Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
J Biol Chem ; 300(2): 105657, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38224949

RESUMEN

The yeast Snf1/AMP-activated kinase (AMPK) maintains energy homeostasis, controlling metabolic processes and glucose derepression in response to nutrient levels and environmental cues. Under conditions of nitrogen or glucose limitation, Snf1 regulates pseudohyphal growth, a morphological transition characterized by the formation of extended multicellular filaments. During pseudohyphal growth, Snf1 is required for wild-type levels of inositol polyphosphate (InsP), soluble phosphorylated species of the six-carbon cyclitol inositol that function as conserved metabolic second messengers. InsP levels are established through the activity of a family of inositol kinases, including the yeast inositol polyphosphate kinase Kcs1, which principally generates pyrophosphorylated InsP7. Here, we report that Snf1 regulates Kcs1, affecting Kcs1 phosphorylation and inositol kinase activity. A snf1 kinase-defective mutant exhibits decreased Kcs1 phosphorylation, and Kcs1 is phosphorylated in vivo at Ser residues 537 and 646 during pseudohyphal growth. By in vitro analysis, Snf1 directly phosphorylates Kcs1, predominantly at amino acids 537 and 646. A yeast strain carrying kcs1 encoding Ser-to-Ala point mutations at these residues (kcs1-S537A,S646A) shows elevated levels of pyrophosphorylated InsP7, comparable to InsP7 levels observed upon deletion of SNF1. The kcs1-S537A,S646A mutant exhibits decreased pseudohyphal growth, invasive growth, and cell elongation. Transcriptional profiling indicates extensive perturbation of metabolic pathways in kcs1-S537A,S646A. Growth of kcs1-S537A,S646A is affected on medium containing sucrose and antimycin A, consistent with decreased Snf1p signaling. This work identifies Snf1 phosphorylation of Kcs1, collectively highlighting the interconnectedness of AMPK activity and InsP signaling in coordinating nutrient availability, energy homoeostasis, and cell growth.


Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato) , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/metabolismo , Inositol/metabolismo , Fosforilación , Polifosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Biochemistry ; 48(27): 6285-7, 2009 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-19518129

RESUMEN

SH2 domain-containing 5-inositol phosphatase (SHIP2) is implicated in the development of type 2 diabetes and cancer. Tyrosine phosphorylation of SHIP2 is shown to enhance its phosphatase activity. Using IP4 as a substrate, we show here that tyrosines 986, 987, and 1135 are critical for EGF-induced stimulation of SHIP2 activity. SHIP2 with a disrupted SH2 domain (R47G mutation) displays higher constitutive activity than wild-type SHIP2. Deletion of the C-terminus region similarly activates SHIP2. Thus, the SH2 domain of SHIP2, in conjunction with the C-terminus, confers an inhibitory effect to maintain a low basal activity, and signal-induced tyrosine phosphorylations overcome this effect to activate SHIP2.


Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Tirosina/metabolismo , Dominios Homologos src , Activación Enzimática , Células HeLa , Humanos , Mutación , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Fosforilación
4.
Cancer Biol Ther ; 3(3): 305-13, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-14726685

RESUMEN

Mammalian cells undergo cell cycle arrest in response to DNA damage due to the existence of multiple checkpoint response mechanisms. One such checkpoint pathway operating at the G(1) phase is frequently lost in cancer cells due to mutation of the p53 tumor suppressor gene. However, cancer cells often arrest at the G(2) phase upon DNA damage, due to activation of another checkpoint pathway that prevents the activation Cdc2 kinase. The kinases, Chk1, Wee1, and Myt1 are key regulators of this G(2) checkpoint, which act directly or indirectly to inhibit Cdc2 activity. Here we show that RNA interference (RNAi)-mediated downregulation of Wee1 kinase abrogated an Adriamycin trade mark -induced G(2) checkpoint in human cervical carcinoma Hela cells that are defective in G(1) checkpoint response. Wee1 downregulation sensitized HeLa cells to Adriamycin trade mark -induced apoptosis. Downregulation of Chk1 kinase in Hela cells also caused a significant amount of cell death in dependent of DNA damage. In contrast, Myt1 downregulation also abrogated Adriamycin trade mark -induced G(2) arrest but did not cause substantial apoptosis. Reduction in Wee1, Chk1, or Myt1 levels did not sensitize normal human mammary epithelial cells (HMEC) cells to Adriamycin trade mark -induced apoptosis unlike the situation in Hela cells. Our study reveals distinct roles for Chk1, Wee1, and Myt1 in G(2) checkpoint regulation. The data reported here support the attractiveness of Wee1 and Chk1 is as molecular targets for abrogating the G(2) DNA damage checkpoint arrest, a situation that may selectively sensitize p53-deficient tumor cells to radiation or chemotherapy treatment.


