RESUMEN
Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-α, TGF-ß, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems.
Asunto(s)
Presión Intraocular/fisiología , Mecanotransducción Celular/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Cadáver , Niño , Cilios/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/fisiopatología , Sensación/fisiología , Malla Trabecular/citología , Malla Trabecular/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Large tumor suppressor (LATS)1/2 protein kinases transmit Hippo signaling in response to intercellular contacts and serum levels to limit cell growth via the inhibition of Yes-associated protein (YAP). Here low serum and high LATS1 activity are found to enhance the levels of the 130-kDa isoform of angiomotin (Amot130) through phosphorylation by LATS1/2 at serine 175, which then forms a binding site for 14-3-3. Such phosphorylation, in turn, enables the ubiquitin ligase atrophin-1 interacting protein (AIP)4 to bind, ubiquitinate, and stabilize Amot130. Consistently, the Amot130 (S175A) mutant, which lacks LATS phosphorylation, bound AIP4 poorly under all conditions and showed reduced stability. Amot130 and AIP4 also promoted the ubiquitination and degradation of YAP in response to serum starvation, unlike Amot130 (S175A). Moreover, silencing Amot130 expression blocked LATS1 from inhibiting the expression of connective tissue growth factor, a YAP-regulated gene. Concordant with phosphorylated Amot130 specifically mediating these effects, wild-type Amot130 selectively induced YAP phosphorylation and reduced transcription of connective tissue growth factor in an AIP4-dependent manner versus Amot130 (S175A). Further, Amot130 but not Amot130 (S175A) strongly inhibited the growth of MDA-MB-468 breast cancer cells. The dominant-negative effects of Amot130 (S175A) on YAP signaling also support that phosphorylated Amot130 transduces Hippo signaling. Likewise, Amot130 expression provoked premature growth arrest during mammary cell acini formation, whereas Amot130 (S175A)-expressing cells formed enlarged and poorly differentiated acini. Taken together, the phosphorylation of Amot130 by LATS is found to be a key feature that enables it to inhibit YAP-dependent signaling and cell growth.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Angiomotinas , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Microscopía Confocal , Mutación , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina/genética , Serina/metabolismo , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Señalizadoras YAPRESUMEN
The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Angiomotinas , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Proteolisis , Proteínas Represoras/genética , Factores de Transcripción , Transcripción Genética/fisiología , Ubiquitina-Proteína Ligasas/genética , Proteínas Señalizadoras YAPRESUMEN
Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of ocrl in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.
Asunto(s)
Cilios/metabolismo , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolasas/análisis , Monoéster Fosfórico Hidrolasas/genética , Animales , Cilios/química , Embrión no Mamífero/metabolismo , Fibroblastos/metabolismo , Genotipo , Humanos , Inmunohistoquímica , Túbulos Renales/metabolismo , Mutación , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Transfección , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
Phosphatases of the regenerating liver (PRL) play oncogenic roles in cancer development and metastasis. Although previous studies indicate that PRL-1 promotes cell growth and migration by activating both the ERK1/2 and RhoA pathways, the mechanism by which it activates these signaling events remains unclear. We have identified a PRL-1-binding peptide (Peptide 1) that shares high sequence identity with a conserved motif in the Src homology 3 (SH3) domain of p115 Rho GTPase-activating protein (GAP). p115 RhoGAP directly binds PRL-1 in vitro and in cells via its SH3 domain. Structural analyses of the PRL-1·Peptide 1 complex revealed a novel protein-protein interaction whereby a sequence motif within the PxxP ligand-binding site of the p115 RhoGAP SH3 domain occupies a folded groove within PRL-1. This prevents the canonical interaction between the SH3 domain of p115 RhoGAP and MEKK1 and results in activation of ERK1/2. Furthermore, PRL-1 binding activates RhoA signaling by inhibiting the catalytic activity of p115 RhoGAP. The results demonstrate that PRL-1 binding to p115 RhoGAP provides a coordinated mechanism underlying ERK1/2 and RhoA activation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/química , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Familia-src Quinasas/metabolismo , Secuencias de Aminoácidos , Animales , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ligandos , Ratones , Unión Proteica , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Polarity proteins promote the asymmetric organization of cells by orienting intracellular sorting mechanisms, such as protein trafficking and cytoskeletal assembly. The localization of individual polarity proteins in turn is often determined by association with factors that mediate contact with other cells or the substratum. This arrangement for the Par and Crb apical polarity complexes at the tight junction is disrupted by the adaptor protein Amot. Amot directly binds the scaffolding proteins Patj and Mupp1 and redistributes them and their binding partners from the plasma membrane to endosomes. However, the mechanism by which Amot is targeted to endosomes is unknown. Here, a novel lipid binding domain within Amot is shown to selectively bind with high affinity to membranes containing monophosphorylated phosphatidylinositols and cholesterol. With similar lipid specificity, Amot inserts into and tubulates membranes in vitro and enlarges perinuclear endosomal compartments in cells. Based on the similar distribution of Amot with cholesterol, Rab11, and Arf6, such membrane interactions are identified at juxtanuclear endocytic recycling compartments. Taken together, these findings indicate that Amot is targeted along with associated apical polarity proteins to the endocytic recycling compartment via this novel membrane binding domain.
