Serine 707 of APPL1 is Critical for the Synaptic NMDA Receptor-Mediated Akt Phosphorylation Signaling Pathway

Abstract

Accumulating evidence indicates that the synaptic activation of N-methyl-D-aspartate receptors (NMDARs) has a neuroprotective effect on neurons. Our previous study demonstrated that APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper motif) mediates the synaptic activity-dependent activation of PI3K-Akt signaling via coupling this pathway with NMDAR-PSD95 (postsynaptic density protein 95) complexes. However, the molecular mechanism underlying this process is still unknown. In the present study, we investigated the interaction of APPL1 with PSD95 using co-immunocytochemical staining and western blotting. We found that the PDZ2 domain of PSD95 is a binding partner of APPL1. Furthermore, we identified serine 707 of APPL1, a predicted phosphorylation site within the PDZ-binding motif at the C-terminus, as critical for the binding of APPL1 to PSD95, as well as for activation of the Akt signaling pathway during synaptic activity. This suggests that serine 707 of APPL1 is a potential phosphorylation site and may be involved in regulating the neuroprotective Akt signaling pathway that depends on synaptic NMDAR activity.

Keywords: APPL1; Akt; NMDA receptors; Neuroprotection; PSD95.

Fig. 1

APPL1 is physically associated with PSD95 in the brain and cultured neurons. (A) Representative 3D-SIM images showing co-localization of APPL1 with PSD95 in cultured hippocampal neurons. Scale bar, 5 μm. (B) Statistics for APPL1 and PSD95 cluster density in dendrites. The mean density of APPL1 clusters was 1.3 ± 0.12 per μm (total 38 neurons, n = 3) and of PSD95 clusters was 1.4 ± 0.14 per μm (total 45 neurons, n =  3). (C) Statistics for APPL1 and PSD95 co-localization. The percentage of APPL1 co-localized with PSD95 was 38 ± 3.61% (total 40 neurons, n =  3), and the that of PSD95 co-localized with APPL1 was 18 ± 1.8% (total 40 neurons, n =  3). (D) APPL1 and PSD95 coexist in the same protein complex in brain tissue. Preparations from adult mouse hippocampus were immunoprecipitated (IP) and blotted with antibodies against PSD95 or APPL1. Three independent co-IP tests were performed for each group.

Fig. 2

Mapping of interaction between APPL1 and PSD95. (A) Schematic of the PSD95 construct and its mutants. (B) APPL1 interacts with the PDZ2 domain of PSD95 in HEK293T cells co-transfected with GFP-APPL1 and mCherry-PSD95, mCherry-PSD95_△PDZ1, or mCherry-PSD95_△PDZ1/2. Three independent co-IP tests were performed. (C) Schematic of the APPL1 construct with serine 707 replaced by alanine. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. (D) APPL1_S707A had a significantly decreased interaction with PSD95 in HEK293T cells co-transfected with mCherry-PSD95 and CFP-APPL1 or CFP-APPL1_S707A or CFP-APPL1_△4. (E) Statistics for (D), **P <0.01, N.S.: no significant difference, n =  4, one-way ANOVA.

Fig. 3

APPL1 serine 707 is critical for synaptic activity-mediated recruitment of APPL1 to PSD95 in cultured neurons. (A) Representative western blot showing that synaptic NMDAR activation significantly increases APPL1 interaction with PSD95 in cultured neurons. (B) Statistics for (A), *P <0.05, n =  3 independent blots, t-test. (C) CFP-APPL1, but not CFP-APPL1_S707A, had significantly increased co-localization with PSD95 in response to synaptic activity in cultured neurons. Scale bars, 20 μm. (D) Statistics for co-localizaiton of APPL1 with PSD95. **P <0.01, ***P <0.001, N.S.: no significant difference, 60 neurons each group, 3 independent cultures, one-way ANOVA.

Fig. 4

APPL1_S707A mutant disrupts synaptic activity-mediated increase in Akt activity. (A) GFP-APPL1_S707A transfection decreased the level of Akt phosphorylation at S473 in response to synaptic activity in cultured neurons. Scale bars, 20 μm. (B) Statistics for intensity of phospho-Akt. *P <0.05, N.S.: no significant difference, 45~50 neurons each group, 3 independent cultures, one-way ANOVA.