RESUMO
KEY MESSAGE: This study provides the first genetic evidence for the role of PP2A in tuberization, demonstrating that the catalytic subunit StPP2Ac2b positively modulates tuber induction, and that its function is related to the regulation of gibberellic acid metabolism. The results contribute to a better understanding of the molecular mechanism controlling tuberization induction, which remains largely unknown. The serine/threonine protein phosphatases type 2A (PP2A) are implicated in several physiological processes in plants, playing important roles in hormone responses. In cultivated potato (Solanum tuberosum), six PP2A catalytic subunits (StPP2Ac) were identified. The PP2Ac of the subfamily I (StPP2Ac1, 2a and 2b) were suggested to be involved in the tuberization signaling in leaves, where the environmental and hormonal signals are perceived and integrated. The aim of this study was to investigate the role of PP2A in the tuberization induction in stolons. We selected one of the catalytic subunits of the subfamily I, StPP2Ac2b, to develop transgenic plants overexpressing this gene (StPP2Ac2b-OE). Stolons from StPP2Ac2b-OE plants show higher tuber induction rates in vitro, as compared to wild type stolons, with no differences in the number of tubers obtained at the end of the process. This effect is accompanied by higher expression levels of the gibberellic acid (GA) catabolic enzyme StGA2ox1. GA up-regulates StPP2Ac2b expression in stolons, possibly as part of the feedback system by which the hormone regulates its own level. Sucrose, a tuber-promoting factor in vitro, increases StPP2Ac2b expression. We conclude that StPP2Ac2b acts in stolons as a positive regulator tuber induction, integrating different tuberization-related signals mainly though the modulation of GA metabolism.
Assuntos
Domínio Catalítico , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Tubérculos/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Solanum tuberosum/enzimologia , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/metabolismo , Giberelinas/farmacologia , Modelos Biológicos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transdução de Sinais/efeitos dos fármacos , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis.
Assuntos
Mitose , Proteína Fosfatase 2/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Anáfase/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Cinética , Mitose/efeitos dos fármacos , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/química , Subunidades Proteicas/metabolismo , Eletricidade Estática , Especificidade por Substrato/efeitos dos fármacos , Fatores de TempoRESUMO
Transcriptional co-activator with PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif (TAZ) regulates in cell proliferation and differentiation. In mesenchymal stem cells it promotes osteogenesis and myogenesis, and suppresses adipogenesis. TAZ activators are expected to prevent osteoporosis, obesity and muscle atrophy. TAZ activation induces epithelial-mesenchymal transition, confers stemness to cancer cells and leads to poor clinical prognosis in cancer patients. In this point of view, TAZ inhibitors should contribute to cancer therapy. Thus, TAZ attracts attention as a two-faced drug target. We screened for TAZ modulators by using human lung cancer A549 cells expressing the fluorescent reporter. Through this assay, we obtained TAZ activator candidates. We unexpectedly found that ethacridine, a widely used antiseptic and abortifacient, enhances the interaction of TAZ and protein phosphatases and increases unphosphorylated and nuclear TAZ. Ethacridine inhibits adipogenesis in mesenchymal C3H10T1/2 cells through the activation of TAZ. This finding suggests that ethacridine is a bona fide TAZ activator and supports that our assay is useful to discover TAZ activators.
Assuntos
Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Etacridina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/agonistas , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/agonistas , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/química , Proteína Fosfatase 2/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAPRESUMO
Protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) are responsible for the dephosphorylation of the majority of phosphoserine/threonine residues in cells. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), polyphenolic constituents of green tea and tannins, inhibit the activity of the PP1 recombinant δ-isoform of the PP1 catalytic subunit and the native PP1 catalytic subunit (PP1c) with IC(50) values of 0.47-1.35 µm and 0.26-0.4 µm, respectively. EGCG and PGG inhibit PP2Ac less potently, with IC(50) values of 15 and 6.6 µm, respectively. The structure-inhibitory potency relationships of catechin derivatives suggests that the galloyl group may play a major role in phosphatase inhibition. The interaction of EGCG and PGG with PP1c was characterized by NMR and surface plasmon resonance-based binding techniques. Competitive binding assays and molecular modeling suggest that EGCG docks at the hydrophobic groove close to the catalytic center of PP1c, partially overlapping with the binding surface of microcystin-LR or okadaic acid. This hydrophobic interaction is further stabilized by hydrogen bonding via hydroxyl/oxo groups of EGCG to PP1c residues. Comparative docking shows that EGCG binds to PP2Ac in a similar manner, but in a distinct pose. Long-term treatment (24 h) with these compounds and other catechins suppresses the viability of HeLa cells with a relative effectiveness reminiscent of their in vitro PP1c-inhibitory potencies. The above data imply that the phosphatase-inhibitory features of these polyphenols may be implicated in the wide spectrum of their physiological influence.
Assuntos
Catequina/análogos & derivados , Taninos Hidrolisáveis/química , Proteína Fosfatase 1/química , Estrutura Terciária de Proteína , Catequina/química , Catequina/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Taninos Hidrolisáveis/metabolismo , Taninos Hidrolisáveis/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Ressonância de Plasmônio de Superfície , Chá/químicaRESUMO
Tuber formation in potato (Solanum tuberosum L.) is regulated by hormonal and environmental signals that are thought to be integrated in the leaves. The molecular mechanisms that mediate the responses to tuberization-related signals in leaves remain largely unknown. In this study we analyzed the roles of protein phosphatase type 2A catalytic subunits (PP2Ac) in the leaf responses to conditions that affect tuberization. The responses were monitored by analyzing the expression of the "tuber-specific" genes Patatin and Pin2, which are induced in tubers and leaves during tuber induction. Experiments using PP2A inhibitors, together with PP2Ac expression profiles under conditions that affect tuberization indicate that high sucrose/nitrogen ratio, which promotes tuber formation, increases the transcript levels of Patatin and Pin2, by increasing the activity of PP2As without affecting PP2Ac mRNA or protein levels. Gibberellic acid (GA), a negative regulator of tuberization, down-regulates the transcription of catalytic subunits of PP2As from the subfamily I and decreases their enzyme levels. In addition, GA inhibits the expression of Patatin and Pin2 possibly by a PP2A-independent mechanism. PP2Ac down-regulation by GA may inhibit tuberization signaling downstream of the inductive effects of high sucrose/nitrogen ratio. These results are consistent with the hypothesis that PP2As of the subfamily I may positively modulate the signaling pathways that lead to the transcriptional activation of "tuber-specific" genes in leaves, and act as molecular switches regulated by both positive and negative modulators of tuberization.