Phosphorylation of p70 ribosomal S6 Kinase (S6K1) by Cdk5 is required for dendritic spine maturation

Abstract

The maturation and maintenance of dendritic spines depends on neuronal activity and protein synthesis. One potential mechanism involves mammalian target of rapamycin (mTOR), which promotes protein synthesis through phosphorylation of 4E-BP and p70S6 kinase (S6K1). Upon extracellular stimulation, mTOR phosphorylates S6K1 at Thr-389 near the catalytic domain. S6K1 also undergoes phosphorylation independently of mTOR at other sites, including four serine residues in the C-terminal auto-inhibitory domain. Despite extensively studied biochemically, the role of phosphorylation in the auto-inhibitory domain on S6K1 kinase activity remains unresolved, and its function in the cellular context has not been explored. Here we focus on Ser-411 within the auto-inhibitory domain, which was shown to be phosphorylated in vitro by multiple kinases. One of them is the proline-directed serine/threonine kinase cyclin-dependent kinase 5 (Cdk5), which has an emerging role in spine morphogenesis and synaptic plasticity. We found that Ser-411 phosphorylation of S6K1 is largely reduced in the brains of Cdk5 knockout mice. Using the azide-bearing noncanonical amino acid for metabolic labeling, we demonstrated that protein synthesis was impaired in Cdk5 knockout neurons. Moreover, S6K1 phosphorylation at Ser-411 is regulated by neuronal activity and BDNF in a Cdk5-dependent manner. Knockdown of S6K1 in hippocampal neurons by RNAi led to the appearance of immature long and thin spines, an effect that mimics neuronal activity blockade by tetrodotoxin. Notably, co-expression of wild-type S6K1, but not the phospho-deficient S411A mutant, could rescue the spine defects. These findings therefore reveal the importance of Cdk5-mediated phosphorylation of S6K1 at Ser-411 in spine maturation driven by spontaneous neuronal activity.