Differential regulation of mTOR‐dependent S6 phosphorylation by muscarinic acetylcholine receptor subtypes

Abstract

Muscarinic receptors subserve many functions in both peripheral and central nervous systems. Some of these processes depend on increases in protein synthesis, which may be achieved by activation of mammalian target of rapamycin (mTOR), a kinase that regulates protein translation capacity. Here, we examined the regulation of mTOR-dependent signaling pathways by muscarinic receptors in SK-N-SH human neuroblastoma cells, and in human embryonic kidney (HEK) cell lines transfected with individual muscarinic receptor subtypes. In SK-N-SH cells, the acetylcholine analog carbachol stimulated phosphorylation of the ribosomal S6 protein, a downstream target of mTOR. The sensitivity of the response to subtype-selective muscarinic receptor antagonists indicated that it was mediated by M3 receptors. Carbachol-evoked S6 phosphorylation was blocked by the mTOR inhibitor rapamycin, but was independent of phosphoinositide 3-kinase activation. The response was significantly reduced by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also inhibited carbachol-evoked S6 phosphorylation in HEK cells expressing M2 receptors, but was ineffective in M3 receptor-expressing HEK cells, although carbachol activated MAPK in both transfected lines. The p90 ribosomal S6 kinase has been implicated in mTOR regulation by phorbol esters, but was not activated by carbachol in any of the cell lines tested. The protein kinase C inhibitor bisindolylmaleimide I reduced carbachol-stimulated S6 phosphorylation in SK-N-SH cells, and in HEK cells expressing M3 receptors, but not in HEK cells expressing M2 receptors. The results demonstrate that multiple muscarinic receptor subtypes regulate mTOR, and that both MAPK-dependent and -independent mechanisms may mediate the response in a cell context-specific manner.

Conversion of short-term potentiation to long-term potentiation in mouse CA1 by coactivation of β-adrenergic and muscarinic receptors

“…Future research is needed to determine the role of Inhibition of mTOR prevented the maintenance of LTP induced by ISO paired with CCh. Although evidence implicating muscarinic receptors in mTOR regulation is sparse, one report has implicated the M3 muscarinic receptor in mTOR regulation (Slack and Blusztajn 2008). As our results have implicated the M1 muscarinic receptor but not M3, this suggests that the b1-AR may be primarily responsible for mTOR regulation (Gelinas et al 2007).…”

Crosstalk between VEGFR2 and muscarinic receptors regulates the mTOR pathway in serum starved SK-N-SH human neuroblastoma cells

“…Since mAchRs signal to the TORC1 target S6 in SK-N-SH cells [26], serum starved cells were treated with the mAchR agonist carbachol and examined over a 30 minute time frame for the phosphorylation of S6K at the TORC1 site Thr389 and Akt activation at Thr308 (Figure 1A). Carbachol elicited opposing effects on S6K and Akt in that an increase in S6K phosphorylation, which peaked at 15 minutes, was accompanied by a concomitant decrease in Akt activation (lane 8).…”

“…In addition, prior evidence shows that PKC modulates the carbachol mediated phosphorylation of S6 and Akt in different cell lines [26,38,39]. Therefore, we addressed a role for PKC in agonist mediated signaling to mTORC1 targets and Akt by preincubating serum starved cells for 2 hours with GFX, an inhibitor of conventional PKC isoforms (α, β, and γ), followed by a 15 minute stimulation with VEGF or carbachol alone or in combination (Figure 3A).…”

“…Akt can potentiate TORC1 signaling by inhibiting the tuberous sclerosis complex (TSC), allowing TORC1 signaling of protein translation via activation of its downstream targets p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6). In neuronal models, agonists for mAchRs were shown to stimulate activation of S6K in 1321 astrocytoma cells [23], SH-SY5Y [24] and PC12 cells [25] and S6 ribosomal protein via PKC in serum-deprived SK-N-SH neuroblastoma cells [26]. …”

Frontier of Epilepsy Research - mTOR signaling pathway

“…PLCβ1 null mice showed epilepsy, and age-dependent hippocampal mossy fiber sprouting (Kim et al, 1997;Böhm et al, 2002). PLCβ1 is coupled to muscarinic receptors, and activation of muscarinic receptors induces mTOR-dependent phosphorylation of ribosomal protein S6 (Popova and Rasenick, 2000;Slack and Blusztajn, 2008). Pilocarpine elicits SE through muscarinic receptors and mGluR5/ PLCβ1 (el-Etri et al, 1993;Liu et al, 2008b).…”