RESUMEN
We recently reported that diacylglycerol kinase (DGK) alpha enhanced tumor necrosis factor-alpha (TNF-alpha)-induced activation of nuclear factor-kappaB (NF-kappaB). However, the signaling pathway between DGKalpha and NF-kappaB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKalpha strongly attenuated protein kinase C (PKC) zeta-dependent phosphorylation of a large subunit of NF-kappaB, p65/RelA, at Ser311 but not PKCzeta-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCzeta suppressed and synergistically enhanced DGKalpha-mediated NF-kappaB activation, respectively. These results strongly suggest that DGKalpha positively regulates TNF-alpha-dependent NF-kappaB activation via the PKCzeta-mediated Ser311 phosphorylation of p65/RelA.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Proteína Quinasa C/metabolismo , Serina/metabolismo , Factor de Transcripción ReIA/metabolismo , Diacilglicerol Quinasa/genética , Humanos , Proteínas I-kappa B/metabolismo , Fosforilación , Interferencia de ARNRESUMEN
The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase eta (DGKeta) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKeta1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKeta serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKeta-silenced HeLa cells, this study revealed that DGKeta activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKeta enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKeta physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKeta-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKeta acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Células COS , Membrana Celular/enzimología , Membrana Celular/genética , Chlorocebus aethiops , Citoplasma/enzimología , Citoplasma/genética , Diacilglicerol Quinasa/antagonistas & inhibidores , Diacilglicerol Quinasa/genética , Dimerización , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Fosforilación/fisiología , Unión Proteica/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , ARN Interferente Pequeño/genéticaRESUMEN
The delta-isozyme (type II) of diacylglycerol kinase (DGK) is known to positively regulate growth factor receptor signaling. DGKdelta, which is distributed to clathrin-coated vesicles, interacts with DGKdelta itself, protein kinase C and AP2alpha. To search for additional DGKdelta-interacting proteins, we screened a yeast two-hybrid cDNA library from HepG2 cells using aa 896-1097 of DGKdelta as a bait. We identified aa 184-317 (WD40 repeats 5-7) of receptor for activated C kinase 1 (RACK1), which interacts with various important signaling molecules, as a novel binding partner of DGKdelta. Co-immunoprecipitation analysis, using COS-7 cells co-expressing RACK1 and DGKdelta, revealed that RACK1 selectively interacted with DGKdelta, but not with type I DGKs, in mammalian cells. The interaction was dynamically regulated by phorbol ester. Intriguingly, DGKdelta appeared to recruit RACK1 to clathrin-coated vesicles and co-localized with RACK1. These results suggest that DGKdelta serves as an adaptor protein to regulate the localization of the versatile scaffold protein, RACK1.
Asunto(s)
Vesículas Cubiertas por Clatrina/metabolismo , Diacilglicerol Quinasa/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Western Blotting , Células COS , Células Cultivadas , Chlorocebus aethiops , Diacilglicerol Quinasa/genética , Proteínas de Unión al GTP/genética , Humanos , Inmunoprecipitación , Riñón/citología , Riñón/enzimología , Microscopía Fluorescente , Proteínas de Neoplasias/genética , Receptores de Cinasa C Activada , Receptores de Superficie Celular/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating (consuming) DAG to yield PA. Ten mammalian DGK isozymes have been identified to date. In addition to two or three cysteine-rich C1 domains (protein kinase C-like zinc finger structures) commonly conserved in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain, a MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation site domain and ankyrin repeats. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of mammalian signal transduction pathways conducting growth factor/cytokine-dependent cell proliferation and motility, seizure activity, immune responses, cardiovascular responses and insulin receptor-mediated glucose metabolism. It is suggested that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes. Unfortunately, there are no DGK isozyme-specific inhibitors/activators at present. Development of these compounds is eagerly awaited for the development of novel drugs targeting DGKs.
Asunto(s)
Diacilglicerol Quinasa/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Transducción de Señal , Animales , Diacilglicerol Quinasa/metabolismo , Diseño de Fármacos , Humanos , Isoenzimas/metabolismo , Ácidos Fosfatidicos/metabolismo , FosforilaciónRESUMEN
Melanosome biogenesis consists of multistep processes that involve synthesis of melanosomal protein, which is followed by vesicle transport/fusion and post-translational modifications such as glycosylation, proteolysis, and oligomerization. Because of its complexity, the details of the molecular mechanism of melanosome biogenesis are not yet fully understood. Here, we report that, in MMAc melanoma cells, wild-type (WT) Rab7 and its dominant-active mutant (Rab7-Q67L), but not its dominant-negative mutant (Rab7-T22N), were colocalized in the perinuclear region with granules containing Stage I melanosomes, where the full-length, immature gp100/Pmel17/Silv was present. It was also found that overexpression of Rab7-Q67L and, to a lesser extent, Rab7-WT increased the amount of proteolytically processed, mature gp100. However, Rab7-T22N did not show such an effect. Moreover, siRNA-mediated Rab7 knockdown considerably inhibited gp100 maturation. These results collectively suggest that the GTP-bound form of Rab7 promotes melanogenesis through the regulation of gp100 maturation in melanoma cells.
