RESUMEN
The CREB-specific coactivator TORC2 (also known as CRTC2) upregulates gluconeogenic gene expression in the liver. Salt-inducible kinase (SIK) family enzymes inactivate TORC2 through phosphorylation and localize it in the cytoplasm. Ser(171) and Ser(275) were found to be phosphorylated in pancreatic beta-cells. Calcineurin (Cn) is proposed as the Ser(275) phosphatase, because its inhibitor cyclosporin A (CsA) stabilizes phospho-Ser(275) and retains TORC2 in the cytoplasm. Because the regulation of dephosphorylation at Ser(171) has not been fully clarified, we performed experiments with a range of doses of okadaic acid (OA), an inhibitor of PP2A/PP1, and with overexpression of various phosphatases and found that PP1 functions as an activator for TORC2, whereas PP2A acts as an inhibitor. In further studies using TORC2 mutants, we detected a disassociation between the intracellular distribution and the transcription activity of TORC2. Additional mutant analyses suggested the presence of a third phosphorylation site, Ser(307). The Ser(307)-disrupted TORC2 was constitutively localized in the nucleus, but its coactivator activity was normally suppressed by SIK1 in COS-7 cells. CsA, but not OA, stabilized the phosphogroup at Ser(307), suggesting that differential dephosphorylation at Ser(171) and Ser(307) cooperatively regulate TORC2 activity and that the nuclear localization of TORC2 is insufficient to function as a coactivator. Because the COS-7 cell line may not possess signaling cascades for gluconeogenic programs, we next examined the importance of Ser(307) and Ser(171) for TORC2's function in mouse liver. Levels of phosphorylation at Ser(171) and Ser(307) changed in response to fasting or fed conditions and insulin resistance of the mouse liver, which were modified by treatment with CsA/OA and by overexpression of PP1/PP2A/Cn. These results suggest that multiple phosphorylation sites and their phosphatases may play important roles in regulating TORC2/CREB-mediated gluconeogenic programs in the liver.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Diabetes Mellitus/metabolismo , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Transactivadores/metabolismo , Animales , Células COS , Chlorocebus aethiops , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Mutagénesis Sitio-Dirigida , Ácido Ocadaico/farmacología , Fosforilación , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina/metabolismo , Factores de TranscripciónRESUMEN
Cyclic AMP responsive element (CRE)-binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133-independent activation. One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 nm staurosporine in LKB1-positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1-defective cells or in staurosporine-treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Quinasas Activadas por AMP , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Citoplasma/metabolismo , Silenciador del Gen , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Ratas , Serina/metabolismo , Transducción de Señal/genética , Estaurosporina/farmacología , Treonina/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Photoinduced charge separation of fullerodendrimers with carboxylates at terminal sites (C60 approximately COO-) has been found in aqueous media. Time-resolved transient absorption and fluorescence measurements of the fullerodendrimers demonstrated that charge separation takes place from the terminal carboxylate anion to the central excited singlet state of C60, generating C60*- approximately COO* with high quantum efficiency in aqueous solution. In the presence of viologen dication and a sacrificial donor, the persistent viologen radical cation was generated.
