RESUMEN
Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) has been shown to play a major role in establishing memories through complex molecular interactions including phosphorylation of multiple synaptic targets. However, it is still controversial whether CaMKII itself serves as a molecular memory because of a lack of direct evidence. Here, we show that a single holoenzyme of CaMKII per se serves as an erasable molecular memory switch. We reconstituted Ca(2+)/Calmodulin-dependent CaMKII autophosphorylation in the presence of protein phosphatase 1 in vitro, and found that CaMKII phosphorylation shows a switch-like response with history dependence (hysteresis) only in the presence of an N-methyl-D-aspartate receptor-derived peptide. This hysteresis is Ca(2+) and protein phosphatase 1 concentration-dependent, indicating that the CaMKII memory switch is not simply caused by an N-methyl-D-aspartate receptor-derived peptide lock of CaMKII in an active conformation. Mutation of a phosphorylation site of the peptide shifted the Ca(2+) range of hysteresis. These functions may be crucial for induction and maintenance of long-term synaptic plasticity at hippocampal synapses.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Fragmentos de Péptidos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cinética , Fragmentos de Péptidos/química , Fosforilación , Unión Proteica , Proteína Fosfatasa 1/metabolismo , Estructura Terciaria de Proteína , Ratas , Receptores de N-Metil-D-Aspartato/química , Células Sf9 , SpodopteraRESUMEN
Secretion of insulin transiently increases after eating, resulting in a high circulating concentration. Fasting limits insulin secretion, resulting in a low concentration of insulin in the circulation. We analyzed transcriptional responses to different temporal patterns and doses of insulin in the hepatoma FAO cells and identified 13 up-regulated and 16 down-regulated insulin-responsive genes (IRGs). The up-regulated IRGs responded more rapidly than did the down-regulated IRGs to transient stepwise or pulsatile increases in insulin concentration, whereas the down-regulated IRGs were repressed at lower concentrations of insulin than those required to stimulate the up-regulated IRGs. Mathematical modeling of the insulin response as two stages-(i) insulin signaling to transcription and (ii)transcription and mRNA stability-indicated that the first stage was the more rapid stage for the down-regulated IRGs, whereas the second stage of transcription was the more rapid stage for the up-regulated IRGs. A subset of the IRGs that were up-regulated or down-regulated in the FAO cells was similarly regulated in the livers of rats injected with a single dose of insulin. Thus, not only can cells respond to insulin but they can also interpret the intensity and pattern of signal to produce distinct transcriptional responses. These results provide insight that may be useful in treating obesity and type 2 diabetes associated with aberrant insulin production or tissue responsiveness.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Insulina/farmacología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Animales , Línea Celular Tumoral , RatasRESUMEN
Sustained contraction of cells depends on sustained Rho-associated kinase (Rho-kinase) activation. We developed a computational model of the Rho-kinase pathway to understand the systems characteristics. Thrombin-dependent in vivo transient responses of Rho activation and Ca2+ increase could be reproduced in silico. Low and high thrombin stimulation induced transient and sustained phosphorylation, respectively, of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) in vivo. The transient phosphorylation of MLC and MYPT1 could be reproduced in silico, but their sustained phosphorylation could not. This discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase indicates that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We found, experimentally, that the sustained phosphorylation of MLC and MYPT1 exhibit all-or-none responses. Bromoenol lactone, a specific inhibitor of Ca2+ -independent phospholipase A2 (iPLA2), inhibited sustained phosphorylation of MLC and MYPT1, which indicates that sustained Rho-kinase activation requires iPLA2 activity. Thus, the systems analysis of the Rho-kinase pathway identified a novel iPLA2-dependent mechanism of the sustained Rho-kinase activation, which exhibits an all-or-none response.
Asunto(s)
Calcio/metabolismo , Simulación por Computador , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfolipasas A/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Fosfolipasas A2 Grupo IV , Humanos , Modelos Biológicos , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosfolipasas A2 , Fosforilación/efectos de los fármacos , Trombina/farmacología , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rhoRESUMEN
The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.