Asunto(s)
Melanosomas/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas de Unión al GTP rab/fisiología , Células Cultivadas , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/análisis , Melanoma/metabolismo , Melanoma/patología , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/metabolismo , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/metabolismo , ARN Interferente Pequeño/farmacología , Antígeno gp100 del Melanoma , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas de Unión a GTP rab7RESUMEN
DGKgamma (diacylglycerol kinase gamma) was reported to interact with beta2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKgamma with beta2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The beta2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKgamma. Moreover, DGKgamma apparently enhanced the beta2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of beta2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of beta2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of beta2-chimaerin interacted with the N-terminal half of catalytic region of DGKgamma. Unexpectedly, the SH2 domain of beta2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKgamma and beta2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas de Neoplasias/química , Ésteres del Forbol/metabolismo , Animales , Células COS , Catálisis , Chlorocebus aethiops , Eliminación de Gen , Humanos , Unión Proteica , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Acetato de Tetradecanoilforbol/farmacología , Zinc/química , Dominios Homologos srcRESUMEN
beta2-Chimaerin, an intracellular receptor for the second messenger diacylglycerol and phorbol esters, is a GTPase-activating protein (GAP) specific for Rac. beta2-Chimaerin negatively controls many Rac-dependent pathophysiological events including tumor development. However, the regulatory mechanism of beta2-chimaerin remains largely unknown. Here we report that beta2-chimaerin is tyrosine-phosphorylated by Src-family kinases (SFKs) upon cell stimulation with epidermal growth factor (EGF). Mutational analysis identified Tyr-21 in the N-terminal regulatory region as a major phosphorylation site. Intriguingly, the addition of SFK inhibitor and the replacement of Tyr-21 with Phe (Y21F) markedly enhanced Rac-GAP activity of beta2-chimaerin in EGF-treated cells. Moreover, the Y21F mutant inhibited integrin-dependent cell spreading, in which Rac1 plays a critical role, more strongly than wild-type beta2-chimaerin. These results suggest Tyr-21 phosphorylation as a novel, SFK-dependent mechanism that negatively regulates beta2-chimaerin Rac-GAP activity.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Tirosina/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Familia-src Quinasas/metabolismo , Sustitución de Aminoácidos , Animales , Células COS , Adhesión Celular/genética , Chlorocebus aethiops , Análisis Mutacional de ADN , Factor de Crecimiento Epidérmico/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Proteínas de Neoplasias/genética , Fenilalanina/metabolismo , Fosforilación , Transfección , Proteínas de Unión al GTP rac/genética , Familia-src Quinasas/análisisRESUMEN
Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. In addition to the C1 domains (protein kinase C-like zinc finger structures) conserved commonly in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain and ankyrin repeats. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways conducting development, neural and immune responses, cytoskeleton reorganization and carcinogenesis. Moreover, there has been rapidly growing evidence indicating that individual DGK isoforms exert their specific roles through interactions with unique partner proteins such as protein kinase Cs, Ras guanyl nucleotide-releasing protein, chimaerins and phosphatidylinositol-4-phosphate 5-kinase. Therefore, an emerging paradigm for DGK is that the individual DGK isoforms assembled in their own signaling complexes should carry out spatio-temporally segregated tasks for a wide range of biological processes via regulating local, but not global, concentrations of DAG and/or PA.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Animales , Diacilglicerol Quinasa/química , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Especificidad por SustratoRESUMEN
We investigated the implication of diacylglycerol kinase (DGK) alpha (type I isoform) in melanoma cells because we found that this DGK isoform was expressed in several human melanoma cell lines but not in noncancerous melanocytes. Intriguingly, the overexpression of wild-type (WT) DGKalpha, but not of its kinase-dead (KD) mutant, markedly suppressed tumor necrosis factor (TNF)-alpha-induced apoptosis of AKI human melanoma cells. In the reverse experiment, siRNA-mediated knockdown of DGKalpha significantly enhanced the apoptosis. The overexpression of other type I isoforms (DGKbeta and DGKgamma) had, on the other hand, no detectable effects on the apoptosis. These results indicate that DGKalpha specifically suppresses the TNF-alpha-induced apoptosis through its catalytic action. We found that the overexpression of DGKalpha-WT, but not of DGKalpha-KD, further enhanced the TNF-alpha-stimulated transcriptional activity of an anti-apoptotic factor, NF-kappaB. Conversely, DGKalpha-knockdown considerably inhibited the NF-kappaB activity. Moreover, an NF-kappaB inhibitor blunted the anti-apoptotic effect of DGKalpha overexpression. Together, these results strongly suggest that DGKalpha is a novel positive regulator of NF-kappaB, which suppresses TNF-alpha-induced melanoma cell apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Diacilglicerol Quinasa/metabolismo , Melanoma/enzimología , Melanoma/patología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Humanos , Isoenzimas/metabolismo , Melanocitos/efectos de los fármacos , Melanocitos/enzimología , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ratas , PorcinosRESUMEN
