RESUMEN
Accumulating evidence suggests that protein S-nitrosylation is enzymatically regulated and that specificity in S-nitrosylation derives from dedicated S-nitrosylases and denitrosylases that conjugate and remove S-nitrosothiols, respectively. Here, we report that mice deficient in the protein denitrosylase SCoR2 (S-nitroso-Coenzyme A Reductase 2; AKR1A1) exhibit marked reductions in serum cholesterol due to reduced secretion of the cholesterol-regulating protein PCSK9. SCoR2 associates with endoplasmic reticulum (ER) secretory machinery to control an S-nitrosylation cascade involving ER cargo-selection proteins SAR1 and SURF4, which moonlight as S-nitrosylases. SAR1 acts as a SURF4 nitrosylase and SURF4 as a PCSK9 nitrosylase to inhibit PCSK9 secretion, while SCoR2 counteracts nitrosylase activity by promoting PCSK9 denitrosylation. Inhibition of PCSK9 by an NO-based drug requires nitrosylase activity, and small-molecule inhibition of SCoR2 phenocopies the PCSK9-mediated reductions in cholesterol observed in SCoR2-deficient mice. Our results reveal enzymatic machinery controlling cholesterol levels through S-nitrosylation and suggest a distinct treatment paradigm for cardiovascular disease.
Asunto(s)
Proproteína Convertasa 9 , S-Nitrosotioles , Ratones , Animales , Proteínas/metabolismo , Oxidorreductasas/metabolismo , S-Nitrosotioles/metabolismo , Homeostasis , Óxido Nítrico/metabolismo , Proteínas de la MembranaRESUMEN
The Golgi apparatus serves a key role in processing and sorting lipids and proteins for delivery to their final cellular destinations. Vesicle exit from the Golgi initiates with directional deformation of the lipid bilayer to produce a bulge. Several mechanisms have been described by which lipids and proteins can induce directional membrane curvature to promote vesicle budding. Here we review some of the mechanisms implicated in inducing membrane curvature at the Golgi to promote vesicular trafficking to various cellular destinations.
Asunto(s)
Aparato de Golgi/patología , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/metabolismo , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas/fisiología , Vesículas TransportadorasRESUMEN
Vesicle budding for Golgi-to-plasma membrane trafficking is a key step in secretion. Proteins that induce curvature of the Golgi membrane are predicted to be required, by analogy to vesicle budding from other membranes. Here, we demonstrate that GOLPH3, upon binding to the phosphoinositide PI4P, induces curvature of synthetic membranes in vitro and the Golgi in cells. Moreover, efficient Golgi-to-plasma membrane trafficking critically depends on the ability of GOLPH3 to curve the Golgi membrane. Interestingly, uncoupling of GOLPH3 from its binding partner MYO18A results in extensive curvature of Golgi membranes, producing dramatic tubulation of the Golgi, but does not support forward trafficking. Thus, forward trafficking from the Golgi to the plasma membrane requires the ability of GOLPH3 both to induce Golgi membrane curvature and to recruit MYO18A. These data provide fundamental insight into the mechanism of Golgi trafficking and into the function of the unique Golgi secretory oncoproteins GOLPH3 and MYO18A.
