Abstract

Synaptic strength at excitatory synapses is determined by the presence of glutamate receptors (i.e. AMPA, NMDA, and kainate receptors) at the synapse. Synaptic strength is modulated by multiple factors including assembly of different receptor subunits, interaction with auxiliary subunits, and post-translational modifications of either the receptors or their auxiliary subunits. Using mass spectrometry, we found that the intracellular region of neuropilin and tolloid-like proteins (Neto) 1 and Neto2, the auxiliary subunits of kainate receptor (KARs), are phosphorylated by multiple kinases in vitro Specifically, Neto2 was phosphorylated at serine 409 (Ser-409) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both in vitro and in heterologous cells. Interestingly, we observed a substantial increase in Neto2 Ser-409 phosphorylation in the presence of CaMKII, and this phosphorylation was reduced in the presence of the KAR subunit GluK1 or GluK2. We also found endogenous phosphorylation of Neto2 at Ser-409 in the brain. Moreover, Neto2 Ser-409 phosphorylation inhibited synaptic targeting of GluK1 because, unlike WT Neto2 and the phosphodeficient mutant Neto2 S409A, the Neto2 S409D phosphomimetic mutant impeded GluK1 trafficking to synapses. These results support a molecular mechanism by which Neto2 phosphorylation at Ser-409 helps restrict GluK1 targeting to the synapse.

Highlights

  • Synaptic strength at excitatory synapses is determined by the presence of glutamate receptors (i.e. AMPA, NMDA, and kainate receptors) at the synapse

  • The autoradiogram shows that the C termini of both Neto1 and Neto2 are phosphorylated by calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), and PKC (Fig. 1, A–C)

  • Our findings show a molecular mechanism by which phosphorylation of the Neto2 C terminus regulates GluK1 synaptic targeting

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Summary

Results

It is well established that the function and trafficking of synaptic proteins are modulated by different post-translational modifications such as phosphorylation. Upon blotting with Ser(P)-409 antibody, a specific signal was observed only with GST-Neto WT when it was incubated with either CaMKII (Fig. 3C) or PKA (Fig. 3D). We observed a substantial increase in Neto Ser-409 phosphorylation both in the presence of CaMKII T286D and with forskolin treatment in heterologous cells using Ser(P)-409 antibody (Fig. 4, A–D), demonstrating the CaMKII- and PKA-mediated phosphorylation of Neto Ser-409 in situ. Neto is an important auxiliary subunit of KARs [5] and affects both the channel gating and synaptic targeting properties of different subunits of KARs. we tested whether Neto Ser-409 phosphorylation is modulated in the presence of KARs. we found that Neto Ser-409 phosphorylation increased considerably in the presence of CaMKII T286D, no such increase was observed when either KAR subunit GluK1 or GluK2 was co-expressed (Fig. 5, A and B). Different phosphorylated (ph) peptides of Neto identified in the reported proteomic screens

Discussion
Constructs and antibodies
Protein purification
In vitro kinase assay
Mass spectrometry
Phosphorylation in heterologous cells
Phosphorylation in brain
Full Text
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