Diacylglycerol kinase (DGK)gamma was shown to act as an upstream suppressor of Rac1. Here we report that, in COS7 cells stimulated with epidermal growth factor (EGF), DGKgamma specifically interacts and co-localizes at the plasma membrane with beta2-chimaerin, a GTPase-activating protein (GAP) for Rac. Moreover, DGKgamma enhanced EGF-dependent translocation of beta2-chimaerin to the plasma membrane. Interestingly, DGKgamma markedly augmented EGF-dependent GAP activity of beta2-chimaerin through its catalytic action. These results indicate that DGKgamma is a novel regulator of beta2-chimaerin, and thus suggest that beta2-chimaerin is an effector molecule, linking DGKgamma functionally with Rac1.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Proteínas de Neoplasias/metabolismo , Animales , Secuencia de Bases , Transporte Biológico Activo , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Diacilglicerol Quinasa/antagonistas & inhibidores , Diacilglicerol Quinasa/genética , Ratones , Células 3T3 NIH , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Lipid phosphate phosphatases (LPPs), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we here report that LPP1 is also associated with lipid rafts distinct from those harboring LPP3. We found that LPP1 was Triton X-100-soluble, but CHAPS-insoluble in LNCaP cells endogenously expressing LPP1 and several LPP1 cDNA-transfected cells including NIH3T3 fibroblasts. In addition to the non-ionic detergent insolubility, LPP1 further possessed several properties formulated for raft-localizing proteins as follows: first, the CHAPS-insolubility was resistant to the actin-disrupting drug cytochalasin D; second, the CHAPS-insoluble LPP1 floated in an Optiprep density gradient; third, the CHAPS insolubility of LPP1 was lost by cholesterol depletion; and finally, the subcellular distribution pattern of LPP1 exclusively overlapped with that of a raft marker, cholera toxin B subunit. Interestingly, confocal microscopic analysis showed that LPP1 was distributed to membrane compartments distinct from those of LPP3. Analysis using various LPP1/LPP3 chimeras revealed that their first extracellular regions determine the different Triton X-100 solubilities. These results indicate that LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions.
Asunto(s)
Microdominios de Membrana/enzimología , Fosfatidato Fosfatasa/metabolismo , Animales , Células COS , Chlorocebus aethiops , Ácidos Cólicos/farmacología , Citocalasina D/farmacología , Humanos , Isoenzimas/metabolismo , Ratones , Microscopía Confocal , Células 3T3 NIH , Octoxinol/farmacología , SolubilidadRESUMEN
Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKgamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.
Asunto(s)
Núcleo Celular/enzimología , Diacilglicerol Quinasa/metabolismo , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/fisiología , Animales , Células CHO , Células COS , Núcleo Celular/genética , Núcleo Celular/fisiología , Tamaño de la Célula , Chlorocebus aethiops , Cricetinae , Cricetulus , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/fisiología , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Ratones , Células 3T3 NIHRESUMEN
Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Here we identified a tenth member of the DGK family designated DGK kappa. The kappa-isozyme (1271 amino acids, calculated molecular mass, 142 kDa) contains a pleckstrin homology domain, two cysteine-rich zinc finger-like structures, and a separated catalytic region as have been found commonly for the type II isozymes previously cloned (DGKdelta and DGKeta). The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N terminus. Reverse transcriptase-PCR showed that the DGK kappa mRNA is most abundant in the testis, and to a lesser extent in the placenta. DGK kappa, when expressed in HEK293 cells, was persistently localized at the plasma membrane even in the absence of cell stimuli. Deletion analysis revealed that the short C-terminal sequence (amino acid residues 1199-1268) is necessary and sufficient for the plasma membrane localization. Interestingly, DGK kappa, but not other type II DGKs, was specifically tyrosine-phosphorylated at Tyr78 through the Src family kinase pathway in H2O2-treated cells. Moreover, H2O2 selectively inhibited DGK kappa activity in a Src family kinase-independent manner, suggesting that the isozyme changes the balance of signaling lipids in the plasma membrane in response to oxidative stress. The expression patterns, subcellular distribution, and regulatory mechanisms of DGK kappa are distinct from those of DGKdelta and DGKeta despite high structural similarity, suggesting unique functions of the individual type II isozymes.