Asunto(s)
Apoptosis/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas Quinasas/biosíntesis , Interferencia de ARN , Factores de Transcripción/biosíntesis , Mama/citología , Proteínas de Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Proteínas de Unión al ADN/farmacología , Regulación hacia Abajo , Células Epiteliales , Femenino , Fase G2 , Células HeLa , Humanos , Proteína Proteolipídica de la Mielina , Proteínas Nucleares , Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas , Factores de Transcripción/farmacología
5.
Nat Med ; 19(3): 313-21, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23396211

RESUMEN

Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IκB kinases IKK-ɛ and TANK-binding kinase 1 (TBK1) are induced in liver and fat by NF-κB activation upon high-fat diet feeding and in turn initiate a program of counterinflammation that preserves energy storage. Here we report that amlexanox, an approved small-molecule therapeutic presently used in the clinic to treat aphthous ulcers and asthma, is an inhibitor of these kinases. Treatment of obese mice with amlexanox elevates energy expenditure through increased thermogenesis, producing weight loss, improved insulin sensitivity and decreased steatosis. Because of its record of safety in patients, amlexanox may be an interesting candidate for clinical evaluation in the treatment of obesity and related disorders.


Asunto(s)
Aminopiridinas/farmacología , Fármacos Antiobesidad/farmacología , Metabolismo Energético/efectos de los fármacos , Quinasa I-kappa B/antagonistas & inhibidores , Resistencia a la Insulina , Obesidad/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Antialérgicos/farmacología , Línea Celular , Dieta Alta en Grasa , Activación Enzimática , Hígado Graso/tratamiento farmacológico , Trastornos del Metabolismo de la Glucosa/tratamiento farmacológico , Quinasa I-kappa B/metabolismo , Resistencia a la Insulina/inmunología , Grasa Intraabdominal/efectos de los fármacos , Grasa Intraabdominal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , FN-kappa B/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/inmunología , Consumo de Oxígeno/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Pérdida de Peso/efectos de los fármacos
6.
J Biol Chem ; 280(13): 13129-36, 2005 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-15668240

RESUMEN

Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3'-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors.


Asunto(s)
Citoesqueleto/metabolismo , Receptores ErbB/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Actinas/metabolismo , Western Blotting , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Citoplasma/metabolismo , Citoesqueleto/ultraestructura , Regulación hacia Abajo , Endocitosis , Silenciador del Gen , Células HeLa , Humanos , Inmunoprecipitación , Insulina/metabolismo , Ligandos , Microscopía Fluorescente , Proteína Oncogénica v-cbl , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/química , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Oncogénicas de Retroviridae/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transfección , Tirosina/metabolismo
7.
J Cell Sci ; 115(Pt 19): 3807-15, 2002 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-12235291

RESUMEN

Inositol phosphatases play an important role in regulation of cellular levels of lipid second messengers. Recently we have reported a novel function for SHIP2 in cell adhesion and spreading. In this study, we further characterize the adhesion-dependent tyrosine phosphorylation of SHIP2 and examine the role of Src family tyrosine kinases in the regulation of SHIP2 function. SHIP2 was tyrosine phosphorylated during cell attachment and spreading on collagen I, but not on fibronectin, collagen IV, laminin or poly-L-lysine. SHIP2 tyrosine phosphorylation, induced by plating on a collagen-I-coated surface but not by epidermal growth factor or insulin treatment of cells, was completely blocked by small molecule inhibitors of Src family kinases. SHIP2 could be phosphorylated in vitro by recombinant Src kinase and tyrosines 986-987 in the NPXY motif of SHIP2 appear to be the major sites of phosphorylation for Src both in vitro and in vivo. An activated form of Src induced strong tyrosine phosphorylation of SHIP2 while a dominant-negative form decreased collagen-I-dependent SHIP2 phosphorylation. SHIP2 associated with the adapter protein Shc via its NPXY motif during cell spreading on collagen I in a Src activity-dependent manner. Expression of SHIP2 with mutated NPXY motif caused deregulation of lamellipodia formation during spreading on collagen I. These observations indicate that SHIP2 is regulated by Src family kinases during cell attachment and spreading on collagen I and suggest an important role for SHIP2 as a part of a signaling pathway that regulates actin cytoskeleton remodeling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Eucariotas/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos/fisiología , Animales , Colágeno Tipo I/metabolismo , Inhibidores Enzimáticos/farmacología , Células Eucariotas/citología , Células HeLa , Humanos , Fosfatos de Inositol/metabolismo , Mutación/fisiología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , Seudópodos/enzimología , Seudópodos/ultraestructura , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA