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1.
Mol Cell ; 83(8): 1210-1215, 2023 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-36990093

RESUMEN

One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.


Asunto(s)
Quinasas raf , Proteínas ras , Dimerización , Consenso , Proteínas ras/genética , Proteínas ras/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismo , Lípidos , Proteínas Proto-Oncogénicas c-raf/metabolismo
2.
Nature ; 576(7787): 482-486, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31827279

RESUMEN

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region1. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins-each capable of transforming cells-are encoded when KRAS is activated by mutation2. No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells3 in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen4 (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes3-5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation-depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.


Asunto(s)
Hexoquinasa/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Regulación Alostérica , Animales , Línea Celular Tumoral , Activación Enzimática , Glucólisis , Guanosina Trifosfato/metabolismo , Hexoquinasa/química , Humanos , Técnicas In Vitro , Isoenzimas/metabolismo , Lipoilación , Masculino , Ratones , Mitocondrias/enzimología , Mitocondrias/metabolismo , Membranas Mitocondriales/enzimología , Membranas Mitocondriales/metabolismo , Neoplasias/enzimología , Neoplasias/metabolismo , Unión Proteica , Transporte de Proteínas
3.
Biochem Soc Trans ; 51(3): 1191-1199, 2023 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-37222266

RESUMEN

RAS proteins are small GTPases that transduce signals from membrane receptors to signaling pathways that regulate growth and differentiation. Four RAS proteins are encoded by three genes - HRAS, KRAS, NRAS. Among them, KRAS is mutated in human cancer more frequently than any other oncogene. The KRAS pre-mRNA is alternatively spliced to generate two transcripts, KRAS4A and KRAS4B, that encode distinct proto-oncoproteins that differ almost exclusively in their C-terminal hypervariable regions (HVRs) that controls subcellular trafficking and membrane association. The KRAS4A isoform arose 475 million years ago in jawed vertebrates and has persisted in all vertebrates ever since, strongly suggesting non-overlapping functions of the splice variants. Because KRAS4B is expressed at higher levels in most tissues, it has been considered the principal KRAS isoform. However, emerging evidence for KRAS4A expression in tumors and splice variant-specific interactions and functions have sparked interest in this gene product. Among these findings, the KRAS4A-specific regulation of hexokinase I is a stark example. The aim of this mini-review is to provide an overview of the origin and differential functions of the two splice variants of KRAS.


Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Animales , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Mutación
4.
Nat Rev Mol Cell Biol ; 13(1): 39-51, 2011 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-22189424

RESUMEN

RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation.


Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas ras/metabolismo , Animales , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ratones
5.
Proc Natl Acad Sci U S A ; 117(50): 31914-31922, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33257571

RESUMEN

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.


Asunto(s)
Membrana Celular/metabolismo , Dimetilaliltranstransferasa/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Neoplasias/patología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores de Estrógenos/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Sistemas CRISPR-Cas/genética , Biología Computacional , Conjuntos de Datos como Asunto , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Neoplasias/genética , Proteínas Asociadas a Matriz Nuclear/genética , Prenilación de Proteína , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores de Estrógenos/genética
6.
Proc Natl Acad Sci U S A ; 116(9): 3524-3529, 2019 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-30733287

RESUMEN

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and phosphoinositol 4,5 bisphosphate but this interaction is inhibited by the N-terminal segment of RIAM. Here we report the structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module. We solved the crystal structure of IN-RA-PH to a resolution of 2.4-Å. The structure reveals that the IN segment associates with the RA segment and thereby suppresses RIAM:RAP1 association. This autoinhibitory configuration of RIAM can be released by phosphorylation at Tyr45 in the IN segment. Specific inhibitors of focal adhesion kinase (FAK) blocked phosphorylation of Tyr45, inhibited stimulated translocation of RIAM to the plasma membrane, and inhibited integrin-mediated cell adhesion in a Tyr45-dependent fashion. Our results reveal an unusual regulatory mechanism in small GTPase signaling by which the effector molecule is autoinhibited for GTPase interaction, and a modality of integrin activation at the level of RIAM through a FAK-mediated feedforward mechanism that involves reversal of autoinhibition by a tyrosine kinase associated with integrin signaling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Integrinas/química , Proteínas de la Membrana/química , Conformación Proteica , Proteínas de Unión a Telómeros/química , Proteínas Adaptadoras Transductoras de Señales/genética , Adhesión Celular/genética , Membrana Celular/química , Cristalografía por Rayos X , Proteína-Tirosina Quinasas de Adhesión Focal/química , Proteína-Tirosina Quinasas de Adhesión Focal/genética , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Proteínas de la Membrana/genética , Fosforilación , Complejo Shelterina , Transducción de Señal , Proteínas de Unión a Telómeros/genética
7.
Clin Immunol ; 217: 108485, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32504780

