Protein Kinase C Promotes N-Methyl-d-aspartate (NMDA) Receptor Trafficking by Indirectly Triggering Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) Autophosphorylation

Abstract

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and longterm potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.The NMDA receptor (NMDAR) 3 plays a critical role in neural development, learning and memory, sensory perception, and synaptic plasticity (1, 2). Therefore, regulation of neuronal NMDARs is of great importance to synaptic transmission. PKC increases the NMDA channel opening rate and delivers new NMDARs to the plasma membrane through regulated exocytosis (3, 4). This potentiation of NMDAR trafficking is not due to phosphorylation of the NMDAR by PKC (5), suggesting that PKC indirectly exerts its effect through interaction with NMDAR-associated signaling and/or trafficking protein(s) (3, 4). Very recently, two elegant studies revealed that a SNARE protein, either SNAP-23 or SNAP-25, is such an NMDAR-associated trafficking protein that is critical to the promotion of NMDAR trafficking by PKC (6, 7). The PKC-dependent activation of the Src family of non-receptor protein kinases enhances NMDAR function mainly through increasing NMDAR channel gating (3, 8 -11). It is still uncertain, however, whether an NMDAR-associated signaling protein that interacts with PKC displays a similar enhancing effect in NMDAR trafficking.Numerous studies performed as early as the late 1980s support the idea of functional and positive cross-talk between CaMKII and PKC (12-15). One speculation based on these findings is that PKC and CaMKII act in series and share, at least partially, a common pathway by convergence in regulating certain common substrates. Interestingly, NMDAR could be such a common molecular target. Both PKC and CaMKII have phosphorylation sites on NMDAR. CaMKII is associated with both NR2A and NR2B NMDAR subunits (16 -18). PKC potentiates NMDAR gating and in turn enhances Ca 2ϩ influx and intracellular Ca 2ϩ /camodulin, which could trigger CaMKII autophosphorylation and increase association with NR2A and NR2B sub...

Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-d-aspartate Receptor Trafficking during Synaptic Plasticity

“…It has been proposed that F-actin polymerization is not only required for the stabilization of early LTP (16) but also supports the eventual capture of plasticity-related proteins involved in the stabilization of late phase LTP (27). NMDAR combined with autophosphorylated CaMKII translocates to the postsynaptic membrane following exposure to LTP-inducing stimuli, including PKC activation and burst stimulation (TBS) (7). Importantly, this NMDARCaMKII complex is proposed to serve as a "seed" for anchoring other plasticity-related proteins, including AMPA receptors (AMPARs) (28,29).…”

“…Regulation of TBS-induced NMDAR Synaptic Incorporation-Although NMDAR trafficking induced by PKC activation has previously been used as a model to investigate NMDAR trafficking (6,7), PKC activation may also exert broad and nonspecific effects on downstream effectors unrelated to NMDAR trafficking (53). Therefore, we examined whether the previously described observations obtained following PKC activation could be reproduced under more physiological conditions.…”

“…insertion of additional NMDARs is responsible for increased NMDAR function (7). Moreover, TBS induced a physiological form of LTP NMDA in hippocampal slices, which was abolished by pretreatment of the slices for 10 min with the myosin IIb inhibitor Blebb (10 M), the actin polymerization inhibitor LatA (10 M), or the MLCK inhibitor ML-7 (10 M) (TBS, 1.74 Ϯ 0.02; n ϭ 4, from four whole-cell recordings of four independent animals; p Ͻ 0.01, paired sample t test; Blebb ϩ TBS, 0.95 Ϯ 0.03; n ϭ 6, from six whole-cell recordings of five independent animals; p Ͼ 0.05, paired sample t test; LatA ϩ TBS, 1.01 Ϯ 0.01; n ϭ 5, from five whole-cell recordings of five independent animals; p Ͼ 0.05, paired sample t test; ML-7 ϩ TBS, 1.01 Ϯ 0.03; n ϭ 4, from four whole-cell recordings of four independent animals; p Ͼ 0.05, paired sample t test; Fig.…”

“…Notably, the insertion or internalization of NMDARs at the postsynaptic membrane is tightly regulated, both during development and in response to synaptic activity and sensory experience (3). PKC activation promotes new NMDARs to the surface of hippocampal neurons (4) and has been used as an important model to investigate NMDAR trafficking (5)(6)(7). Rapid channel insertion occurs through SNARE-dependent exocytosis and does not require the direct phosphorylation of NMDAR subunits; instead, it requires a receptor anchoring and/or trafficking protein (8), which suggests that PKC indirectly exerts its effect through interactions with NMDAR-associated signaling and/or protein trafficking (3).…”

“…We pretreated slices with PMA (0.5 M) for 20 min, which produces LTP NMDA with a saturated potentiation level (7). When the potentiation level reached a stable plateau after 20 min, the loading of recorded neurons with the active form of MLCK through the recording pipette to further induce MLCK activation failed to produce additional potentiation (1.00 Ϯ 0.02; n ϭ 7, from seven whole-cell recordings of six independent animals; p Ͼ 0.05, paired sample t test; Fig.…”