Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca2+/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.
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We are grateful to A. Sanz-Clemente and A. Scimemi for technical assistance and for discussions on the project and manuscript. We thank the NINDS sequencing facility and light imaging facility for their expertise. This research was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (M.A.B., T.H., J.D.B., Y.L., J.S.D. and K.W.R.) and the National Institute of Mental Health grant number 5 R37 MH038256 (S.L.S., B.E.H. and R.A.N.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) GST-NL-1 or GluA1 were incubated with purified CaMKII and [γ-P32]ATP and analyzed by autoradiography. Reactions were stopped at their marked time. (b) Protein concentrations are plotted as a ratio of phosphorylated NL-1 (P-32) to total NL-1 (CBB) normalized to the 1 min reaction condition. Saturation of phosphorylated NL-1 and GluA1 occurred at approximately 10 min. Total protein was visualized by CBB protein staining, in a,b. Full-length blots are presented in Supplementary Figure 4 when applicable.
Paired-pulse ratio (PPR), second EPSC over first EPSC for consecutive stimuli separated by 40 ms. Example traces normalized at first EPSC for (a) NL-1. (b) NL-1 T739A. (c) Second EPSC over first ± SEM. NL-1 and NL-1 T739A had similar PPRs (P > 0.05, n = 5). Scale bars represent 25 ms.
(a) Protein concentrations from adult WT or NL-1 KO brains were titrated with pT739-Ab. (b) Protein concentrations are plotted as ratio of phosphorylated (IP) to total NL-1 (input) normalized to the 0.2 mg protein condition for an individual brain. Saturation of the pT739-Ab occurs between 0.5 and 1 mg of protein.
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Bemben, M., Shipman, S., Hirai, T. et al. CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses. Nat Neurosci 17, 56–64 (2014). https://doi.org/10.1038/nn.3601
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