PKA-Dependent Phosphorylation of Ribosomal Protein S6 Does Not Correlate with Translation Efficiency in Striatonigral and Striatopallidal Medium-Sized Spiny Neurons

Abstract

Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant D-amphetamine (D-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by D-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5Ј terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency.

Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains

“…Our finding of no detectable difference in global protein synthesis in the dentate gyrus despite robust induction of p-rpS6 is in line with other reports (Biever et al 2015a). However, the lack of increase in global protein synthesis does not rule out the possibility that p-rpS6 may be involved in the preferential translation of specific mRNAs (Jefferies 1997).…”

“…It has been reported that ser240/244 is predominately mTOR-dependent being phosphorylated by S6K1 and S6K2 (Pende et al 2004). In contrast, ser235/236 has been implicated as a target for multiple signaling pathways including MAPK/ERK via RSK (Roux et al 2007) and PKA (Gobert et al 2008;Moore et al 2009;Biever et al 2015a). Thus, the relatively higher activation in the region of active synapses for p-ser235/236 could be because it integrates signals from multiple pathways.…”

Ribosomal Protein S6 Phosphorylation

“…This phosphorylation is carried out through activation of the cAMP signaling, as was indicated by elevated Ser235/236 phosphorylation upon stimulation of PKA in cultured striatal neurons (Valjent et al, 2011). Likewise, stimulation of cAMP production by forskolin, an adenylate cyclase activator, in mouse striatal slices led to a marked increase in the phosphorylation of S235/236, which was attenuated by pretreatment with Rp-cAMP, a PKA inhibitor (Biever et al, 2015) (Figure 1). …”

G protein‐dependent signaling triggers a β‐arrestin‐scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation

“…In agreement, our previous data indicate that FSH activates p70S6K by a PKA-dependent mechanism that could be involved in the mechanism reported herein (6, 7). Likewise, in the brain, forskolin stimulation enhances rpS6 phosphorylation on S235/S236 residues (54,72), presumably reflecting p70S6K activity. We show here that endogenous b-arrestins 1 and 2 form a constitutive complex with p70S6K, independent on the translocation of b-arrestin to the FSHR upon hormone binding.…”

“…However, its upstream mechanisms of regulation and its precise role in translation are still poorly understood and have been a matter of many controversial studies (53,79). For example, a recent report suggested that stimulation of dopamine receptors in the striatum leads to rpS6 phosphorylation that depends on cAMP/PKA-but not on p70S6K-mediated signaling and that is not correlated to translation efficiency nor 59TOP mRNA translation (72). By quantitative confocal microscopy, we show that p70S6K and rpS6 colocalize to a lesser extent in MEFs from b-arrestin 1,2 knockout mice.…”