A number of these chemicals have also exhibited detrimental outcomes such as cellular toxicity and immuno-suppression. The impact of small molecule inhibitors of IKK catalytic activity will therefore be reappraised, examining the advantages and potential disadvantages to this type of intervention strategy in the treatment of diseases such as arthritis, intestinal inflammation and cancer. Furthermore, we will outline some emerging strategies, particularly the disruption of protein-protein interactions within the IKK complex, as an alternative route towards the development of novel pharmacological agents. Whether these alternatives may negate the limitations of ATP-competitive molecules and potentially avoid the issues of toxicity will be discussed.
Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Animales , Humanos , Quinasa I-kappa B/metabolismo , Inflamación/metabolismo , FN-kappa B/metabolismo , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Hepatitis C virus (HCV) translation initiation is mediated by a highly structured and conserved RNA, termed the Internal Ribosome Entry Site (IRES), located at the 5'-end of its single stranded RNA genome. It is a key target for the development of new antiviral compounds. Here we made use of the recently developed HCV cell culture system to test the antiviral activity of artificial ribonucleases consisting of imidazole(s) linked to antisense oligodeoxynucleotides targeting the HCV IRES. Results from the cell culture model indicate that the naked antisense oligodeoxynucleotide displayed an efficient antiviral activity. Despite the increased activity observed with the addition of imidazole moieties when tested with the cell-free system, it appears that these improvements were not reproduced in the cellular model.