Abstract
Ca2+ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors plays a pivotal role in synaptic plasticity during brain development as well as in mature brain. Cyclic AMP-dependent protein kinase (PKA) and members of the protein kinase C (PKC) family are also essential for various forms of synaptic plasticity and regulate the activity of different ion channels including NMDA and non-NMDA receptors. We now demonstrate that PKA and various PKC isoforms phosphorylate the NMDA receptor in vitro. The stoichiometry of [32P]phosphate incorporation per [3H]MK-801 binding site is greater than 1 for both PKA and PKC. Double immunoprecipitation experiments show that all three NMDA receptor subunits that are prevalent in the cortical structures, NR1, NR2A, and NR2B, are substrates for PKA as well as PKC. Two-dimensional phosphopeptide mapping reveals that the major phosphorylation sites for PKA and PKC differ for all three subunits. We provide evidence that some if not most of these sites are phosphorylated in the central nervous system of rats in vivo. The results presented in this article together with earlier electrophysiological experiments demonstrating that PKA and PKC activation increases the activity of NMDA receptors indicate that NMDA receptor potentiation can be mediated by direct phosphorylation by PKA and PKC. Collectively, these results strongly suggest that NMDA receptor functions such as control of neuronal development or expression of synaptic plasticity are modulated by PKA- and PKC-mediated phosphorylation of NMDA receptors.
Highlights
Analogous to other ligand-gated ion channels of the nicotinic acetylcholine receptor superfamily, glutamate receptors are thought to consist of five different subunits that are homolo
NMDA receptor-mediated currents were potentiated by protein kinase C (PKC) activators, and this potentiation was prevented by PKC inhibitors (H7, staurosporin, or sphingosine) in trigeminal [29, 30], spinal cord dorsal horn [31], and hippocampal neurons (Ref. 32, but see Ref. 33)
Two-dimensional Phosphopeptide Mapping—The method for the generation of phosphopeptide maps [47] was modified as follows: NMDA receptors were solubilized from rat brain membranes with deoxycholate, immunoprecipitated with ␣R1, phosphorylated with protein kinase (PKA) or PKC, and dissociated with SDS, and NR1, NR2A, or NR2B was reprecipitated with ␣R1, ␣2A, or ␣2B, respectively, as described above
Summary
No previous evidence has demonstrated that NMDA receptors are directly phosphorylated by PKA, it has been shown earlier by Tingley et al [16] that NR1 is phosphorylated by PKC in vitro and in cell culture upon stimulation with phorbol ester. It has been unknown whether PKC phosphorylates NR2 subunits. Back-phosphorylation of NMDA receptors immunoisolated from rapidly collected and homogenized rat brain in the absence and presence of phosphatase inhibitors shows that NR1 and NR2 subunits are phosphorylated in vivo at those sites recognized by PKA and PKC in vitro
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