RESUMEN

T cell co-stimulation is important for the maintenance of immunologic tolerance. Co-inhibitory receptors including programmed cell death-1 (PD-1) confer peripheral tolerance to prevent autoimmunity. SAP (SH2D1A) is an adaptor molecule that is important in T cell signaling and has been shown to interact with signaling lymphocytic activation molecule (SLAM) family receptors also in the context of self-tolerance. We recently reported that SAP interferes with PD-1 function. In the current study, we investigated the levels of SAP and PD-1 in patients with rheumatoid arthritis (RA) to further understand what role they play in disease activity. We observed increased SAP levels in lymphocytes of RA patients and found that PD-1 levels correlated positively with RA disease activity. Additionally, we found that SAP interacts with CD28 to inhibit T cell signaling in vitro. This work demonstrates a putative molecular mechanism for SAP mediated PD-1 inhibition.


Asunto(s)
Artritis Reumatoide/inmunología , Antígenos CD28/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/metabolismo , Linfocitos T/inmunología , Adulto , Anciano , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Autoinmunidad/genética , Autoinmunidad/fisiología , Línea Celular Tumoral , Femenino , Humanos , Células Jurkat , Masculino , Persona de Mediana Edad , Fosforilación , Receptor de Muerte Celular Programada 1/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/genética , Adulto Joven
8.
J Biol Chem ; 293(45): 17574-17581, 2018 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-30228186

RESUMEN

Ras proteins participate in multiple signal cascades, regulating crucial cellular processes, including cell survival, proliferation, and differentiation. We have previously reported that Ras proteins are modified by sumoylation and that Lys-42 plays an important role in mediating the modification. In the current study, we further investigated the role of Lys-42 in regulating cellular activities of K-Ras. Inducible expression of K-RasV12 led to the activation of downstream components, including c-RAF, MEK1, and extracellular signal-regulated kinases (ERKs), whereas expression of K-RasV12/R42 mutant compromised the activation of the RAF/MEK/ERK signaling axis. Expression of K-RasV12/R42 also led to reduced phosphorylation of several other protein kinases, including c-Jun N-terminal kinase (JNK), Chk2, and focal adhesion kinase (FAK). Significantly, K-RasV12/R42 expression inhibited cellular migration and invasion in vitro in multiple cell lines, including transformed pancreatic cells. Given that K-Ras plays a crucial role in mediating oncogenesis in the pancreas, we treated transformed pancreatic cells of both BxPC-3 and MiaPaCa-2 with 2-D08, a small ubiquitin-like modifier (SUMO) E2 inhibitor. Treatment with the compound inhibited cell migration in a concentration-dependent manner, which was correlated with a reduced level of K-Ras sumoylation. Moreover, 2-D08 suppressed expression of ZEB1 (a mesenchymal cell marker) with concomitant induction of ZO-1 (an epithelial cell marker). Combined, our studies strongly suggest that posttranslational modification(s), including sumoylation mediated by Lys-42, plays a crucial role in K-Ras activities in vivo.


Asunto(s)
Movimiento Celular , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonas/farmacología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Ratones , Células 3T3 NIH , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Sumoilación/efectos de los fármacos , Sumoilación/genética , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo
9.
Mol Cell ; 41(2): 173-85, 2011 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-21255728

RESUMEN

A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines.


Asunto(s)
Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína 1A de Unión a Tacrolimus/fisiología , Acilación , Animales , Lipoilación , Células PC12 , Transporte de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/química , Ratas , Transducción de Señal , Proteína 1A de Unión a Tacrolimus/metabolismo
10.
Proc Natl Acad Sci U S A ; 112(45): 13916-21, 2015 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-26512117

RESUMEN

The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.