Asunto(s)
Endosomas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Angiomotinas , Animales , Sitios de Unión , Fenómenos Biofísicos , Compartimento Celular , Línea Celular , Polaridad Celular/fisiología , Colesterol/metabolismo , Perros , Endocitosis/fisiología , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/genética , Membranas Intracelulares/metabolismo , Liposomas , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Filogenia , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.
Asunto(s)
Glucógeno/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Familia de las Proteínas 8 Relacionadas con la Autofagia , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Chlorocebus aethiops , Glucógeno/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de la Membrana/genética , Ratones , Mutación Puntual , Estructura Terciaria de Proteína , RatasRESUMEN
Glycogen, a branched polymer of glucose, acts as an intracellular carbon and energy reserve in many tissues and cell types. An important pathway for its degradation is by transport to lysosomes in an autophagy-like process. It has been proposed that starch-binding domain-containing protein 1 (Stbd1) may participate in this mechanism by anchoring glycogen to intracellular membranes. In addition, Stbd1 has been reported to interact with a known autophagy protein, GABARAPL1, a member of the Atg8 family. Here, we confirm this interaction and identify an Atg8 interacting motif (AIM) in Stbd1 necessary for GABARAPL1 binding as judged by co-immunoprecipitation from cell extracts and co-localization in cells as evidenced by immunofluorescence microscopy. The AIM sequence of Stbd1 (200)HEEWEMV(206) lies within a predicted disordered region of the molecule and fits the consensus of other AIM sequences in cargo-specifying proteins such as p62 and Nix. Mutation of the AIM, including single point mutations of either W203 or V206, eliminated the co-localization of Stbd1 with both over-expressed and endogenous GABARAPL1. Stbd1 may therefore function as a novel cargo binding protein that delivers glycogen to lysosomes in an autophagic pathway that could be termed "glycophagy".
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucógeno/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Musculares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Familia de las Proteínas 8 Relacionadas con la Autofagia , Línea Celular , Humanos , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Mutación PuntualRESUMEN
Actin-dependent mechanisms drive the nuclear translocation of Yap1 to enable its co-activation of transcription factors that induce pro-growth and survival programs. While Rho GTPases are necessary for the nuclear import of YAP1, the relevant Guanine Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs) that connect this process to upstream signaling are not well defined. To this end, we measured the impact of expressing sixty-seven RhoGEFs and RhoGAPs on the YAP1 dependent activity of a TEAD element transcriptional reporter. Robust effects by all three members of the regulator of G-protein signaling (RGS) domain containing RhoGEFs (ArhGEF1, ArhGEF11 and ArhGEF12) prompted studies relating their known roles in serum signaling onto the regulation of Yap1. Under all conditions examined, ArhGEF12 preferentially mediated the activation of YAP1/TEAD by serum versus ArhGEF1 or ArhGEF11. Conversely, ArhGEF1 in multiple contexts inhibited both basal and serum elevated YAP1 activity through its GAP activity for Gα13. The sensitivity of such inhibition to cellular density and to low states of serum signaling supports that ArhGEF1 is a context dependent regulator of YAP1. Taken together, the relative activities of the RGS-RhoGEFs were found to dictate the degree to which serum signaling promotes YAP1 activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Transcripción/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de la Mama/genética , Proteínas Activadoras de GTPasa/genética , Células HEK293 , Humanos , Inmunoprecipitación , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Transcripción/genética , Proteínas Señalizadoras YAP , Quinasas Asociadas a rho/genéticaRESUMEN