Asunto(s)
Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Sanguíneas/química , Western Blotting , Células COS , Catálisis , Dominio Catalítico , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Clonación Molecular , Cisteína/química , ADN Complementario/metabolismo , Diacilglicerol Quinasa/metabolismo , Diglicéridos/química , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Humanos , Peróxido de Hidrógeno/farmacología , Immunoblotting , Inmunoprecipitación , Lípidos/química , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , Estrés Oxidativo , Ácidos Fosfatidicos/química , Fosfoproteínas/química , Fosforilación , Plásmidos/metabolismo , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Transducción de Señal , Porcinos , Testículo/metabolismo , Distribución Tisular , Tirosina/química , Dedos de ZincRESUMEN
Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGKalpha, -epsilon, -zeta, and -iota was detected in the ovary and placenta. Intense expression signals for DGKzeta and -alpha were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGKepsilon were seen more intensely in granulosa cells. In the placenta, signals for DGKalpha and -iota were observed in the junctional zone, whereas those for DGKzeta were detected in the labyrinthine zone. At higher magnification, the signals for DGKalpha were mainly discerned in giant cytotrophoblasts, and those for DGKiota were found in small cytotrophoblasts of the junctional zone. DGKzeta signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGKepsilon signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Ovario/enzimología , Placenta/enzimología , Animales , Northern Blotting , Ciclo Estral , Femenino , Hibridación in Situ , Isoenzimas/metabolismo , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKalpha, -epsilon, and -zeta were detected in the lung. By immunohistochemical examination, DGKalpha and -zeta were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKalpha in the cytoplasm and DGKzeta in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKalpha and -zeta was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKepsilon was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Pulmón/embriología , Pulmón/enzimología , Animales , Diacilglicerol Quinasa/genética , Isoenzimas , Macrófagos Alveolares/citología , Macrófagos Alveolares/enzimología , Alveolos Pulmonares/citología , Alveolos Pulmonares/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Ovarian cancer is the most frequent cause of cancer death among all gynecologic cancers. We demonstrate here that lysophosphatidic acid (LPA)-induced ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) is a critical to tumor formation in ovarian cancer. We found that among the epidermal growth factor receptor (EGFR) family of growth factors, HB-EGF gene expression in cancerous tissues and HB-EGF protein levels in patients' ascites fluid were significantly elevated. The human ovarian cancer cell lines SKOV3 and RMG-1 form tumors in nude mice. Tumor formation of these cells was enhanced by exogenous expression of pro-HB-EGF and completely blocked by pro-HB-EGF gene RNA interference or by CRM197, a specific HB-EGF inhibitor. Transfection with mutant forms of HB-EGF indicated that the release of soluble HB-EGF is essential for tumor formation. LPA, which is constitutively produced by ovarian cancer cells, induced HB-EGF ectodomain shedding in SKOV3 and RMG-1 cells, resulting in the transactivation of EGFR and the downstream kinase extracellular signal-regulated kinase/mitogen-activated protein kinase. LPA-induced transactivation was abrogated by HB-EGF gene RNA interference or by CRM197. Introduction of lipid phosphate phosphohydrolase, which hydrolyzes LPA, decreased the constitutive shedding of HB-EGF, EGFR transactivation, and the tumorigenic potential of SKOV3 and RMG-1 cells. These results indicate that HB-EGF is the primary member of the EGFR family of growth factors expressed in ovarian cancer and that LPA-induced ectodomain shedding of this growth factor is a critical step in tumor formation, making HB-EGF a novel therapeutic target for ovarian cancer.