Asunto(s)
Aparato de Golgi/metabolismo , Liposomas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismo , Vías Secretoras , Células HEK293 , Células HeLa , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Proteínas de la Membrana/química , Miosinas/metabolismo , Fosfatidilinositoles/química , Unión Proteica , Dominios ProteicosRESUMEN
The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Factores de Transcripción E2F/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Animales , Sitios de Unión , Ciclo Celular , Línea Celular Tumoral , Regulación de la Expresión Génica , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Células 3T3 NIH , Fosfoproteínas/genética , Regiones Promotoras GenéticasRESUMEN
The discovery of the phosphatidylinositol-3-kinase (PI3K) pathway was a major advance in understanding growth factor signaling. The high frequency of PI3K pathway mutations in many cancers has encouraged a new field targeting cancer driver mutations. Although there have been many successes, targeting PI3K itself has proven challenging, in part because of its multiple isoforms with distinct roles. Despite promising preclinical results, development of PI3K inhibitors as pharmacologic anticancer agents has been limited by modest single-agent efficacy and significant adverse effects. If we could overcome these limitations, PI3K inhibitors would be a powerful cancer-fighting tool. Data from phase III clinical trials yields insight into some of the problems with PI3K inhibitors. Recent advances have shed light on the mechanisms of tumor resistance to PI3K inhibitors via feedback pathways that cause elevated insulin levels that then activate the same PI3K pathways that are the targets of inhibition. Improving our understanding of the complex regulatory feedback pathways that activate in response to PI3K inhibition will reveal ways to increase the efficacy of PI3K inhibitors and reduce adverse effects, increasing the usefulness of this class as a treatment option for multiple cancer types.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
GOLPH3 is a peripheral membrane protein localized to the Golgi and its vesicles, but its purpose had been unclear. We found that GOLPH3 binds specifically to the phosphoinositide phosphatidylinositol(4)phosphate [PtdIns(4)P], which functions at the Golgi to promote vesicle exit for trafficking to the plasma membrane. PtdIns(4)P is enriched at the trans-Golgi and so recruits GOLPH3. Here, a GOLPH3 complex is formed when it binds to myosin18A (MYO18A), which binds F-actin. This complex generates a pulling force to extract vesicles from the Golgi; interference with this GOLPH3 complex results in dramatically reduced vesicle trafficking. The GOLPH3 complex has been identified as a driver of cancer in humans, likely through multiple mechanisms that activate secretory trafficking. In this review, we summarize the literature that identifies the nature of the GOLPH3 complex and its role in cancer. We also consider the GOLPH3 complex as a hub with the potential to reveal regulation of the Golgi and suggest the possibility of GOLPH3 complex inhibition as a therapeutic approach in cancer.
Asunto(s)
Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Transporte Biológico , HumanosRESUMEN
Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of â¼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ésteres/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinolinas/química , Quinolinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Simulación del Acoplamiento Molecular , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Conformación Proteica , Quinolinas/metabolismoRESUMEN
MYO18A is a divergent member of the myosin family characterized by the presence of an amino-terminal PDZ domain. MYO18A has been found in a few different complexes involved in intracellular transport processes. MYO18A is found in a complex with LURAP1 and MRCK that functions in retrograde treadmilling of actin. It also has been found in a complex with PAK2, ßPIX, and GIT1, functioning to transport that protein complex from focal adhesions to the leading edge. Finally, a high proportion of MYO18A is found in complex with GOLPH3 at the trans Golgi, where it functions to promote vesicle budding for Golgi-to-plasma membrane trafficking. Interestingly, MYO18A has been implicated as a cancer driver, as have other components of the GOLPH3 pathway. It remains uncertain as to whether or not MYO18A has intrinsic motor activity. While many questions remain, MYO18A is a fascinatingly unique myosin that is essential in higher organisms.
Asunto(s)
Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Miosinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Transporte Biológico Activo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/genética , Aparato de Golgi/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Miosinas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Phosphatidylserine (PtdSer) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) have been implicated in the maintenance of caveolae, but direct evidence that these lipids are required for normal caveolar structure and dynamics in living cells has been lacking. A new study by Fairn and colleagues uses sophisticated tools to perturb specific lipids in living cells to assess the consequences for caveolae. This study demonstrates disparate roles for these lipids in the stability and mobility of caveolae and points the way for future work to understand how these lipids contribute to the biology of caveolae.
Asunto(s)
Caveolas/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Caveolas/química , Caveolinas/química , Caveolinas/metabolismo , Membrana Celular/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas de Unión a Fosfato , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilserinas/química , Multimerización de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Transporte VesicularRESUMEN
The Golgi is generally recognized for its central role in the secretory pathway to orchestrate protein post-translational modification and trafficking of proteins and lipids to their final destination. Despite the common view of the Golgi as an inert sorting organelle, emerging data demonstrate that important signaling events occur at the Golgi, including those that regulate the trafficking function of the Golgi. The phosphatidylinositol-4-phosphate/GOLPH3/MYO18A/F-actin complex serves as a hub for signals that regulate Golgi trafficking function. Furthermore, the Golgi is increasingly appreciated for its important role in cell growth and in driving oncogenic transformation, as illuminated by the discovery that GOLPH3 and MYO18A are cancer drivers.