Like most solid tumors, glioblastoma multiforme (GBM) harbors multiple overexpressed and mutated genes that affect several signaling pathways. Suppressing tumor growth of solid tumors like GBM without toxicity may be achieved by small molecules that selectively modulate a collection of targets across different signaling pathways, also known as selective polypharmacology. Phenotypic screening can be an effective method to uncover such compounds, but the lack of approaches to create focused libraries tailored to tumor targets has limited its impact. Here, we create rational libraries for phenotypic screening by structure-based molecular docking chemical libraries to GBM-specific targets identified using the tumor's RNA sequence and mutation data along with cellular protein-protein interaction data. Screening this enriched library of 47 candidates led to several active compounds, including 1 (IPR-2025), which (i) inhibited cell viability of low-passage patient-derived GBM spheroids with single-digit micromolar IC50 values that are substantially better than standard-of-care temozolomide, (ii) blocked tube-formation of endothelial cells in Matrigel with submicromolar IC50 values, and (iii) had no effect on primary hematopoietic CD34+ progenitor spheroids or astrocyte cell viability. RNA sequencing provided the potential mechanism of action for 1, and mass spectrometry-based thermal proteome profiling confirmed that the compound engages multiple targets. The ability of 1 to inhibit GBM phenotypes without affecting normal cell viability suggests that our screening approach may hold promise for generating lead compounds with selective polypharmacology for the development of treatments of incurable diseases like GBM.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Transcriptoma/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The family of Angiomotin (Amot) proteins regulate several biological pathways associated with cellular differentiation, proliferation, and migration. These adaptor proteins target proteins to the apical membrane, actin fibers, or the nucleus. A major function of the Amot coiled-coil homology (ACCH) domain is to initiate protein interactions with the cellular membrane, particularly those containing phosphatidylinositol lipids. The work presented in this article uses several ACCH domain lysine/arginine mutants to probe the relative importance of individual residues for lipid binding. This identified four lysine and three arginine residues that mediate full lipid binding. Based on these findings, three of these residues were mutated to glutamates in the Angiomotin 80 kDa splice form and were incorporated into human mammary cell lines. Results show that mutating three of these residues in the context of full-length Angiomotin reduced the residence of the protein at the apical membrane. These findings provide new insight into how the ACCH domain mediates lipid binding to enable Amot proteins to control epithelial cell growth.
RESUMEN
The Hippo pathway coordinates extracellular signals onto the control of tissue homeostasis and organ size. Hippo signaling primarily regulates the ability of Yap1 to bind and co-activate TEA domain (TEAD) transcription factors. Yap1 tightly binds to TEAD4 via a large flat interface, making the development of small-molecule orthosteric inhibitors highly challenging. Here, we report small-molecule TEADâ Yap inhibitors that rapidly and selectively form a covalent bond with a conserved cysteine located within the unique deep hydrophobic palmitate-binding pocket of TEADs. Inhibition of TEAD4 binding to Yap1 by these compounds was irreversible and occurred on a longer time scale. In mammalian cells, the compounds formed a covalent complex with TEAD4, inhibited its binding to Yap1, blocked its transcriptional activity, and suppressed expression of connective tissue growth factor. The compounds inhibited cell viability of patient-derived glioblastoma spheroids, making them suitable as chemical probes to explore Hippo signaling in cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cisteína/química , Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Humanos , Simulación de Dinámica Molecular , Proteínas Musculares/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Factores de Transcripción de Dominio TEA , Termodinámica , Factores de Transcripción/antagonistas & inhibidores , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.
Asunto(s)
Citoesqueleto/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas/fisiología , Tirosina 3-Monooxigenasa/metabolismo , Proteínas 14-3-3 , Actinas/fisiología , Animales , Diferenciación Celular/genética , Tamaño de la Célula/genética , Células Cultivadas , Análisis por Conglomerados , Biología Computacional , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoesqueleto/genética , Cartilla de ADN , ADN Complementario/genética , Perros , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Espectrometría de Masas , Ratones , Fosforilación , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Factores de Intercambio de Guanina Nucleótido Rho , TransfecciónRESUMEN
OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Imidazoles/uso terapéutico , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Temozolomida/uso terapéutico , Animales , Neoplasias Encefálicas/patología , Terapia Combinada , Modelos Animales de Enfermedad , Glioblastoma/patología , Humanos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. RESULTS: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. CONCLUSION: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.