Asunto(s)
Ciclo Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Estatmina/metabolismo
11.
Proc Natl Acad Sci U S A ; 112(3): 779-84, 2015 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-25561545

RESUMEN

The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti-K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants.


Asunto(s)
Genes ras , Neoplasias/genética , Empalme del ARN , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa
12.
Blood ; 126(25): 2695-703, 2015 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-26324702

RESUMEN

Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Linfocitos B/inmunología , Quimiotaxis de Leucocito/inmunología , Proteínas de la Membrana/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Adhesión Celular/inmunología , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Linfocitos T/citología , Linfocitos T/metabolismo , Proteínas de Unión al GTP rap1/inmunología , Proteínas de Unión al GTP rap1/metabolismo
13.
Proc Natl Acad Sci U S A ; 110(51): 20593-8, 2013 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-24297914

RESUMEN

K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho-K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL-dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP3 regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors.


Asunto(s)
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína bcl-X/metabolismo , Animales , Calcio/metabolismo , Muerte Celular/fisiología , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Supervivencia Celular/fisiología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Inositol 1,4,5-Trifosfato/genética , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones , Membranas Mitocondriales/metabolismo , Fosforilación/fisiología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Células Sf9 , Spodoptera , Proteína bcl-X/genética
14.
J Cell Physiol ; 230(3): 610-9, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25158650

RESUMEN

Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C-terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H-Ras and N-Ras) localize on the Golgi apparatus whereas K-Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub-organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live-cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H-Ras is distributed throughout the Golgi stacks, the singly palmitoylated N-Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants, we show that the different sub-Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane, but also their distribution within the Golgi stacks.


Asunto(s)
Compartimento Celular/genética , Genes ras , Aparato de Golgi/ultraestructura , Proteínas ras/genética , Línea Celular , Aparato de Golgi/genética , Humanos , Lipoilación/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/genética , Transducción de Señal , Proteínas ras/ultraestructura
15.
Proc Natl Acad Sci U S A ; 109(27): 10843-8, 2012 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-22711838

RESUMEN

Members of the RAS small GTPase family regulate cellular responses to extracellular stimuli by mediating the flux through downstream signal transduction cascades. RAS activity is strongly dependent on its subcellular localization and its nucleotide-binding status, both of which are modulated by posttranslational modification. We have determined that RAS is posttranslationally acetylated on lysine 104. Molecular dynamics simulations suggested that this modification affects the conformational stability of the Switch II domain, which is critical for the ability of RAS to interact with guanine nucleotide exchange factors. Consistent with this model, an acetylation-mimetic mutation in K-RAS4B suppressed guanine nucleotide exchange factor-induced nucleotide exchange and inhibited in vitro transforming activity. These data suggest that lysine acetylation is a negative regulatory modification on RAS. Because mutations in RAS family members are extremely common in cancer, modulation of RAS acetylation may constitute a therapeutic approach.


Asunto(s)
Genes ras/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas ras/química , Proteínas ras/metabolismo , Acetilación , Animales , Células COS , Chlorocebus aethiops , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Mutagénesis Sitio-Dirigida , Prenilación/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/fisiología , Relación Estructura-Actividad , Proteínas ras/genética
16.
Nat Cell Biol ; 9(6): 713-9, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17486117

RESUMEN

Ras activation as a consequence of antigen receptor (T-cell receptor; TCR) engagement on T lymphocytes is required for T-cell development, selection and function. Lymphocyte function-associated antigen-1 (LFA-1) mediates lymphocyte adhesion, stabilization of the immune synapse and bidirectional signalling. Using a fluorescent biosensor we found that TCR activation with or without costimulation of CD28 led to activation of Ras only on the Golgi apparatus, whereas costimulation with LFA-1 induced Ras activation on both the Golgi and the plasma membrane. Ras activation on both compartments required RasGRP1, an exchange factor regulated by calcium and diacylglycerol (DAG), but phospholipase C (PLC) activity was required only for activation on the Golgi. Engagement of LFA-1 increased DAG levels at the plasma membrane by stimulating phospholipase D (PLD). PLD2 and phosphatidic acid phosphatase (PAP) were required for Ras activation on the plasma membrane. Thus, LFA-1 acts through PLD2 to reshape the pattern of Ras activation downstream of the TCR.