Asunto(s)
Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/fisiología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Animales , Proteínas Bacterianas/farmacología , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/biosíntesis , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Receptores ErbB/metabolismo , Líquido Extracelular/metabolismo , Femenino , Expresión Génica , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Lisofosfolípidos/fisiología , Ratones , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , Receptores de Superficie Celular/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Proteína Quinasa C/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Factor de Crecimiento Epidérmico/fisiología , Humanos , Leucemia de Células Plasmáticas , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Nine diacylglycerol kinase (DGK) isozymes have been identified. However, our knowledge of their individual functions is still limited. Here, we demonstrate the role of DGKgamma in regulating Rac1-governed cell morphology. We found that the expression of kinase-dead DGKgamma, which acts as a dominant-negative mutant, and inhibition of endogenous DGKgamma activity with R59949 induced lamellipodium and membrane ruffle formation in NIH3T3 fibroblasts in the absence of growth factor stimulation. Reciprocally, lamellipodium formation induced by platelet-derived growth factor was significantly inhibited upon expression of constitutively active DGKgamma. Moreover, the constitutively active DGKgamma mutant suppressed integrin-mediated cell spreading. These effects are isoform-specific because, in the same experiments, none of the corresponding mutants of DGKalpha and DGKbeta, closely related isoforms, affected cell morphology. These results suggest that DGKgamma specifically participates in the Rac1-mediated signaling pathway leading to cytoskeletal reorganization. In support of this, DGKgamma co-localized with dominant-active Rac1 especially in lamellipodia. Moreover, we found that endogenous DGKgamma was physically associated with cellular Rac1. Dominant-negative Rac1 expression blocked the lamellipodium formation induced by kinase-dead DGKgamma, indicating that DGKgamma acts upstream of Rac1. This model is supported by studies demonstrating that kinase-dead DGKgamma selectively activated Rac1, but not Cdc42. Taken together, these results strongly suggest that DGKgamma functions through its catalytic action as an upstream suppressor of Rac1 and, consequently, lamellipodium/ruffle formation.
Asunto(s)
Diacilglicerol Quinasa/fisiología , Seudópodos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Western Blotting , Células COS , Movimiento Celular , Diacilglicerol Quinasa/metabolismo , Fibronectinas/metabolismo , Genes Dominantes , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Mutación , Células 3T3 NIH , Piperidinas/farmacología , Plásmidos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pruebas de Precipitina , Unión Proteica , Quinazolinas/farmacología , Quinazolinonas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Diacylglycerol kinase (DGK) catalyzes phosphorylation of a second messenger diacylglycerol (DG) to phosphatidic acid in cellular signal transduction. Previous studies have revealed that DGK consists of a family of isozymes including our rat clones. In this study we isolated from rat brain cDNA library the cDNA clones for a rat homologue of DGKiota (rDGKiota-1) that contains two zinc finger-like sequences, the highly conserved DGK catalytic domain, a bipartite nuclear localization signal, and four ankyrin repeats at the carboxyl terminus. In addition, we found novel splice variants, which contain either insertion 1 (71 bp) or insertion 2 (19 bp) or both in the carboxyl-terminal portion. Each of the insertions causes a frameshift, and the resultant premature stop codons produce two truncated forms (termed rDGKiota-2 and -iota-3), the former lacking the ankyrin repeats at the carboxyl terminus and the latter lacking a part of the catalytic domain and the ankyrin repeats. Truncation of the carboxyl-terminal portion clearly exerts effects on the detergent solubility and enzymatic activity of the splice variants, although all three variants showed similar cytoplasmic localization in cDNA-transfected cultured neurons despite the continued presence of the nuclear localization signal sequence. Immunoblot analysis using anti-rDGKiota antibody raised against the common amino-terminal portion clearly shows that these rDGKiota variants are indeed expressed in the brain. These results suggest that the carboxyl-terminal truncated forms of rDGKiota-2 and -iota-3 that exhibit reduced enzymatic activities might show a dominant negative effect against the intact rDGKiota-1, and that the modulation of signal transduction by the splice variants may play some roles in the physiologic and/or pathologic conditions of neurons.
Asunto(s)
Encéfalo/enzimología , Diacilglicerol Quinasa/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Diacilglicerol Quinasa/análisis , Regulación Enzimológica de la Expresión Génica , Isoenzimas/análisis , Isoenzimas/genética , Datos de Secuencia Molecular , Neuronas/metabolismo , Especificidad de Órganos , RatasRESUMEN
The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins. Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates. However, it is unclear how immature protein dynamics are regulated. In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase. The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy. The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP. Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature. This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion. Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching. In contrast, terminally misfolded tyrosinase synthesized in cells in which alpha-glucosidases were inhibited showed extensive immobilization. Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.