RESUMO
Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small-molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5-bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug-resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5-bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux.
Assuntos
Aminopiridinas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Fosfatos de Fosfatidilinositol/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Retroviridae/efeitos dos fármacos , Retroviridae/metabolismo , Aminopiridinas/química , Animais , Transporte Biológico/efeitos dos fármacos , Biomarcadores/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Inibidores Enzimáticos/química , Compostos Heterocíclicos com 3 Anéis/química , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Células NIH 3T3RESUMO
Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.
Assuntos
Células Cromafins/fisiologia , Exocitose/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Bovinos , Humanos , Técnicas In Vitro , Camundongos , Sistemas Neurossecretores/efeitos dos fármacos , Sistemas Neurossecretores/fisiologia , Células PC12 , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibition of p38 and the induction of MKP-1 by dexamethasone occurred with similar dose dependence and kinetics. No other known p38 phosphatases were induced by dexamethasone, and other cell types which failed to express MKP-1 also failed to inhibit p38 in response to dexamethasone. The proinflammatory cytokine interleukin 1 (IL-1) induced MKP-1 expression in a p38-dependent manner and acted synergistically with dexamethasone to induce MKP-1 expression. In HeLa cells treated with IL-1 or IL-1 and dexamethasone, the dynamics of p38 activation mirrored the expression of MKP-1. These observations suggest that MKP-1 participates in a negative-feedback loop which regulates p38 function and that dexamethasone may inhibit proinflammatory gene expression in part by inducing MKP-1 expression.