Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis

Abstract

Gardner TW, Abcouwer SF, Losiewicz MK, Fort PE. Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis. Am J Physiol Endocrinol Metab 309: E546 -E556, 2015. First published July 21, 2015; doi:10.1152/ajpendo.00180.2015.-Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of D-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2␣ phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1.retinal protein synthesis; glycolysis; Akt/mTOR pathway; protein phosphatases NORMAL RETINAL FUNCTION requires high cellular metabolic activity (3, 47) to perform phototransduction, regenerate photoreceptor outer segments, maintain electrical gradients across membranes of unmyelinated neuronal axons, and conduct bidirectional macromolecular transport through ganglion cell axons to the lateral geniculate nucleus of the thalamus. Moreover, unlike skeletal muscle, liver, or adipose tissue, the retina has little capacity to locally store protein, glycogen, or lipids to respond to fluctuations of metabolic demands, which makes it highly dependent on glucose availability. We (5) previously suggested that the high metabolic activity required to maintain visual function coupled with a low intrinsic storage capacity may compromise the retina's adaptability to metabolic stress.The metabolic features of the retina include an exceptionally high dependence on glycolysis, with 90% o...

AMPK and Akt/mTOR signalling pathways participate in glucose‐mediated regulation of hepatitis B virus replication and cellular autophagy

“…Glycolysis is considered as a "central" carbon metabolic pathway because it is the backbone of several metabolic pathways and is pivotal for energy homeostasis (Akram, 2014;Dashty, 2013;Kornberg, 2000). On the other hand, 2-DG may markedly inhibit glycolysis that is essential for viral replication (Gardner, Abcouwer, Losiewicz, & Fort, 2015;Maus et al, 2006;Muaddi et al, 2010). This observation highlights the notion that HBV replication requires glycolysis in host cells.…”

Reprogramming towards anabolism impedes degeneration in a preclinical model of retinitis pigmentosa

“…7B). Conversely, mTOR, p-mTOR and their downstream targets were upregulated in the knockout mice, including pS6, glucose transporter 1 (GLUT1) and phosphorylated 4E binding protein-1 (p4EBP1), which promotes protein synthesis (25,26). Conversely, no difference in expression was found in unphosphorylated S6 and 4EBP1 in the Tsc1 loxP/loxP Pde6b H620Q/ H620Q versus the Tsc1 -/-Pde6b H620Q/H620Q mice.…”

Serine-threonine protein phosphatases: Lost in translation

“…Thus, inhibitory FGF signaling in chondrocytes stimulated PP2A activity without any effect on mTORC1 activity [51]. Similarly, in insulin stimulated rat retinas treated with a glycolytic inhibitor, 4E-BP was rapidly dephosphorylated with no reduction in mTORC1 activity detected [54]. The cases like these can serve as experimental models to digest the mechanism of PP2A activation.…”

“…Gardner et al reported that 4E-BP is rapidly dephosphorylated in the presence of glycolytic inhibitor in ex vivo rat retinas stimulated with insulin [54]. This dephosphorylation was mostly prevented by either 1µ M OA (PP1/PP2A inhibition) or by cadmium (PPM1 inhibition) suggesting that more than one phosphatase might be involved in dephosphorylation of all four residues, or maybe, 4E-BP phosphatase has to be activated (directly or not) by another phosphatase.…”