GDF15 regulates Kv2.1-mediated outward K+ current through the Akt/mTOR signalling pathway in rat cerebellar granule cells

摘要

GDF15 (growth/differentiation factor 15), a novel member of the TGFβ (transforming growth factor β) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the I K (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the I K is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the I K channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK (extracellular-signal-regulated kinase), Akt and mTOR (mammalian target of rapamycin), but the GDF15-induced I K densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFβR2 (TGFβ receptor 2), not TGFβR1, abrogated the effect of GDF15 on I K amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFβRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFβRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling.Abbreviations: 4-AP, 4-aminopyridine; CGN, cerebellar granule neuron; CHX, cycloheximide; DIC, days in culture; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular-signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDF15, growth/differentiation factor 15; IA, fast-transient outward K+ current; IK, delayed-rectifier outward K+ current; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MIC-1, macrophage inhibitory cytokine-1; mTOR, mammalian target of rapamycin; NP40, Nonidet P40; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; qPCR, quantitative real-time PCR; R-Smad, receptor-regulated Smad; TGFβ, transforming growth factor β; TGFβR, TGFβ receptor; TTX, tetrodotoxin