Editors' ChoiceCancer Metabolism

Releasing aldolase from the cytoskeleton

Sci. Signal.  02 Feb 2016:
Vol. 9, Issue 413, pp. ec21
DOI: 10.1126/scisignal.aaf3599

Insulin-stimulated cells activate phosphoinositide 3-kinase (PI3K) that in turn initiates downstream signaling events to promote cell growth and metabolism. Hu et al. identified a mechanism by which PI3K signaling promoted glycolysis. Mammary epithelial MCF10A cells exposed to inhibitors targeting specific enzymes in the PI3K signaling pathway exhibited different effects on the ratio of NADH (an electron donor)/NAD+ (an electron acceptor). Whereas drugs that inhibited PI3K activity produced a sustained decrease in the ratio of NADH/NAD+, inhibitors of either the downstream PI3K-dependent kinase AKT or the AKT-activated kinase mTOR resulted in a transient decrease in the NADH/NAD+ ratio. Because the NADH/NAD+ ratio is a proxy for glycolytic rate, a decrease in this ratio indicates a decrease in glycolysis. The glycolytic metabolic intermediates glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone-phosphate (DHAP), which are both products of reactions catalyzed by aldolase, were reduced in HCC1937 breast cancer cells exposed to PI3K inhibitors compared with cells exposed to an AKT inhibitor. Aldolase functions not only as a metabolic enzyme but also interacts with several proteins that control actin polymerization. To explore if insulin and PI3K signaling affected the solubility of aldolase, MCF10A cells were exposed to insulin and then permeabilized to enable quantification of soluble aldolase. Exposure of the cells to PI3K inhibitors, but not to an AKT inhibitor or an mTOR inhibitor, reduced the amount of aldolase activity released from the permeabilized cells. Fractionation of MCF10A revealed that aldolase shifted from the cytoskeletal to cytoplasmic fraction in response to insulin, and PI3K inhibition prevented this shift. Immunofluorescence analysis of aldolase in mammary epithelial cell lines showed that aldolase colocalized with F-actin and insulin led to the PI3K-dependent diffuse cytosolic localization of aldolase. Fluorescence correlation spectroscopy analysis confirmed colocalization of aldolase with F-actin and the lack of colocalization upon insulin stimulation in MCF10A cells, consistent with sequestration of aldolase with polymerized actin in the absence of insulin stimulation. In MCF10A cells, inhibition of PI3K, but not of AKT or mTOR, prevented insulin-mediated activation of the guanosine triphosphatase (GTPase) Rac1, a protein involved in cytoskeletal rearrangement, and the binding of active GTP-bound Rac1 to p21-activated protein kinase (PAK), a kinase activated by this GTPase. In MCF10A cells, the insulin-induced phosphorylation of PAK and aldolase release from the cytoskeleton was reduced when mutated Rac1 was overexpressed or when Rac1 was knocked down. In a mouse model of breast cancer given radiolabeled glucose to enable monitoring of glycolytic metabolism, administration of a PI3K inhibitor produced a decrease in the abundance of the aldolase product DHAP in the tumor tissue compared with the abundance of DHAP in tumors from mice that were not given the inhibitor. The abundance of aldolase was unchanged by administration of the PI3K inhibitor, which is consistent with PI3K stimulating the redistribution of aldolase to promote glycolysis. This study, therefore, suggests an AKT-independent role of PI3K in altering glycolysis through spatial regulation of aldolase.

H. Hu, A. Juvekar, C. A. Lyssiotis, E. C. Lien, J. G. Albeck, D. Oh, G. Varma, Y. P. Hung, S. Ullas, J. Lauring, P. Seth, M. R. Lundquist, D. R. Tolan, A. K. Grant, D. J. Needleman, J. M. Asara, L. C. Cantley, G. M. Wulf, Phosphoinositide 3-kinase regulates glycolysis through mobilization of aldolase from the actin cytoskeleton. Cell 164, 433–446 (2016). [PubMed]