RESUMEN
To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.
Asunto(s)
Colesterol/metabolismo , Prolina/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/fisiología , Capacitación Espermática/fisiología , Espermatozoides/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , 2-Hidroxipropil-beta-Ciclodextrina , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Bucladesina/farmacología , Butadienos/farmacología , Bovinos , AMP Cíclico/agonistas , Flavonoides/farmacología , Humanos , Masculino , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mitosis , Nitrilos/farmacología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Albúmina Sérica Bovina/farmacología , Bicarbonato de Sodio/farmacología , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
Capacitation is a complex series of molecular events that occurs in sperm after epididymal maturation and confers on sperm the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of oviductal fluid. In most cases, capacitation media contain energy substrates, such as pyruvate, lactate and glucose, a cholesterol acceptor (usually serum albumin), NaHCO(3), Ca(2+), low K(+), and physiological Na(+) concentrations. The mechanism of action by which these compounds promote capacitation is poorly understood at the molecular level; however, some molecular events significant to the initiation of capacitation have been identified. For example, capacitation correlates with cholesterol efflux from the sperm plasma membrane, increased membrane fluidity, modulations in intracellular ion concentrations, hyperpolarization of the sperm plasma membrane and increased protein tyrosine phosphorylation. These molecular events are required for the subsequent induction of hyperactivation and the acrosome reaction. This review discusses the recent progress that has been made in elucidating mechanisms which regulate sperm capacitation.
Asunto(s)
Transducción de Señal/fisiología , Capacitación Espermática/fisiología , Animales , Bicarbonatos/metabolismo , Señalización del Calcio , Colesterol/metabolismo , AMP Cíclico/metabolismo , Femenino , Humanos , Técnicas In Vitro , Transporte Iónico , Masculino , Potenciales de la Membrana , Modelos Biológicos , Fosforilación , Interacciones Espermatozoide-Óvulo/fisiología , Tirosina/metabolismoRESUMEN
Significant stimulation of protein kinase C-alpha (PKCalpha) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO). The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED(50)) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol. Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity. (31)P NMR experiments did not reveal formation of non-lamellar phases with octanol. Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PKC-bilayer binding.
Asunto(s)
Alcoholes/farmacología , Isoenzimas/metabolismo , Lípidos/química , Proteína Quinasa C/metabolismo , Alcoholes/química , Unión Competitiva/efectos de los fármacos , Diglicéridos/química , Dimiristoilfosfatidilcolina/química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Isoenzimas/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Fluidez de la Membrana , Octanoles/farmacología , Pentanoles/farmacología , Ésteres del Forbol/metabolismo , Fosfatidilserinas/química , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/química , Proteína Quinasa C-alfa , TemperaturaRESUMEN
In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.
Asunto(s)
Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , Animales , Cristalización , Diglicéridos/farmacología , Activación Enzimática , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Cinética , Liposomas , Oligopéptidos/química , Oligopéptidos/metabolismo , Fosfatidilcolinas/farmacología , Fosfatidilserinas/farmacología , Proteína Quinasa C/química , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C-alfa , Proteína Quinasa C-delta , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Spodoptera , Especificidad por Sustrato , Transfección , Unitiol/farmacologíaRESUMEN
Three two-dimensional (2D) crystal forms of protein kinase C (PKC) alpha and three of PKC delta have been grown on lipid monolayers composed of dioleoylphosphatidylcholine: dioleoylphosphatidylserine: (45:50:5 molar ratio). In the absence of DO, two additional 2D crystals of PKC delta are seen, suggesting that the presence of diolein (DO) alters the conformation of intact PKC at the lipid surface. Reconstructions of electron micrographs of these eight lattices show good reproducibility and indicate that several are appropriate for three-dimensional reconstruction to 20 A resolution.
Asunto(s)
Isoenzimas/aislamiento & purificación , Isoenzimas/ultraestructura , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C/ultraestructura , Cristalización , Diglicéridos , Procesamiento de Imagen Asistido por Computador , Isoenzimas/química , Microscopía Electrónica , Fosfatidilcolinas , Fosfatidilserinas , Conformación Proteica , Proteína Quinasa C/química , Proteína Quinasa C-alfa , Proteína Quinasa C-delta , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructura , Análisis Espectral , Rayos XRESUMEN
Lysophosphatidic acid (LPA) has attracted recent attention as a major serum-derived regulator implicated in responses to vascular injury and inflammation, in tumour invasiveness and in neuronal signalling and remodelling. Although the possibility of a specific G-protein-coupled LPA receptor protein has been suggested, characterization of such a receptor is lacking. Since LPA can activate protein kinase C (PKC) pathways in many cells and PKC activators mimic many LPA effects, the possibility of more direct LPA effects on PKC was investigated. Phosphatidylcholine (PC)/phosphatidylserine (PS)/diacylglycerol (DAG) lipid vesicles of defined acyl chain composition were used to activate the enzyme. At total concentrations of saturated PC/PS + DAG vesicles (2-3 mM) that provided maximal PKC activation, 1-10 mol % [18:1]-LPA led to a further approx. 2-fold activation of PKC alpha. At lower lipid concentrations, a greater increase was observed with LPA concentrations up to 16-20 mol %. Higher concentrations of LPA were inhibitory. The LPA activation of PKC was dependent on the presence of DAG, PS and Ca2+. [18:1]-Lysophosphatidylcholine produced similar PKC activation in PC/PS/DAG vesicles. [14:0]-LPA was less effective, and longer-chain saturated lysolipids were ineffective. In unsaturated PC/PS vesicles, very little to no effect of LPA was discernable. These results suggest that physiologically or pathologically relevant concentrations of LPA can contribute to PKC activation depending on the composition of the lipid membrane. We hypothesize that LPA may affect the formation of lipid domains that are recognized by the enzyme.