Asunto(s)
Membrana Celular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Fosfolipasa D/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteínas ras/metabolismo , Animales , Antígenos CD28/metabolismo , Comunicación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Diglicéridos/metabolismo , Activación Enzimática/fisiología , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/metabolismo
17.
Curr Opin Cell Biol ; 18(2): 162-7, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16488589

RESUMEN

The discovery that Ras proteins are modified by enzymes restricted to the endoplasmic reticulum and Golgi apparatus and that, at steady state, a significant pool of Ras is localized on the Golgi has led to the hypothesis that Ras can become activated on and signal from intracellular membranes. Fluorescent probes capable of showing when and where in living cells Ras becomes activated together with studies of Ras proteins stringently tethered to intracellular membranes have confirmed this hypothesis. Thus, recent studies of Ras have contributed to the rapidly expanding field of compartmentalized signaling.


Asunto(s)
Aparato de Golgi/metabolismo , Transducción de Señal/fisiología , Proteínas ras/fisiología , Animales , Humanos , Modelos Biológicos , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas ras/metabolismo
19.
Res Sq ; 2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36711820

RESUMEN

KRAS, HRAS and NRAS oncogenes belong to a family of 40 highly homologous genes, which in turn are a subset of a superfamily of >160 genes encoding small GTPases. RAS oncoproteins consist of a globular G-domain (aa1-166) and a 22-23aa unstructured hypervariable region (HVR) that mediates membrane targeting. The evolutionarily origins of the RAS isoforms, their HVRs and alternative splicing of the KRAS locus has not been explored. We found that KRAS is basal to the oncogene family and its duplication generated HRAS in the common ancestor of vertebrates. In a second round of duplication HRAS generated NRAS and KRAS generated an additional RAS gene we have designated KRASBL, absent in mammals and birds. KRAS4A arose through a duplication and insertion of the 4th exon of NRAS into the 3rd intron of KRAS. We found evolutionarily conservation of a short polybasic region (PBR1) in HRAS, NRAS and KRAS4A, a second polybasic region (PBR2) in KRAS4A, two neutralized basic residues (NB) and a serine in KRAS4B and KRASBL, and a modification of the CaaX motif in vertebrates with farnesyl rather than geranylgeranyl polyisoprene lipids, suggesting that a less hydrophobic membrane anchor is critical to RAS oncoprotein function. The persistence of four RAS isoforms through >400 MY of evolution argues strongly for differential function.

20.
Oncogene ; 42(21): 1741-1750, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37031342

RESUMEN

KRAS, HRAS and NRAS proto-oncogenes belong to a family of 40 highly homologous genes, which in turn are a subset of a superfamily of >160 genes encoding small GTPases. RAS proteins consist of a globular G-domain (aa1-166) and a 22-23 aa unstructured hypervariable region (HVR) that mediates membrane targeting. The evolutionary origins of the RAS isoforms, their HVRs and alternative splicing of the KRAS locus has not been explored. We found that KRAS is basal to the RAS proto-oncogene family and its duplication generated HRAS in the common ancestor of vertebrates. In a second round of duplication HRAS generated NRAS and KRAS generated an additional RAS gene we have designated KRASBL, absent in mammals and birds. KRAS4A arose through a duplication and insertion of the 4th exon of NRAS into the 3rd intron of KRAS. We found evolutionary conservation of a short polybasic region (PBR1) in HRAS, NRAS and KRAS4A, a second polybasic region (PBR2) in KRAS4A, two neutralized basic residues (NB) and a serine in KRAS4B and KRASBL, and a modification of the CaaX motif in vertebrates with farnesyl rather than geranylgeranyl polyisoprene lipids, suggesting that a less hydrophobic membrane anchor is critical to RAS protein function. The persistence of four RAS isoforms through >400 million years of evolution argues strongly for differential function.


Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras , Animales , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Isoformas de Proteínas/genética , Proteínas ras/genética , Proteínas ras/metabolismo , Genes ras , Mamíferos/genética
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