Asunto(s)
Aparato de Golgi/metabolismo , Metabolismo de los Lípidos , Transporte de Proteínas , Transducción de Señal , Animales , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The mechanism of directional cell migration remains an important problem, with relevance to cancer invasion and metastasis. GOLPH3 is a common oncogenic driver of human cancers, and is the first oncogene that functions at the Golgi in trafficking to the plasma membrane. Overexpression of GOLPH3 is reported to drive enhanced cell migration. Here we show that the phosphatidylinositol-4-phosphate/GOLPH3/myosin 18A/F-actin pathway that is critical for Golgi-to-plasma membrane trafficking is necessary and limiting for directional cell migration. By linking the Golgi to the actin cytoskeleton, GOLPH3 promotes reorientation of the Golgi toward the leading edge. GOLPH3 also promotes reorientation of lysosomes (but not other organelles) toward the leading edge. However, lysosome function is dispensable for migration and the GOLPH3 dependence of lysosome movement is indirect, via GOLPH3's effect on the Golgi. By driving reorientation of the Golgi to the leading edge and driving forward trafficking, particularly to the leading edge, overexpression of GOLPH3 drives trafficking to the leading edge of the cell, which is functionally important for directional cell migration. Our identification of a novel pathway for Golgi reorientation controlled by GOLPH3 provides new insight into the mechanism of directional cell migration with important implications for understanding GOLPH3's role in cancer.
Asunto(s)
Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Polaridad Celular , Citoesqueleto , Células HeLa , Humanos , Proteínas de la Membrana/fisiología , Miosinas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas/fisiología , Transducción de SeñalRESUMEN
The Golgi protein GOLPH3 binds to PtdIns(4)P and MYO18A, linking the Golgi to the actin cytoskeleton. The GOLPH3 pathway is essential for vesicular trafficking from the Golgi to the plasma membrane. A side effect of GOLPH3-dependent trafficking is to generate the extended ribbon shape of the Golgi. Perturbation of the pathway results in changes to both Golgi morphology and secretion, with functional consequences for the cell. The cellular response to DNA damage provides an example of GOLPH3-mediated regulation of the Golgi. Upon DNA damage, DNA-PK phosphorylation of GOLPH3 increases binding to MYO18A, activating the GOLPH3 pathway, which consequently results in Golgi fragmentation, reduced trafficking, and enhanced cell survival. The PtdIns(4)P/GOLPH3/MYO18A/F-actin pathway provides new insight into the relationship between Golgi morphology and function, and their regulation.
RESUMEN
Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.
Asunto(s)
Enfermedades Cerebelosas/genética , Cerebelo/patología , Lisosomas/metabolismo , Fagosomas/metabolismo , Nexinas de Clasificación/genética , Ataxias Espinocerebelosas/genética , Animales , Atrofia/genética , Autofagia , Preescolar , Femenino , Frecuencia de los Genes , Humanos , Lactante , Escala de Lod , Enfermedades por Almacenamiento Lisosomal/genética , Masculino , Mutación , Síndrome , Pez CebraRESUMEN
GOLPH3 is the first example of an oncogene that functions in secretory trafficking at the Golgi. The discovery of GOLPH3's roles in both cancer and Golgi trafficking raises questions about how GOLPH3 and the Golgi contribute to cancer. Our recent investigation of the regulation of GOLPH3 revealed a surprising response by the Golgi upon DNA damage that is mediated by DNA-PK and GOLPH3. These results provide new insight into the DNA damage response with important implications for understanding the cellular response to standard cancer therapeutic agents.
Asunto(s)
Daño del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteínas de la Membrana/genética , Neoplasias/genética , Movimiento Celular/genética , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Neoplasias/patología , Transporte de Proteínas/genética , Transducción de Señal/genéticaRESUMEN
The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas overexpression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.
Asunto(s)
Daño del ADN , Proteína Quinasa Activada por ADN/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Miosinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Humanos , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Fosforilación , Ratas , Alineación de SecuenciaRESUMEN
GOLPH3 is a phosphatidylinositol-4-phosphate (PI4P) effector that plays an important role in maintaining Golgi architecture and anterograde trafficking. GOLPH3 does so through its ability to link trans-Golgi membranes to F-actin via its interaction with myosin 18A (MYO18A). GOLPH3 also is known to be an oncogene commonly amplified in human cancers. GOLPH3L is a GOLPH3 paralogue found in all vertebrate genomes, although previously it was largely uncharacterized. Here we demonstrate that although GOLPH3 is ubiquitously expressed in mammalian cells, GOLPH3L is present in only a subset of tissues and cell types, particularly secretory tissues. We show that, like GOLPH3, GOLPH3L binds to PI4P, localizes to the Golgi as a consequence of its PI4P binding, and is required for efficient anterograde trafficking. Surprisingly, however, we find that perturbations of GOLPH3L expression produce effects on Golgi morphology that are opposite to those of GOLPH3 and MYO18A. GOLPH3L differs critically from GOLPH3 in that it is largely unable to bind to MYO18A. Our data demonstrate that despite their similarities, unexpectedly, GOLPH3L antagonizes GOLPH3/MYO18A at the Golgi.
Asunto(s)
Aparato de Golgi/ultraestructura , Proteínas de la Membrana/metabolismo , Miosinas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Línea Celular , Glicosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteínas de la Membrana/genética , Ratones , Miosinas/genética , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño , Alineación de Secuencia , Transducción de SeñalRESUMEN
The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants we identified the phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, we examined PtdInsP-binding of Pbl and the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pbl(ΔPH) mutants. Taken together, our results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mesodermo/metabolismo , Animales , Sitios de Unión , Membrana Celular/metabolismo , Movimiento Celular , Drosophila/genética , Drosophila/metabolismo , Guanosina Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.
Asunto(s)
Aparato de Golgi/metabolismo , Hepacivirus/metabolismo , Hepatitis C/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Liberación del Virus/fisiología , Transporte Biológico Activo/genética , Línea Celular Tumoral , Aparato de Golgi/genética , Aparato de Golgi/virología , Hepacivirus/genética , Hepatitis C/genética , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virología , Proteínas de la Membrana/genética , Miosinas/genética , Miosinas/metabolismo , Fosfatos de Fosfatidilinositol/genética , Virión/genética , Virión/metabolismoRESUMEN
Tumor inflammation promotes angiogenesis, immunosuppression, and tumor growth, but the mechanisms controlling inflammatory cell recruitment to tumors are not well understood. We found that a range of chemoattractants activating G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and Toll-like/IL-1 receptors (TLR/IL1Rs) unexpectedly initiate tumor inflammation by activating the PI3-kinase isoform p110γ in Gr1+CD11b+ myeloid cells. Whereas GPCRs activate p110γ in a Ras/p101-dependent manner, RTKs and TLR/IL1Rs directly activate p110γ in a Ras/p87-dependent manner. Once activated, p110γ promotes inside-out activation of a single integrin, α4ß1, causing myeloid cell invasion into tumors. Pharmacological or genetic blockade of p110γ suppressed inflammation, growth, and metastasis of implanted and spontaneous tumors, revealing an important therapeutic target in oncology.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Inflamación/etiología , Neoplasias/patología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Interleucina-1/fisiología , Receptores Toll-Like/fisiología , Animales , Adhesión Celular , Movimiento Celular , Progresión de la Enfermedad , Humanos , Integrina alfa4beta1/fisiología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias/prevención & control , Proteínas ras/fisiologíaRESUMEN
Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIbeta and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.