Asunto(s)
Clonación Molecular/métodos , Vectores Genéticos , Proteómica/métodos , Sitios de Empalme de ARN , Recombinación Genética , Línea Celular , Humanos , Integrasas , Sistemas de Lectura Abierta , Empalme del ARN , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas ViralesRESUMEN
Protein tyrosine phosphatases (PTPs) are potential therapeutic targets for many diseases. Unfortunately, despite considerable drug discovery efforts devoted to PTPs, obtaining selective and cell permeable PTP inhibitors remains highly challenging. We describe a strategy to explore the existing drug space for previously unknown PTP inhibitory activities. This led to the discovery of cefsulodin as an inhibitor of SHP2, an oncogenic phosphatase in the PTP family. Crystal structure analysis of SHP2 interaction with cefsulodin identified sulfophenyl acetic amide (SPAA) as a novel phosphotyrosine (pTyr) mimetic. A structure-guided and SPAA fragment-based focused library approach produced several potent and selective SHP2 inhibitors. Notably, these inhibitors blocked SHP2-mediated signaling events and proliferation in several cancer cell lines. Thus, SPAA may serve as a new platform for developing chemical probes for other PTPs.
RESUMEN
Transforming growth factor-ß (TGF-ß) regulates the expression of genes supporting breast cancer cells in bone, but little is known about prostate cancer bone metastases and TGF-ß. Our study reveals that the TGFBR1 inhibitor SD208 effectively reduces prostate cancer bone metastases. TGF-ß upregulates in prostate cancer cells a set of genes associated with cancer aggressiveness and bone metastases, and the most upregulated gene was PMEPA1. In patients, PMEPA1 expression decreased in metastatic prostate cancer and low Pmepa1 correlated with decreased metastasis-free survival. Only membrane-anchored isoforms of PMEPA1 interacted with R-SMADs and ubiquitin ligases, blocking TGF-ß signaling independently of the proteasome. Interrupting this negative feedback loop by PMEPA1 knockdown increased prometastatic gene expression and bone metastases in a mouse prostate cancer model.
Asunto(s)
Neoplasias Óseas/secundario , Proteínas de la Membrana/metabolismo , Neoplasias de la Próstata/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/prevención & control , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Pteridinas/farmacología , Transducción de Señal , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
The Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase associated with various kinds of leukemia and solid tumors. Thus, there is substantial interest in developing SHP2 inhibitors as potential anticancer and antileukemia agents. Using a structure-guided and fragment-based library approach, we identified a novel hydroxyindole carboxylic acid-based SHP2 inhibitor 11a-1, with an IC50 value of 200 nM and greater than 5-fold selectivity against 20 mammalian PTPs. Structural and modeling studies reveal that the hydroxyindole carboxylic acid anchors the inhibitor to the SHP2 active site, while interactions of the oxalamide linker and the phenylthiophene tail with residues in the ß5-ß6 loop contribute to 11a-1's binding potency and selectivity. Evidence suggests that 11a-1 specifically attenuates the SHP2-dependent signaling inside the cell. Moreover, 11a-1 blocks growth factor mediated Erk1/2 and Akt activation and exhibits excellent antiproliferative activity in lung cancer and breast cancer as well as leukemia cell lines.
Asunto(s)
Antineoplásicos/química , Indoles/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática , Humanos , Indoles/síntesis química , Indoles/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Simulación del Acoplamiento Molecular , Terapia Molecular Dirigida , Conformación Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
The asymmetric organization of epithelial cells is a basic counter to cellular proliferation. However, the mechanisms whereby pro-growth pathways are modulated by intracellular factors that control cell shape are not well understood. This study demonstrates that the adaptor protein Amot, in addition to its established role in regulating cellular asymmetry, also promotes extracellular signal-regulated kinase 1 and 2 (ERK1/2)-dependent proliferation of mammary cells. Specifically, expression of Amot80, but not a mutant lacking its polarity protein interaction domain, enhances ERK1/2-dependent proliferation of MCF7 cells. Further, expression of Amot80 induces nontransformed MCF10A cells to overgrow as disorganized cellular aggregates in Matrigel. Conversely, Amot expression is required for proliferation of breast cancer cells in specific microenvironmental contexts that require ERK1/2 signaling. Thus, Amot is proposed to coordinate the dysregulation of cell polarity with the induction of neoplastic growth in mammary cells.