Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies.
Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.
Tingley
W G
WG
Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Ehlers
M D
MD
Kameyama
K
K
Doherty
C
C
Ptak
J B
JB
Riley
C T
CT
Huganir
R L
RL
D000595:Amino Acid Sequence; D000906:Antibodies; D001665:Binding Sites / Q000276:immunology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017459:Receptors, Amino Acid / Q000378:metabolism*
D000906:Antibodies; D017459:Receptors, Amino Acid; C031959:aspartic acid receptor; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C