RESUMEN
A macromolecular complex of the enzymes involved in human de novo purine biosynthesis, the purinosome, has been shown to consist of a core assembly to regulate the metabolic activity of the pathway. However, it remains elusive whether the core assembly itself can be selectively controlled in the cytoplasm without promoting the purinosome. Here, we reveal that pharmacological inhibition of the cytoplasmic activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1) selectively promotes the formation of the core assembly, but not the purinosome, in cancer cells. However, alternative signaling cascades that are associated with the plasma membrane-bound PDK1 activity, including Akt-mediated cascades, regulate neither the core assembly nor the purinosome in our conditions. Along with immunofluorescence microscopy and a knock-down study against PDK1 using small interfering RNAs, we reveal that cytoplasmic PDK1-associated signaling pathways regulate subcellular colocalization of three enzymes that form the core assembly of the purinosome in an Akt-independent manner. Collectively, this study reveals a new mode of compartmentalization of purine biosynthetic enzymes controlled by spatially resolved signaling pathways.
Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Purinas/biosíntesis , Transducción de Señal , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Biomarcadores , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Inmunohistoquímica , Redes y Vías Metabólicas , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells.